Accessory Folding Proteins (Advances in Protein Chemistry; V. 44)

ADVANCES IN PROTEIN CHEMISTRY Volume 44

Accessory Fold ing Proteins

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ADVANCES IN PROTEIN CHEMISTRY EDITED BY C. B. ANFINSEN

JOHN T. EDSALL

Department of Biology The Johns Hopkins University Baltimore, Maryland

Department of Biochemistry and Molecular Biology Harvard University Cambridge, Massachusetts

FREDERIC M. RICHARDS

DAVID S. EISENBERG

Department of Molecular Biophysics and Biochemistry Yale University New Haven, Connecticut

Department of Chemistry and Biochemistry University of California, Los Angeles Los Angeles, California

VOLUME 44

Accessory Folding Proteins EDITED BY GEORGE LORIMER E. 1. du Pont de Nemours & Co. Wilmington, Delaware

ACADEMIC PRESS, INC. Harcourt Brace & Company San Diego New York Boston London Sydney Tokyo Toronto

This book is printed on acid-free paper. @

Copyright 0 1993 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

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9 3 9 4 9 5 9 6 9 1 9 8

MM

9 8 1 6 5 4 3 2 1

CONTENTS

ix

PREFACE

Mechanism of Enzymatic and Nonenzymatic Prolyl cis-trans lsomerization

Ross L. STEIN I. 11. 111. IV.

Introduction . Nonenzymatic Prolyl Isomerization . Enzymatic Prolyl Isomerization . Mechanism of Enzymatic Prolyl Isomerization: Catalysis by Distortion . References .

1 2

9 21 22

Prolyl lsomerases: Role in Protein Folding

FRANZ X. SCHMID, LORENZ M. MAYR,MATTHIASMUCKE, AND E. RALFSCHONBRUNNER I. 11. 111. IV. V. VI.

VII. VIII. IX.

Introduction . Prolyl Isomerization . Prolyl Isomerases . RNase T1 as Model System to Probe Catalysis of Folding . . Catalysis of Folding in Absence of Disulfide Bonds Catalysis of Prolyl Isomerization during Unfolding and Refolding . Simultaneous Action of Prolyl Isomerase and Protein Disulfide-Isomerase as Catalysts of Folding . . Role of Prolyl Isomerase for Cellular Folding Conclusions . References . Note Added in Proof .

25 26

31 36 42 44 51 54 59 62 65

vi

CONTENTS

Structure and Mechanism of 70-kDa Heat-Shock-Related Proteins

DAVIDB. MCKAY Overview of Stress-70 Proteins . Biochemical Activities of Stress-70 Proteins . Structure of Stress-70 Proteins . Enzymatic Mechanism of Stress-70 Proteins Modulators of Stress-70 Protein Activity . Epilogue . References .

I. 11. HI. IV. V. VI.

67 69 73 80 89 92 93

.

PapD and Superfamily of Periplasmic Immunoglobulin-like Pilus Chaperones SCOTTJ. HULTGREN, FRANCOISE JACOB-DUBUISSON, C. HALJONES, CARL-IVAR BRANDEN

I. 11. 111. IV. V.

. General Perspective Introduction . pup Gene Cluster . Postsecretional Assembly . Summary References . Note Added in Proof

AND

99 100 101 104 120 121 123

.

Protein Disulfide-lsomerase: Role in Biosynthesis of Secretory Proteins

NEILJ. BULLEID Introduction . Catalytic Properties of Protein Disulfide-Isomerase Cellular Properties of Protein Disulfide-Isomerase Role of Protein Disulfide-Isomerase in Intracellular Protein Folding . V. Multifunctionality of Protein Disulfide-Isomerase VI. Conclusions . References .

I. 11. 111. IV.

. . .

125 126 131 133 139 147 148

CONTENTS

vii

SecB: A Molecular Chaperone of Escherichia coli Protein Secretion Pathway DAVIDN. COLLIER I. Overview . Introduction 11. 111. Precursor Conformation Governing Signal Peptide Function . . SecB as Component of Secretion Machinery IV. . V. Properties o f SecB VI. SecB and Its Ligands Forming Isolable Complexes . VII. Nature of SecB Binding Sites . Other Chaperones and Protein Secretion VIII. IX. Recapitulation and Speculation . References .

152 152 155 157 162 169 171 180 184 189

AUTHOR INDEX

.

195

SUBJECT INDEX

.

209

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PREFACE

Anson and Mirsky in the 1930s and Lumry and Eyring in the 1950s had already demonstrated the reversibility of protein denaturation. However, a detailed analysis of the phenomenon was, of course, not possible in view of the complete absence of detailed information on the covalent structure of these macromolecules. In the late 1950s detailed structures of proteins began to appear, and a number of colleagues in my laboratory, including Michael Sela, Fred White, and Edgar Haber, could begin to examine the refolding of protein polypeptide chains, first with pancreatic ribonuclease (with its eight half-cysteine residues, which allowed 105 potential isomeric refolded molecules with four disulfide cross-linkages). Earlier studies on the incorporation of radioactive amino acids into the sequence of RNase had indicated that the time required for the total synthesis of the native, active enzyme was of the order of two minutes. However, regeneration of native, fully active protein required many hours during which slow reshuffling of disulfide bonds took place. A moderate shortening of this process could be achieved by the addition of catalytic amounts of mercaptoethanol. Robert Goldberger, Charles Epstein, and I finally resorted to the classical biochemist’s friend, homogenized rat’s liver, and obtained a purified and reasonably characterized enzyme, now called PDI, or protein disulfide-isomerase, which promoted the reshuffling process to yield the desired two-minute renaturation. The much more convenient staphylococcal nuclease, consisting of 149 residues in a single chain free of half-cysteine residues, could be shown to assume its fully active, mature conformation in less than 500 msec when the random chain at pH 3 was brought to pH 7 in a rapid mixing apparatus. The single tryptophan residue at position 145 in the chain was almost instantaneously brought from its initial hydrophilic to its native hydrophobic environment, permitting kinetic measurements of the extremely rapid increase in emission fluorescence at the low wavelength characteristic of full shielding from the aqueous environment. These observations led to the study of what has now become a field of wide interest-the “pathway(s) in the folding process.” This volume of Advances in Protein Chemistry contains discussions of a number of ancillary catalytic systems that may help us understand the great rapidity and steric specificity exhibited during the transition from unidimensional chaos to three-dimensional, functional structure. Several useful approaches have already begun to help clarify the problem. T h e “Chu-Fasman” rules offer a powerful initial screening in the ix

X

PREFACE

search for self-contained initiating sites, such as sections of helical and pleated sheet structure, that may undergo local assembly in an autonomous manner. A second approach involves the use of antibodies, whose antigenic-binding sites are so highly selective in terms of their stereospecificity for ligands. Some years ago David Sachs, Ann Eastlake, Alan Schechter, and I began to attack the problem using antibodies against staphylococcal nuclease. The numerous antibodies formed in sheep against the antigenic determinants of nuclease were first isolated on an affinity chromatography column of native nuclease attached to an agarose support. Using a series of subsequent columns bearing, as ligands, various fragments of the polypeptide sequence it was possible to isolate a nonprecipitating, enzyme-inactivating antibody against the fragment (99- 126) which, in the native protein, contains two short helical regions. T h e inactive nuclease-anti (99- 126) complex released free, active nuclease when the sterically random fragment 99- 149 (containing the 99- 126 determinant) was added. It would appear that the 99-126 portion “remembered” its original native format and could effectively compete with the intact nuclease structure for the antigenic site in question. Analysis of the stoichiometry between antibody, nuclease, and competing fragment indicated that the fragment was able to assume a native conformation about 0.02% of the time. Such a value, although low, is quite large relative to the likelihood of a peptide fragment of a protein being found in the native format on the basis of chance alone. It would seem likely that contiguity of a number of such selfdetermined nucleation sites might well result in an accelerating cascade of interactions that could help account for the remarkable speed of protein folding. Understanding of the additional control and facilitation of the overall process by catalysts of the sort described in this volume might soon lead to a true understanding of this fundamental problem.

C.B. ANFINSEN

MECHANISM OF ENZYMATIC AND NONENZYMATIC PROLYL CIS-TRANS ISOMERlZATlON By ROSS L. STEIN Department of Enzymology, Merck, Sharp and Dohme Research Laboratories, Rahway, New Jersey 07065

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

............................

2

11. Nonenzymatic Prolyl Isomerization

C. Acid and B D. Substituent

........................ cts . . . . . . . . . . .

IV. Mechanism of Enzymatic Prolyl Isomer References . . . . . . . . . . . . . . . . . . . . . . . .

I. INTRODUCTION The cis-trans isomerization of proline imidic peptide bonds (Scheme I) is a reaction of biochemical interest due to its often rate-limiting role in protein folding (see Schmid et al., this volume, Chapter 2). T h e central role that this reaction plays in biochemistry is further highlighted by the existence of a ubiquitous enzyme that catalyzes this reaction. This enzyme, peptidylprolyl cis-trans-isomerase (PPI, EC 5.2.1.Q was first described in 1984 (Fischer et al., 1984) and catalyzes prolyl isomerization in both peptides (Fischer et al., 1984) and proteins. Interest in PPI was sparked by the discovery that cyclophilin (CyP), the binding protein for the immunosuppresant drug cyclosporin A, is identical to PPI (Fischer et al., 1989b; Takahashi et al., 1989). Remarkably, FKBP, the binding protein for another immunosupressant, FK506, also possesses PPI activity (Harrison and Stein, 1990a; Sierkierka et al., 1989). Furthermore, the PPI activities of CyP and FKBP are potently inhibited by their respective ligands with no cross-inhibition (Fischer et al., 1989b; Harding et al., 1989; Harrison and Stein, 1990b; Sierkierka et al., 1989; Takahashi et al., 1989). In this review, I will describe our current understanding of the mechanism of both enzymatic and nonenzymatic prolyl isomerization. I will first review nonenzymatic prolyl isomerization and rotation about amide ADVANCES IN PROTEIN CHEMISTRY, Vol. 44

1

Copyright Q 1993 by Academic Press, Inr. All rights of reproduction in any form reserved.

2

ROSS L. STEIN

;+;g

trans

CIS

H

-

*

p2’

0

Rl

p,’

SCHEME 1. Prolyl isomerization

bonds. As we will see, amide rotation is mechanistically related to prolyl isomerization and serves as a useful model for prolyl isomerization. Here I will propose a common mechanism for these reactions and a structure for the rate-limiting transition state. This will be followed by a discussion of what is known of enzyme-catalyzed prolyl isomerization. A theme that will be developed is that the chemistry of nonenzymatic prolyl isomerization and the mechanisms of the nonenzymatic catalysis of this reaction dictate the catalytic strategy that is used by prolyl isomerases. Finally, this will lead to a mechanistic proposal for enzymatic prolyl isomerization. 11.

NONENZYMATIC PROLYLISOMERIZATION

To supplement the data on prolyl isomerization, I will draw on the literature describing rotation about the C-N bond in secondary amides. Early studies in this field were described by Stewart and Siddall in an excellent 1970 review. As we will see, these reactions are related to prolyl isomerization and support the mechanism to be proposed for prolyl isomerization. The mechanism is based on results from a variety of experimental approaches. In all cases, experiments employing kineticbased probes will be used to obtain an accurate picture of the activated complex in the rate-limiting transition state. The experiments that will be described include thermodynamics, in which activation parameters (i.e., AG$, AH$, and ASS) will be described; solvent effects, in which the influence of organic solvents and deuterium oxide will be reviewed; acid-base catalysis; substituent effects; and secondary deuterium isotope effects. A.

Thermodynamics

For a number of prolyl-containing peptides and secondary amides, the kinetics of C-N bond rotation were determined as a function of temperature and allowed the determination of the activation parameters: A&, AH*,and ASS (see Table I). The results clearly indicate that the

3

PROLYL ISOMERIZATION

TABLE I Activation Parameters for Prolyl Cis-to-Tram Isomerization Peptide

A& (kcal/mol)

AH* (kcal/mol)

(1) Suc-Ala-Leu-Pro-Phe-pNA (2) Suc-Ala-Ala-Pro-Phe-pNA (3) Gly-Gly-Pro-Ala (4) N,N-Dimethylacetarnide (5) Suc-Ala-Trp-Pro-Phe-pNA (6) Suc-Ala-Gly-Pro-Phe-pNA (7) Ac-Sar-OCH3

19.3 19.3 19.2 19.3 19.9 19.5 19.7

21.3 20.2 19.5 19.1 19.1 18.6 18.2

6.6 3.1 0.9 - 0.8 -2.6 -3.0 -4.9

(8)Gly-Pro ( 9 ) Ala-Pro (10) Val-Pro (11) His-Pro (12) Gly-Gly-Lys-Phe-Pro

20.0 21.0 21.2 21.6 21.7

20.0 18.9 18.8 18.9 16.1

0.0 -7.0 -8.1 -8.9 - 18.9

ASS (e.u.)

Ref. a a b C

a a d e

f

f g

h

" Harrison and Stein (1992).

'Grastoph and Wurthrich (1981). Drakenberg el al. (1972). Love et al. (1972). ' Cheng and Bovey (1977). Jacobson et al. (1984). R Galardy and Liakopoulou-Kyriakides (1982). Lin and Brandts (1983).

barrier to C-N bond rotation is entirely enthalpic: although the mean AH$ is 19 +: 1 kcal/mol, the mean -TASS is 0.0 ? 1.0 kcal/mol [T = 298°C; only compounds 1-7 (Table I ) were used in this average]. An entropy of activation of zero is consistent with a unimolecular reaction and a transition state with no solvent participation and little solvent reorganization. Although the data indicate that all of these reactions are enthalpy driven, they also suggest that there may be a mechanistic difference between isomerization of proline-containing oligopeptides and dipeptides, because for dipeptides cis-to-trans isomerization rates are lower (larger A&; see Table I). As Table I indicates, this is principally a consequence of more negative ASS values. T h e slower isomerization rates for Xaa-Pro dipeptides can be explained in terms of a ground state structure stabilized by intramolecular interactions between the charged amino and carboxy termini (Brandts et al., 1977; Evans and Rabenstein, 1975) and a transition state for isomerization in which these attractive interactions must be destroyed. These negative entropy of activation values may reflect an increase in solvation of the ionic amino and carboxylate groups. That this phenomenon may be related to intramolecular

4

ROSS L. STEIN

hydrogen bonding or ionic interactions is supported by the very negative value of AS$ for isomerization of Gly-Gly-Lys-Phe-Pro. For this peptide, the interaction between the &-aminoof Lys with the carboxylate of the C-terminal Pro may mimic the ionic interaction in dipeptides. The mechanistic difference between the di- and oligopeptides is graphically illustrated in the enthalpy-entropy compensation plot (Exnor, 1973) of Fig. 1. For the oligopeptides, the slope of the correlation, or the critical temperature T, (Exnor, 1973), is 232 ? 29 K , whereas for the dipeptides T, = 212 iz 27 K. These results support the notion that these reactions may fall into two classes, depending on the position of the proline residue. B . Solvent Effects

T w o types of solvent effects have been determined for prolyl isomerization and amide rotation: (1) the effect of solvent deuterium on reaction rate and (2)the effect of organic solvents on reaction rate. Solvent deuterium isotope effects are useful tools in probing the role of proton transfer

- - r 22 21

-E

-

n

0

20-

-r

0 0

19-

Y 18-

I -4

1716 -20

-15

-10

-5

0

5

10

FIG. Enthalpy-entropy compensation for nonenzymatic prolyl isomerization (see Table I for literature references). 0 , Proline-containing oligopeptides; A, dipeptide; A, Gly-Gly-Lys-Phe-Pro. 1, Suc-Ala-Leu-Pro-Phe-pNA; 2, Suc-Ala-Ala-Pro-Phe-pNA; 3, Gly-Gly-Pro-Ala;4, N,N-dimethylacetamide;5, Suc-Ala-Trp-Pro-Phe-pNA; 6, Suc-Ala-GlyPro-Phe-pNA; 7, Gly-Pro; 8, Ala-Pro; 9, Val-Pro; 10, His-Pro; 11, Gly-Gly-Lys-Phe-Pro. Linear regression analysis of the data for compounds 1-6 yields a slope or critical temperature, Tc,of 232 f 29 K; analysis of the data for compounds 7-11 yields T, = 2 12 & 27 K.

PROLYL ISOMERIZATION

5

in reactions (Quinn and Sutton, 1991). For prolyl isomerization, the effect of heavy water on reaction rates is negligible, with kHpO/kDgO equal to 1.1 0.1 (Harrison and Stein, 1990a, 1992). Along with the absence of a pH effect between 5 and 9 (Harrison and Stein, 1990a, 1992) (see below), these results indicate that prolyl isomerization proceeds without general-acid/general-base catalysis or the involvement of solvent. T h e effect of organic solvents on the rate constant for amide rotation in N,N-dimethylacetamide (DMA) has also been investigated (Drakenberg et al., 1972). As the solvent is changed from water to acetone to cyclohexane, first-order rate constants for rotation increase from 0.025 to 0.33 to 1.5 sec-'. This observation that nonpolar solvents increase reaction rates indicates that the transition state for amide rotation is nonpolar relative to the reactant state and, thus, is stabilized in nonpolar solvents. This transition state is presumably characterized by partial rotation about the amide bond. In this transition state, polar resonance structures for the amide bond no longer exist and, thus, the transition state is less polar than the reactant state. The 60-fold rate acceleration that accompanies transfer of DMA from water to cyclohexane will provide an important clue in understanding enzymatic prolyl isomerization (see below).

*

C . Acid and Base Catalysis

Rate constants for prolyl isomerization are independent of pH from pH 5 to 9 (Harrison and Stein, 1990a, 1992) and support a transition state structure for amide bond rotation that is characterized by partial rotation about the bond with no nucleophilic participation by solvent and no significant solvent reorganization. However, at extremes of pH, this mechanism changes. At acid pH, both cis-trans prolyl isomerization (Berger et al., 1959; Steinberg et al., 1960) and C-N bond rotation in DMA are accelerated (Gerig, 1971). For example, the free energy barrier to rotation in DMA decreases from 19.3 kcal/mol at pH 7.0 to 16.4 kcall mol at pH 1.8 (Gerig, 1971), which translates to a 130-fold increase in rate constant from 0.051 to 6.7 sec-' (T = 25°C). Although the carbonyl oxygen is thought to be the predominant site of protonation in acid (Challis and Challis, 1979; Homer and Johnson, 1970), the mechanism for acid-catalyzed C-N bond rotation must involve the relatively rare Nprotonated species (Martin, 1972) in which rotation becomes freer due to the loss of double-bond character that the C-N bond experiences on N protonation. This is illustrated in Scheme 11, where K, is the acid dissociation constant for the amide protonated on the oxygen, K,,,,, is the equilibrium constant for the tautomerization of the protonated amide and equals [NH+]/[OH+],and KNH+is the acid dissociation constant for

6

ROSS L. STEIN

'OH

0 h u t .

R R'

Ka

R'

It

L SCHEME 11. Mechanism for acid-catalyzed prolyl isomerization.

the amide protonated on the nitrogen and equals K,/KtaU,,. Typical values of K, range from 1 to 10' M (-2 < pK, < 0) and we can assume that K,,,,, is less than 0.1. Thus, KNH+will probably be greater than 10' M (PKNH+< -2)Now, if we take amide rotation in N,N-dimethylacetamide as an example, we can assume that ko = 0.05 sec-', which is the observed rate constant at neutral pH. Knowing that at pH 1.8 kObs= 6.7 sec-' allows us to calculate that k+/KNH+ = 420 M - ' sec-' [see Eq. (l)].

Assuming that K N H + is greater than lo2 M we see that k+ will be greater than or equal to 42,000 sec-'. Thus, k+/k, 2 lo6, which represents the rate acceleration from acid catalysis. This will become important when we discuss the origins of enzymatic catalytic power for prolyl isomerases. At alkaline pH, an interesting situation occurs. The activation parameters for C-N rotation in DMA at pH 11.8 are AGS = 19.0 kcal/mol, AH$ = 16.3 kcal, -TASS = 2.7 kcal/mol (Gerig, 1971). T h e large contribution to ACS that is made from the entropy term may indicate nucleophilic participation of hydroxyl leading to the formation of a tetrahedral adduct. At the stage of this tetrahedral species, the C-N bond has lost all double-bond character and rotation will be energetically more favorable. This may be reflected in the low AH$value.

7

PKOLYL ISOMERIZATION

D. Substituent Effects

To probe the transition state structure for these reactions further, the effect of para substituents on amide rotation rates was measured for a series of N,N-dimethylbenzamides (Berarek, 1973). When the data are correlated with wp (Ritchie and Sager, 1964), a p value of - 1.14 0.06 is obtained (see Fig. 2). The negative p value indicates that electrondonating substituents accelerate the reaction. This can rationalized in the context of Scheme 111, where resonance forms for these substrates are shown. The rotational barrier about the C-N bond is decreased as resonance forms I and I11 predominate. If R is electron donating, these resonance forms will contribute more to the structure of the amide than will I1 and C-N rotation will therefore be accelerated.

*

E . Secondary Deuterium Isotope Effects The single most revealing mechanistic parameter for prolyl isomerization and amide rotation is the secondary deuterium isotope effect. In general for such studies, the hydrogens on the carbon that is bonded to the carbonyl carbon of the amide or imide (the "P-hydrogens") are substituted with deuterium and reaction rate constants are measured for

1.5' I -1 .o

I

-0.5

I

0.0

I

0.5

I

1 .o

FIG.2. Linear free energy correlation for amide rotation in N,N-dimethylbenzamides. Rate constants were taken from the work of Berarek (1973) and correlated with upvalues (Ritchie and Sager, 1964) to obtain a p value of - 1.14 ? 0.06 (I" = 0.986).

8

ROSS L. STEIN

+ k = ( =y - - x - Q ( N-

N-

:+x-

+N-

I

1

I I11

I

I1

SCHEME 111. Resonance forms for 4-substituted N,N-dimethylbenzamides.

the normal and isotopically substituted compounds. The ratio of rate constants, k,lkD, is a sensitive indicator of transition state structure. Secondary deuterium isotope effects have been measured for the cisto-trans prolyl isomerization of Suc-Ala-Gly-cis-Pro-Phe-pNA (where pNA is p-nitroanilide) and C-N rotation in DMA. In the former case, the isotope effect for the two hydrogens of glycine is 1.05 2 0.02 ( Fischer et al., 1989a; Harrison et al., 1990; Harrison and Stein, 1990a), and for C-N rotation in DMA, the isotope effect for the three hydrogens of the acetyl moiety is 1.10 0.05. Significantly, the effect for two deuteriums in DMA can be calculated to be 1.05 (Fujihara and Schowen, 1985). These isotope effects indicate a transition state in which the force field associated with the 0-hydrogens is weakened relative to the force field of the reactant state. In the framework of the hyperconjugation model of 0-deuterium isotope effects, these isotope effects suggest that the hyperconjugation between the 0-hydrogens and the carbonyl group is enhanced in the transition state relative to the reactant state. This situation would obtain if the transition state were characterized by partial rotation about the C-N bond (see Scheme IV). Partial rotation would destroy the double-bond character of the C-N bond, enhance the doublebond and “ketone” character of the carbonyl, and, thus, enhance the hyperconjugative interaction between the 0-hydrogens and the carbonyl group. These isotope effects allow us to eliminate mechanisms involving nucleophilic participation by solvent because isomerization by any of these mechanisms would involve the intermediacy of a tetrahedral adduct and a transition state that would be characterized by a decreased ability of the @-hydrogensto hyperconjugate. Mechanisms of this sort generate

*

SCHEME IV. Transition state structure for prolyl isomerization.

PROLYL ISOMERIZATION

9

inverse isotope effects (i.e., k , / k , < 1) and are the rule in acyl addition and amide and ester hydrolyses.

F. Mechanistic Proposal for Prolyl Isomerization The available data suggest that in aqueous solution and at neutral pH prolyl isomerization proceeds according to a simple, one-step mechanism. Solvent water does not participate in the reaction and there is no accumulation of intermediates. T h e energy barrier to isomerization is enthalpic and represents the energy of resonance stabilization that is possessed by the C-N imide bond. The energy barrier to isomerization is lowered when substrates are transferred either to acidic or organic solutions. In acidic solution, rate acceleration occurs because an alternate, low-energy pathway is provided by protonation to produce a substrate in which the C-0 bond is more ketonelike and the C-N bond more aminelike, thereby destroying resonance stabilization. In organic solutions, rate acceleration occurs as a result of transferring the substrate from a nonpolar transition state to a hydrophobic environment. We will see below that enzymatic strategies for catalysis may exploit both of these chemical mechanisms.

111. ENZYMATIC PROLYL ISOMERIZATION

As indicated above, the barrier to prolyl cis-trans isomerization is the resonance stabilization energy that is possessed by the C-N imide bond. The task of a prolyl isomerase is, therefore, to develop an enzymatic-chemical strategy that will result in the lowering of this barrier. When one reflects on the strategies that might be used by an enzyme, one realizes that there are two general mechanisms: catalysis by distortion and nucleophilic catalysis (see Scheme V). In a mechanism involving nucleophilic catalysis, the enzyme would promote nucleophilic attack on the carbonyl carbon to produce a tetrahedral intermediate. In this intermediate, resonance stabilization of the C-N bond has been destroyed and the barrier to rotation about the C-N bond greatly reduced. Collapse of the tetrahedral intermediate with expulsion of nucleophile can produce either the cis or trans Xaa-Pro peptide. In the original work on PPI (Fischer et al., 1984), results were presented that indicated that an enzyme sulfhydryl group was required for activity. This result was later interpreted to support a mechanism involving nucleophilic catalysis (Fischer et al., 1989a,b). In contrast, according to mechanisms involving catalysis by distortion,

10

[B-:,~s ROSS L. STEIN

#

Pistortionby Catalvsis

//

\\

SCHEME V. Mechanistic alternatives for prolyl isomerases.

the enzyme induces strain or distortion in the substrate. This can be the result of geometric, desolvation, or electrostatic destabilization but is dependent on the binding energy between enzyme and substrate, since such destabilization that is induced in the substrate will only result in rate enhancement if it is “paid” for by binding energy. In this section, we review the available data that address questions of the enzyme mechanism for prolyl isomerization. As we will see, these data overwhelmingly support a mechanism involving catalysis by distortion.

PROLYL ISOMERIZATION

11

A . Secondary Deuterium Isotope Effects As we saw with nonenzymatic prolyl isomerization, the most compelling mechanistic information for the enzymatic reaction comes from the secondary deuterium isotope effect determinations. The first isotope effect in this area was reported to be 0.91 ? 0.01 for the CyP-catalyzed cis-totrans isomerization of Suc-Ala-Gly-(L,L)-cis-Pro-Phe-pNA (L = H, D) (Fischer et al., 1989a). An inverse isotope effect of this magnitude was correctly interpreted by Fischer to indicate a mechanism of isomerization involving nucleophilic catalysis. However, this isotope effect has never been repeated. We found this isotope effect to be large and normal and equal to 1.13 2 0.01 (Harrison and Stein, 1990a). This value was later confirmed by a double-label technique to be 1.11 2 0.02 (Harrison et al., 1990) and was found to be temperature independent and equal to 1.14 2 0.02, 1.13 ? 0.01, and 1.14 ? 0.03 at 2, 10, and 30°C, respectively (Harrison and Stein, 1992). Furthermore, an isotope effect of 1.12 ? 0.02 has been determined for the FKBP-catalyzed reaction (Dr. David Livingston, personal communication, 1992).Large, normal isotope effects of this magnitude rule out mechanisms involving nucleophilic catalysis but favor catalysis by distortion in which the enzyme induces strain or distortion in the substrate (Harrison and Stein, 1990a). While the discrepancy still remains between the original inverse and later normal isotope effects, the normal isotope effects are more consistent with the growing body of data that will be presented below. Thus, we are compelled to assign the isotope effect for both CyP and FKBP catalysis a value of 1.12. An important point of mechanistic interest is the difference in magnitude between the isotope effects for the nonenzymatic and enzymecatalyzed reactions, which are 1.05 and 1.12, respectively. Because the reactant state is the same for both reactions, that is, free, uncomplexed substrate, the difference in isotope effect must signal a difference in transition state structure. The larger isotope effect for the enzymecatalyzed reaction indicates that the transition state force field associated with the hydrogens of the glycine is looser relative to the nonenzymatic reaction. This translates into greater hyperconjugative delocalization of P-CH electrons in the transition state for the enzymatic reaction. One explanation for this involves a model in which aqueous solvation impedes hyperconjugation of the P-CH electrons into the carbony1 group. Loss of solvation on binding of the substrate in a hydrophobic enzyme active site would then increase hyperconjugation. In a purely aqueous environment, the transition state would still be solvated and the loss of hyperconjugation would not be as great as in the enzymatic case. Solvation isotope

12

ROSS L. STEIN

effects such as this have been observed on transfer of carbonyl compounds from water to hydrophobic solvents (Kovach and Quinn, 1983). In these cases, normal P-deuterium isotope effects on the order of 1-2% per deuterium were measured. The investigators suggest that these effects originate from a smaller hyperconjugative release of P-CH electrons into the carbonyl center in water than in organic solvents. For prolyl isomerase, transfer of substrate to a hydrophobic active site will not only explain our large, normal isotope effects but also helps to account for how the enzyme effects catalysis, since, as we pointed out above, this will stabilize the apolar transition state and thus lead to rate enhancement (Drakenberg et al., 1972; Radzicka et al., 1988).

B . Additional Probes of Mechanism I . Substrate Specifcity Studies on the substrate specificity of an enzyme aim to probe the structural requirements for efficient catalysis. In such studies, it is best to correlate the kinetic parameter K,IK, with substrate structure and not K,, because the latter is often compromised by nonproductive binding (Fersht, 1985). For the PPIs, specificity studies have probed three general structural domains of simple peptide substrates: (1) length of the substrate, (2) P,’ residue’ (i.e., Pro replacements), and, (3) P, residue. In the earliest study on the PPI activity of CyP (Fischer et al., 1984), Fischer made the observation that the length of the peptide substrate is important for efficient catalysis. In the series Glt-(Ala),,-Pro-Phe-pNA, k,lK, values are similar for n = 2 and 3, but are about 10-fold larger than n = 1. These results suggest the existence of distinct subsites at the active site of CyP that are able to interact with the amino acid residues of the substrate. Energy released from favorable interactions of the substrate at these remote subsites is then used for catalysis. Thus, CyP utilizes binding energy to stabilize catalytic transition states (Jencks and Page, 1972).

Fischer has investigated the effect of proline replacements on isomer-

’

For reference to amino acid residues of prolylisornerase substrates and their corresponding enzyme subsites, we will adopt the nomenclature system of Schecter and Berger (1967) that is commonly used in protease chemistry. According to this system, if isomerization occurs at the P,-PI’ bond, and Pi‘is Pro, the amino acid residues of the peptide . .P,, while the corresponding enzyme substrate are named P,. . .P3-P,-Pl-Pl’-P,’-P3’. subsites are named S,; .S3-S2-Si-SI’-S,’-S3’. . .S,.

13

PKOLYL ISOMEKIZATION

TABLE I1 Rate Cosntants for Enzymatic and Uncatalyzed Prolyl Isomerization in Peptides of General Structure: Suc-Ala-Ala-Xaa-Phe-pNA Xaa

-?

k,,,,,

(sec-I)

0.12

k,lK,,, ( p M - ' sec-I) 0.01

0

2?

0.0 1

11

0

9 0

0.1

0.0 1

0.03

0.01

ization rate constants (Dr. Gunter Fischer, personal communication). His findings are summarized in Table I1 for both uncatalyzed isomerization and catalysis by CyP and indicate that this enzyme is highly specific for the five-membered ring of proline. It is interesting that inherent reactivity toward isomerization is not manifested at all in enzymatic rate constants; that is, while isomerization about the Ala-Pro bond proceeds with the slowest uncatalyzed rate, it is catalyzed by prolyl isomerase to the greatest extent. This is another reflection of the finely tuned specificity of this enzyme. Finally, several investigators have examined the reaction of these prolyl isomerases toward substrates of general structure Suc-Ala-Xaa-Pro-PhepNA. In a 1989 paper, Fischer investigated CyP-catalyzed isomerization for Xaa = Ala, Gly, Ser, and Val and found values of k,lK, of 6.6, 2.1, 5.4, and 8.8 p M - ' sec-', respectively. He interpreted these results in terms of a very broad specificity for CyP. These results were later confirmed by a larger set of substrates (Harrison and Stein, 1990b, 1992) (see Table 111).

14

ROSS L. S T E I N

TABLE 111 Specificities of PPI Activities of Cyclophilin and FKBP toward Substrates of Structure: Suc-Ala-Xaa-Pro-PhepNA" k,lk,, (d-' sec-I)

Xaa

CYP

FKBP

Gly Ala Val 1 le Nle Leu Phe TrP His

1200 3200 3200

1.2 53 170 320 330 640 620 110 28 28 0.6

LYS

Glu "

2700 1400 360 600 920 2100

pH 7.8, T = 10°C.

In contrast to these results for CyP, FKBP has a quite narrow P, specificity" and reacts fastest with substrates having a hydrophobic residue at Xaa (Harrison and Stein, 1990b, 1992) (see Table 111). We attempted to quantitate this correlation by calculating high-performance liquid chromatography (HPLC) capacity factors, R' (McCall, 1975), for each of the peptides. For a given substance, R' is equal to (TK - To)/To, where T , is the elution time of a retained substance (the peptide) and Tois the elution time of an unretained substance, in this case simply the solvent front. R' reflects the hydrophobicity of a substance: the larger the value of R' for a peptide, the more hydrophobic is the peptide. Thus, we anticipated that the logarithm of R', which is proportional to the free energy of transfer from the aqueous to the hydrophobic phase, might correlate with the logarithm of k,lK,. This correlation is shown in Fig. 3 and, when the outliers Xaa = Gly and T r p are excluded, is quite good (slope = 3.5 0.1; y intercept = 2.5 5 0.2).

*

2 . pH Dependence The pH dependence of kinetic parameters frequently provides useful mechanistic information about enzymatic reactions (Cleland, 1975,

* These data are for the native human FKBP and were reproduced by Schreiber and colleagues for the human recombinant FKBP (Albers el al., 1990).

15

PROLYL ISOMERIZATION

1982). At the very least, these studies tell us the ionization state that the enzyme and/or substrate must be in for catalysis and will often reveal the identity of important ionizing active site residues and changes in ratelimiting step and mechanism. For the CyP- and FKBP-catalyzed isomerization of Suc-Ala-Xaa-Pro-Phe-pNA (Xaa = Ala and Leu, respectively), k,lK, is independent of pH from 5 to 10 (Harrison and Stein, 1990a, 1992). This result is inconsistent with a mechanism involving nucleophilic catalysis because this is the pH range where most enzyme active site nucleophiles (e.g., His, Cys, and Lys) normally ionize. However, one cannot exclude unusual ionizations of these residues outside this pH range. This result is also inconsistent with highly basic nucleophiles, such as the hydroxyl of Ser, whose nucleophilic attack would be expected to be subject to general-base catalysis. In these cases, ionization of the general base would also be expected to occur in this pH range and reveal itself kinetically.

3. Solvent Deuterium Isotope Effects Enzymatic reactions conducted in heavy water frequently proceed with rate constants that differ from those of reactions in normal water, and the ratio of these rate constants, kHpO/kD20,o r the solvent deuterium

I

I

-0.2 0.0

I

I

I

I

I

0.2

0.4

0.6

0.8

1.0

1.2

Iog (R') FIG.3. Dependence of k,lK, for the FKBP-catalyzed cis-to-trans isomerization of SucAla-Xaa-cis-Pro-Phe-pNA on the hydrophobicity of o f Xaa. Hydrophobicity was quantitated as R ' , the HPLC capacity factor (McCall, 1975). Data for the outliers, Xaa = Gly and Trp, were not used in the linear regression analysis (shown as the solid line).

16

KOSS L. STEIN

isotope effect, is diagnostic of reaction mechanism (Quinn and Sutton, 1991). Solvent deuterium isotope effects on k J K , for the CyP-catalyzed cis-to-trans isomerization of Suc-Ala-Ala-cis-Pro-Phe-pNA and the FKBP-catalyzed cis-to-trans isomerization of Suc-Ala-Leu-cis-Pro-PhepNA (Harrison and Stein, 1990a, 1992) as well as the solvent isotope effect on k, for the Cy P-catalyzed cis-to-trans isomerization of Suc-AlaAla-cis-Pro-Phe-pNA (Kofron et al., 1991) are all near unity. A solvent isotope effect of unity indicates that in the rate-limiting transition state, proton transfer o r reorganization is not occurring and suggests the absence of general-acid/general-base catalysis. Again, this is inconsistent with mechanisms for PPI involving nucleophilic catalysis in which the attack of the nucleophile is general-base catalyzed. 4 . Thermodynamics

To probe the mechanistic origins of the substrate specificity differences at P, between CyP and FKBP, activation parameters were determined for reaction of these enzymes with several substrates (Harrison and Stein, 1992). These parameters were calculated from Eyring plots that were based on the temperature dependence of k,/K,. Although the plots were linear for reactions of FKBP, and thus readily provided values of AH* and ASS, they were curved for reactions of CyP (Harrison and Stein, 1992). A detailed analysis of the Eyring plots for CyP catalysis suggests that the nonlinearity can be accounted for by a mechanism in which the CyP exists in two interconvertible forms, only one of which is enzymatically active. This analysis allowed values of AGS, AH$, and ASS to be extracted from the experimental data (Table IV).

TABLE 1V Thermodynamic Parameters for PPI-Catalyzed Cis-to-Trans lsomerizalion of

Suc-Ala-Xaa-cis-Pro-Phe-pNA AH* (kcal/mol)

ASS

Enzyme

Xaa

CYP CYP

GlY Ala

CyP CyP

=rP

FKBP FKBP FKBP

TrP Ala

11

- 25

12

-21

Leu

15

'I

Leu

3.2 4.3 7.5 7.9

Calculated with T = 283 K.

(e.u.)

- 47

-41 - 34 - 29

- 8.6

ACS" (kcal/mol) 17 16 17 16 18 18 17

PROLYL ISOMERIZATION

17

As previously outlined, values of k,/K, for reactions of FKBP increase with increasing hydrophobicity of the P, residue. When A& values for these reactions are dissected into AH$ and ASS values (see Table IV), we see that these reactions proceed with large values of AHf and ASS. In contrast, k,lK, values for reactions catalyzed by CyP have no significant dependence on the PI residue and these reactions proceed with more negative values of AH$ and ASS. Now, if we assume that the active sites of these enzymes have a hydrophobic pocket at S, as well as discrete subsites for substrate amino acids, we can explain these results by assigning different levels of importance to these different modes of interaction for the two enzymes. T o account for the PI specificity of FKBP, we not only assume a more prominent role for PI-S, interactions but also that these interactions are characterized by dehydration of the Michaelis complex, E:S, as it proceeds to the transition state, [E:S]*. What we are suggesting here is that in E:S, the PI residue is not yet buried in S, and that the active site and the substrate are still at least partially solvated. As E:S proceeds to [E:S]S, the PI residue becomes buried in the S, pocket and the residual water of solvation is expelled from the active site. This scenario can reasonably account for the large values of AH$ and AS* that we observe for reactions of FKBP, since the formation of hydrophobic contacts between apolar groups in aqueous solution is known to be accompanied by positive enthalpy and entropy changes (Nemethy, 1967). Likewise, to account for the lack of PI specificity for CyP, we assume that subsite interactions play a more prominent role than do P,-Sl interactions. Thus, the PI-S, hydrophobic interactions that dominate the thermodynamic parameters for FKBP have a smaller role for this enzyme. Another point of mechanistic interest is the enthalpy-entropy compensation that we observe for reactions of both CyP and FKBP (Fig. 4). This compensation even includes a reaction that is catalyzed by recombinant human FKBP (rhFKBP) (Albers et al., 1990). The molecular origins of enthalpy-entropy compensation in enzyme catalysis are unclear (Exnor, 1973; Leffler and Grunwald, 1963; Lumry and Rajender, 1970; Schowen, 1967). T h e simplest explanation that one can advance involves a situation in which stronger transition state interactions between enzyme and substrate, which will manifest in lower AH* values, are accompanied by greater restrictions of translational and rotational freedom, which will manifest in more negative ASS values (Leffler and Grunwald, 1963; Schowen, 1967). That there are two separate correlations for the two enzymes suggests consistency with the mechanistic and specificity differences that we described above.

18

ROSS L. STEIN

20

I

I

I

I

I

Leu 15-

-

Ala

TrP 10-

50 --

-

//I

GlY AII a

I

I

I

-

FIG. 4. Enthalpy-entropy compensation for PPI catalysis. Data taken from Table IV. 0, F K B P 0, rhFKBP (Albers et al., 1990);A, CyP.

5. Inhibition Studies CyP and FKBP are potently inhibited by cyclosporin A and FK506, respectively (see Scheme VI for structures), and although a great deal has been learned about the interaction of these enzymes and inhibitors (Jorgensen, 1991; Wuthrich et al., 1991),it is unclear if these observations have provided any insights into the catalytic mechanism. However, one interesting case wherein inhibition data may provide insight into catalysis is a I3C NMR experiment performed by Schreiber and colleagues (Rosen et al., 1990). These investigators demonstrated that [8,9-'3C]FK506 does not undergo a hybridization change from sp2 to sp3 at either the C-8 or C-9 carbonyl positions on its binding to FKBP. Given the assumption that the interaction of one of these carbon atoms with FKBP mimics the interaction of the carbonyl carbon of Xaa-Pro of substrates, this experiment argues against nucleophilic catalysis. If FKBP enlisted an active site nucleophile during catalysis, one might have anticipated that this nucleophile would have attacked C-8 or C-9 with the resultant formation of a tetrahedral, sp3-hybridized adduct. 6 . Structural Studies As outlined above, a variety of mechanistic probes have been applied to the prolyl isomerases, and while they allowed us to eliminate many of the standard ways that enzymes effect catalysis (e.g., general-acid/

PROLYL ISOMERIZATION

19

FK506

Cyclosporin

SCHEME VI. Structures of FK506 and cyclosporin A.

general-base catalysis),these studies still did not provide a clear mechanistic picture. The problem is that the reaction catalyzed by immunophilins provides no mechanistic “handles”; that is, enzymatic prolyl isomerization is not accompanied by covalent bond changes, accumulation of intermediates, spectral changes of cofactors, nor any other signature of mechanism. However, the three-dimensional structures of these proteins, and the structures of these proteins complexed with substrates or inhibitors, may lead to the identity of catalytic active site residues and, therefore, to a clearer picture of mechanism. Such structures are being studied: both the solution structure of the FKBP (Michnick et al., 1991; Moore et al., 1991) and the crystal structure of the complex of FKBP and FK506 (Van Duyne et al., 1991) have been reported. The NMR and molecular dynamic studies that resulted in the solution structures of FKBP reveal several unusual features (Michnick el al., 1991;

20

ROSS L. STEIN

Moore et al., 1991). The secondary structure of FKBP is about 35% p sheet and less than 10% helix. FKBP has an unprecedented antiparallel P-sheet folding topology that results in the crossing of the two loops that connect strands of the sheet. This structural motif creates a hydrophobic cavity that is lined by a conserved array of six of the nine aromatic amino acids of the proteins. This cavity is the binding site for FK506 and probably is the isomerase active site. Although the structure of uncomplexed FKBP is clearly informative, one can argue that the structure that is most likely to provide mechanistic insights is the structure of the FKBP:FK506 complex (Van Duyne et al., 1991). Central to our understanding of how FKBP might catalyze cis-trans isomerization is the way in which this protein binds FK506 at the pipecolic amide (C-1 to C-8) and keto (C-9) moieties. This structural unit is buried in the hydrophobic cavity of FKBP, with the pipecolinyl ring buttressed u p against the indole ring of Trp-59 and the oxygen of the amide carbonyl of C-8 hydrogen bonded to the ring OH of Tyr-82. Of greatest significance is the disposition of the C-9 carbonyl. In solution, the C-8 and C-9 carbonyls are orthogonal and this is maintained in the structure of the complex. In this complex, there are no hydrogen bonds to the C-9 keto oxygen, but rather this oxygen finds itself buried among the three hydrophobic rings of the conserved amino acids Phe-99, Tyr26, and Phe-36. This binding configuration immediately suggests a mechanism for catalysis (Stein, 1991). According to this mechanism, when a prolinecontaining peptide, R-Xaa-Pro-R', binds to FKBP, the amide oxygen of Xaa is inserted into the same hydrophobic hole that contains the keto oxygen of C-9. Transfer from aqueous solution to this very hydrophobic environment destabilizes amide resonance structures in which the oxygen is negatively charged and favors resonance structures in which the amide carbonyl is more ketonelike. This is consistent with the observations that the rate constant for C-N bond rotation in DMA is accelerated in nonpolar solvents (Drakenberg et al., 1972). At the same time, the nitrogen atom of the proline ring, N-7, loses sp' character and resembles a cyclic tertiary amine. The picture is complete when Tyr-82 donates a hydrogen bond to N-7, thereby enforcing the developing sp3 character of this atom. This is equivalent to the observed acid catalysis for both prolyl cis-trans isomerization (Berger et al., 1959; Steinberg et al., 1960) and C-N bond rotation in DMA (Cerig, 1971). With these interactions, the bond between Xaa and Pro will have lost resonance stabilization and double-bond character. The energy barrier to rotation about this bond will be much lower than rotation about an amide bond and, thus, FKBP will have effected catalysis. This mechanistic hypothesis has value in that it explains the perplexing

PROLYL ISOMEKIZATION

21

observation noted above that the secondary deuterium isotope effect for enzymatic prolyl isomerization [K,lK, = 1.13 for cyclophilin (Harrison et al., 1990; Harrison and Stein, 1990a) and k H / k , = 1.12 for FKBP (D. Livingston, personal communication, 1992)]is unaccountably larger than the isotope effect for nonenzymatic isomerization [k,/k, = 1.05 (Fischer el al., 1989a; Harrison et al., 1990; Harrison and Stein, 1990a)l. The hydrophobic environment in which the amide bond finds itself in the enzyme active site will stabilize a transition state with less polar character than the transition state for reaction in solution. As we discussed above, this will magnify the enzymatic isotope effect (Kovach and Quinn, 1983).

7. Mutagenesis T o test the hypothesis that a cysteine is essential for catalysis by CyP, the four cysteines at positions 52, 62, 115, and 161 of human CyP were mutated individually to alanine (Liu et al., 1990). For all four mutants, k J K , for prolyl cis-to-trans isomerization of Suc-Ala-Ala-cis-Pro-PhepNA is equal to about 15 pM-' sec-'. From these results it is clear that the cysteines play no essential role in catalysis and thus rule out mechanisms involving nucleophilic catalysis in which a tetrahedral hemithioorthoamide is formed (Fischer et al., 1989a,b). Note, however, that these mutagenesis experiments alone cannot rule out mechanisms involving other nucleophiles. MECHANISMOF ENZYMATIC PROLYL ISOMERIZATION: CATALYSIS BY DISTORTION T h e catalytic strategy that an enzyme develops over evolutionary time is dictated by the chemistry of the reaction being catalyzed. The prolyl isomerases that have been studied to date are able to simply stabilize the nonenzymatic transition state without formation of covalent intermediates. Based on a k,,, value of lo4 sec-' for CyP (Harrison and Stein, sec-' for the cis-to-trans 1992; Kofron et al., 1991) and a k,,,., of isomerization of Suc-Ala-Ala-cis-Pro-Phe-pNA, we calculate an acceleration factor, k,,t/k,,,,,, of lo6, which corresponds to a transition state stabilization free energy of over 8 kcal/m01.~In this final section, we will summarize how the prolyl isomerases bring about this acceleration. IV.

Kofron and co-workers report a catalytic acceleration factor of 5 X loti (Kofron et al., 1991). from which they erroneously calculate an enormous stabilization energy of 16 kcal/mol (T = O O C ) . The actual stabilization energy is calculated using a simple expression: AAGZ = AG$,,,,- A&, = RT[ln(k,,,/k,,,,,)] = RT ln(5 x lo6) = 8.4 kcal/mol (T = 0°C).

22

ROSS L. S I E I N

The available data support a mechanism involving catalysis by distortion in which the enzyme binds and stabilizes a transition state that is characterized by partial rotation about the C-N amide bond. The energy that is required to distort this bond out of planarity with the C=O bond, thereby destroying the resonance stabilization of the amide linkage, is supplied by favorable transition state binding interactions between enzyme and substrate. As Lumry states (1986), “mechanical distortion as a source of small-molecule reactivity is attractive as a basis for enzymatic catalysis. It is quite realistic to assume that a distorted substrate will have enhanced reactivity, either because its ground state or the activated complex for its chemical reaction or both are altered by strain and stress in the protein conformation.” However, as mentioned previously, this distortion need not be the result of mechanical deformation but could also be the result of desolvation or electrostatic destabilization. In fact, the current data support contributions from all three mechanisms for distortion. A role for mechanical distortion is consistent with the existence of an extended substrate binding site (Fischer et al., 1984; Fischer, 1989) and an S, pocket with a defined shape (Harrison and Stein, 1990b; 1992). Desolvation as a mechanism for inducing distortion is supported by the following data: (1) the entropy of activation for the reactions of FKBP (Harrison and Stein, 1992), (2) the acceleration of C-N rotation that occurs when DMA is transferred from aqueous to hydrophobic solvents (Drakenberg et d., 1972), and (3) the crystal structure of the FKBP:FK506 complex (Van Duyne et al., 199 l), which suggests that the carbonyl carbon in the P, residue is transferred from an aqueous to a very hydrophobic pocket lined by Phe-99, Tyr-26, and Phe-36 of FKBP. Finally, distortion induced by electrostatics is consistent with (1) observations of acid catalysis of both C-N rotation in DMA (Gerig, 1971) and prolyl isomerization (Berger et al., 1959; Steinberg et al., 1960) and (2) the crystal structure of the FKBP:FK506 complex (Van Duyne et al., 1991), which suggests that Tyr-82 may be able to donate a hydrogen bond to the nitrogen of the proline ring. In all the above mechanisms, the energetic price to distort the Xaa-Pro bond out of planarity must ultimately be paid by favorable transition state interactions. These enzymes waste little binding energy on stabilizing the Michaelis complex or other stable state complexes, as evidenced by the relatively high K , of 1 mM (Harrison and Stein, 1992; Kofron et al., 1991).

REFERENCES Albers, M. W., Walsh, C. T., and Schreiber, S. L. (1990).J.Org. Chem. 55,4984-4986. Berarek, V. (1973). Actu Chin. Pulacki. Olomuc., Fac. Rerum Nut. 41, 11 1-1 15.

PROLYL ISOMERIZATION

23

Berger, A., Loewenstein, A., and Meiboom, S. (1959).J. Am. Chem. SOC. 81,62-67. Brandts, J. F., Brennan, M., and Lin, L. N. (1977). Proc. Natl. Acad. Sci. U.S.A. 74, 7312-7317. Challis, B. C., and Challis, J. A. (1979). “Nitrogen Compounds, Carboxylic Acids, Phosphorous Compounds,” pp. 957- 1067. Pergamon, Oxford. Cheng, H. N., and Bovey, F. A. (1977). Biopolymers 16, 1465-1472. Cleland, W. W. (1975). Adv. Enzymol. 45, 273-387. Cleland, W. W. (1982). In “Methods in Enzymology” (D. Purich, ed.), Vol. 87, pp. 390-405. Academic Press, New York. Drakenberg, T., Dahlquist, H.-I., and Forsen, S. (1972).J. Phys. Chem. 76, 2178-2183. Evans, C. A., and Rabenstein, D. L. (1975).J. Am. Chem. SOC.96, 7312-7317. Exnor, 0. (1973). Prog. Phys. Org. Chem. 10, 411-482. Fersht, A. (1985). “Enzyme Structure and Function,” pp. 109-1 1 1 . Freeman, New York. Fischer, C. (1989). Nova Acta Leopold. 61, 35-53. Fischer, G., Bang, H., and Mech, C. (1984). Ezomed. Eiochzm. Acta 43, 1101-1 1 1 1 . Fischer, G., Berger, E., and Bang, H. (1989a). FEES Lett. 250, 267-270. Fischer, G., Wittmann-Leibold, B., Lang, K., Kiefhaber, T., and Schmid, F. X. (1989b). Nature (London) 337, 476-478. Fujihara, H., and Schowen, R. L. (1985). Eioorg. Chem. 13, 57-61. Galardy, R. E., and Liakopoulou-Kyriakides, M. (1982).Int. J . P e p . ProleinRes. 20,144-148. Gerig, J. T. (1971). Eiopolymers 10, 2443-2453. Grastoph, G., and Wurthrich, K. (1981). Eiopolymers 20, 2623-2633. Harding, M. W., Galat, A,, Uehling, D. E., and Schreiber, S. L. (1989). Nature (London) 341, 758-760. Harrison, R. K., and Stein, R. L. (1990a). Biochemistry 29, 1684-1689. Harrison, R. K., and Stein, R. L. (1990b). Biochemistry 29, 3813-3816. Harrison, R. K., and Stein, R. L. (1992).J. Am. Chem. SOC.114, 3464-3471. Harrison, R. K., Caldwell, C. G., Rosegay, A., Melillo, D., and Stein, R. L. (1990).J . Am. Chem. Soc. 112, 7063-7064. Homer, R. B., and Johnson, R. B. (1970). In “The Chemistry of Amides” (J. Zabicky, ed.), pp. 187-245. Wiley, New York. Jacobson, J., Melander, W., Vaisnys, G., and Horvath, C. (1984). J . Phys. Chem. 88, 4536-4542. Jencks, W. P., and Page, M. I. (1972). Proc. FEES Meet. 29, 45-58. Jorgensen, W. L. (1991). Science 254, 954-955. Kofron, J. L., Kuzmic, P., Kishore, V., Colbn-Bonilla, E., and Rich, D. H. (1991).Biochemistry 30,6127-6134. Kovach, I. M., and Quinn, D. M. (1983).J. Am. Chem. SOC.105, 1947-1950. Leffler, J. E., and Grunwald, E. (1963). “Rates and Equilibrium in Organic Reactions,” pp. 321, 325, 358. Wiley, New York. Lin, L.-N., and Brandts, J. F. (1983). Biochemistry 22, 553-559. Liu, J., Albers, M. W., Chen, C. M., Schreiber, S. L., and Wakh, C. T. (1990). Proc. Natl. Acad. Sci. U.S.A. 87, 2304-2308. Love, A. L., Alger, T. D., and Olsen, R. K. (1972).J. Phys. Chem. 76, 853-855. Lumry, R. (1986). I n “The Fluctuating Enzyme” (G. R. Welch, ed.), p. 6. Wiley, New York. Lumry, R., and Rajender, S. (1970). Eiopolymers 9, 1125-1227. Martin, R. B. (1972).J . Chem. Soc., Chem. Commun. pp. 793-794. McCall, J. M. (1975).J. Med. Chem. 18, 549-552. Michnick, S. W., Rosen, M. K., Wandless, T. J., Karplus, M., and Schreiber, S. L. (1991). Science 252, 836-839.

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Moore, J. M., Peattie, D. A., Fitzgibbon, M. J., and Thompson, J. A. (1991).Nature (London) 351,248-250. Nemethy, G . (1967). Angew. Chem., Int. Ed. Engl. 6 , 195-206. Quinn, D. M., and Sutton, L. D. (1991). In “Enzyme Mechanism from Isotope Effects” (P. F. Cook, ed.), pp. 73-126. CRC Press, Boston. Radzicka, A., Perersen, L., and Wolfenden, R. (1988). Biochemistry 27, 4538-4541. Ritchie, C. D., and Sager, W. F. (1964). Prog Phys. Org. Chem. 2, 323. Rosen, M. K., Standaert, A. G., Galat, A., Nakatsuka, M., and Schreiber, S. L. (1990). Science 248,863-866. Schowen, R. L. (1967).J. Pharm. Sci. 56,931-943. Sierkierka, J. J., Hung, S. H. Y., Poe, M., Lin, C., and Sigal, N. H. (1989).Nature (London) 341,755-757. Stein, R. L. (1991). Curr. Biol. 1, 234-236. Steinberg, I . Z., Harrington, W. F., Berger, A., Sela, M., and Katchalski, E. (l960).J. Am. Chem. Sac. 82, 5263-5279. Stewart, W. E., and Siddall, T. H. (1970). Chem. Rev. 70, 517-551. Takahashi, N., Hayano, T., and Suzuki, M. (1989). Nature (London) 337, 473-475. Van Duyne, G. D., Standaert, R. F., Karplus, P. A., Schreiber, S. L., and Clardy, J. (1991). Science 252, 839-842. Wuthrich, K., von Freyberg, B., Weber, C., Wider, G., Traber, R., Widmer, H., and Braun, W. (1991). Science 254, 953-954.

PROLYL ISOMERASES: ROLE IN PROTEIN FOLDING By FRANZ X. SCHMID, LOREN2 M. MAYR, MATTHIAS MUCKE, and E. RALF SCHONBRUNNER Laboratorium fiir Biochemie, Universitlt Bayreuth, D-W-8580 Bayreuth, Germany

I.

Introduction . . . . . .

.......................

11. Prolyl Isomerization

A. Fast and Slow Protein Folding Reactions . . . . . . . . . . . . . . . . , . . . . . . . B. Prolyl Peptide Bonds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Prolyl Isomerization in Protein Folding . . . . 111. Prolyl Isomerases

A. B. C. D.

Discovery of P Three-Dimensional Structure of Cyclophilin Cyclophilin Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Catalysis of Slow-Folding Steps

IV.

V.

VI. B. Dependence on Substrate Concentration of Folding Catalysis C . Efficiency of Catalysis . . . . D. Native State Isomerization

......

25 26 26 27 28 30 31 31 33 33 34 36 36 37 40 42 42 44 44 46 48 51

VII. VIII.

IX.

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Note Added in Proof

51 54 54 55 59 59 60 61 62 65

I . INTRODUCTION Cis-trans isomerizations of Xaa-Pro peptide bonds are involved in the refolding reactions of many proteins. These processes are slow and they frequently determine the overall rate of folding, particularly of small monomeric proteins. Enzymes that catalyze prolyl isomerizations in short ADVANCES ljV PROTEIN CHEMISTRY, Vol. 44

25

C;op)righi 0 1W1 b y Acadeniic Press. Inc. All righis of reproductioti in any form reserved.

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FRANZ X. SCHMID E T AL.

oligopeptides, peptidylprolyl cis-trans-isomerases (PPIs, EC 5.2.1.8), were discovered in 1984 by Fischer and co-workers. In addition to this activity toward small substrates, prolyl isomerases were also found to accelerate slow steps in the folding of several proteins (Lang et al., 1987; Lin et al., 1988). This function of prolyl isomerases in protein folding is the major topic of this review. Mechanistic aspects of the enzymes and the catalyzed reactions are described by Stein elsewhere in this volume. Here we discuss first the importance of prolyl isomerization reactions as slow, rate-limiting steps of folding and their interdependence with other events in protein folding. Then, experimental data on the catalysis by prolyl isomerases of various slow in vitro protein folding reactions are reviewed. Finally, we describe results that possibly suggest a role for prolyl isomerases in cellular folding and a close interrelationship with disulfide bond formation. It is still largely unknown whether the catalysis of slow steps in folding is in fact a major function of prolyl isomerases in the cell, whether additional functions are carried by these enzymes, and whether the PPI activity plays a role in signal transduction pathways. Given the rapid development in this area and the high degree of interest from workers in different fields, such as protein folding and immunosuppression, we are likely to experience a rapid improvement in our understanding of the prolyl isomerases and of their biological functions. 11. PROLYLISOMERIZATION

A. Fast and Slow Protein Folding Reactions In 1973, Garel and Baldwin made the major discovery that unfolded ribonuclease A (RNase A) consists of a kinetically heterogeneous mixture of molecules that differ vastly in the rate of refolding. The respective fast-folding (U,) and slow-folding (Us) species coexist in a slow equilibrium and they give rise to parallel fast (in the time range of milliseconds) and slow (in the time range of minutes) phases in the refolding of RNase A. Subsequently, similar U, and Us species have been detected in the

trans FIG. 1. Cis

cis

trans isornerization of' a prolyl peptide bond.

PROLYL ISOMERASES

27

folding of many other proteins (for reviews, see Kim and Baldwin, 1982, 1990; Schmid, 1992).A plausible molecular explanation for this phenomenon was provided by the proline hypothesis of Brandts et al. (1975). They suggested that the fast- and slow-folding molecules differ in the cis-trans isomeric state of one or more Xaa-Pro peptide bonds (cf. Fig. 1).

B . Prolyl Peptide Bonds Peptide bonds are planar and can be either in the trans or in the cis conformation with respect to the two successive C, positions. These conformations are equivalent to dihedral angles w of 180" and 0", respectively. The trans state is strongly favored for peptide bonds that d o not involve proline residues. The cis conformation has not been detected in unstructured, linear oligopeptides, and the equilibrium population of the cis form is believed to be less than 0.1% (Brandts et al., 1975,Jorgensen and Gao, 1988). Very few nonproline cis peptide bonds have been found in native, folded proteins by X-ray crystallography (Stewart et al., 1990; MacArthur and Thornton, 1991). Unlike other peptide bonds, those between proline and its preceding amino acid (Xaa-Pro bonds, Fig. 1) typically exist as a mixture of cis and trans isomers in solution, unless structural constraints, such as in folded proteins, stabilize one of the two isomers. T h e trans isomer is usually favored slightly over the cis isomer in the absence of ordered structure and in short linear peptides. Frequently, cis contents of 10-30% are found (Cheng and Bovey, 1977; Grathwohl and Wuthrich, 1976a,b, 1981).The cis-trans equilibrium depends to some extent on the chemical nature of the flanking amino acids and on the charge distribution around the Xaa-Pro bond. T h e cis trans isomerization is an intrinsically slow reaction (time constants in the range of 10 to 100 sec are frequently observed at 25°C) with a high activation energy ( E A= 85 kJ/mol) because it involves rotation about a partial double bond. In native proteins of known three-dimensional structure about 7% of all prolyl peptide bonds are cis (Stewart et al., 1990; MacArthur and Thornton, 1991). Usually, the conformational state of each peptide bond is clearly defined. It is either cis o r trans in every molecule, depending on the structural framework imposed by the folded protein chain. There are a few exceptions to this rule. In the native states of staphylococcal nuclease (Evans et al., 1987), insulin (Higgins et al., 1988), and calbindin (Chazin et al., 1989) cis-trans equilibria at particular Xaa-Pro bonds have been detected in solution by NMR. In staphylococcal nuclease, the cis conformer of the Lys 116-Pro117bond can be selectivelystabilized by bind-

28

FRANZ X. SCHMID ET AL.

ing of Ca2+ ions and the inhibitor thymidine 3‘,5‘-phosphate (Evans et al., 1987; Alexandrescu et al., 1989, 1990).

C . Prolyl Isomerization in Protein Folding Normally, the native protein, N , has each prolyl peptide bond in a unique arrangement, either cis or trans. After unfolding [N-U,, Eq. (l)], however,

N

eU,e

Usi

(1)

the conformational restraints of the native state disappear and, in the U,’ reaction, these bonds become free to isomerize slowly as in U, short oligopeptides. This leads to an equilibrium mixture of a single unfolded species with correct prolyl isomers, U,, and one or more unfolded species with incorrect prolyl isomers, U;. Refolding of the U, molecules is fast, since they have their prolyl peptide bonds in the nativelike conformation. Usi molecules refold slowly, because refolding involves reisomerizations of the incorrect prolyl bonds. Nonnative isomers do not necessarily block refolding, and reisomerization is not required to be the first step of folding as suggested initially. This implies that refolding of U,’ (under native solvent conditions) usually does not occur by a reversal of the unfolding mechanism [i.e., reisomerization, followed by folding; cf. Eq. (l)]. The extended two-state mechanism in Eq. ( 1 ) is valid only under unfolding conditions and within the transition region, where partially folded species are unstable. Theoretical models for the kinetic analysis of folding under such conditions and the interrelationship with one or two prolyl isomerization reactions have been worked out and tested for several model proteins (Hagerman, 1977; Kiefhaber et al., 1992a; Kiefhaber and Schmid, 1992). Under solvent conditions that strongly favor folded structure (“strongly native conditions”), chains with certain incorrect isomers can rapidly form intermediates, lsi, which are partially nativelike [Eq. (2)] well before prolyl peptide bond reisomerization occurs (Cook et al., 1979;

fast folding

Usi

slow prolyl

I,’Nisomerization

Schmid and Blaschek, 1981; Goto and Hamaguchi, 1982; Kelley et al., 1986; Kiefhaber et al., 1990b; Nall, 1990). T h e rate and the extent of this rapid structure formation depends primarily on two factors: the location

PROLYL ISOMERASES

29

of the nonnative prolyl isomers in the structure and the solvent conditions selected for folding. Generally, incorrect prolyl isomers located at the surface of the folded protein or in flexible chain regions will allow more extensive structure formation of the intermediates Isi. Likewise, solvent conditions that strongly stabilize folded proteins will also stabilize partially folded structure in intermediates with incorrect isomers. The importance of prolyl peptide bond isomerizations for protein folding is indicated by the following experimental observations. 1. The fraction of Us molecules depends on the number of proline residues and on their isomeric state in the native protein. In particular, the presence of cis-prolyl peptide bonds in the folded molecules leads to a high fraction of Us, since in unfolded proteins the cis state is populated to a small extent only. Adler and Scheraga (1990) showed by NMR that in heat-unfolded RNase A the nonnative trans isomers predominate at both Pro93 and Proll4. The U, molecules dominate in the unfolded state of proteins that have only trans-prolyl peptide bonds, such as lysozyme (Kato et al., 1981, 1982), cytochrome c (Ridge et al., 1981; Nall, 1990), and thechymotrypsin inhibitor C12 (Jackson and Fersht, 1991a,b). It was questioned recently whether the small slow phase observed in the folding of lysozyme involves prolyl isomerization at all (Herning et al., 1991). RNase A (Garel and Baldwin, 1973) and RNase T1 (Kiefhaber et al., 1990a,b) have two cis-prolyl peptide bonds each and they show low fractions of U, of 0.20 and 0.04, respectively. An immunoglobulin fragment with a single czs-prolyl peptide bond displays 20% U, after unfolding (Goto and Hamaguchi, 1982). Brandts et al. (1977) and Lin and Brandts (1978) have studied the refolding of three homologous carp parvalbumins. Two of them contain one proline residue, and they show a small, slow refolding reaction. Such a reaction is not found in the folding of the third parvalbumin variant that lacks proline. 2. T h e U, 2 Us reactions in unfolded proteins have properties that are characteristic of prolyl peptide bond isomerizations in small peptides. The equilibrium is independent of temperature (Schmid, 1982) and independent of the concentration of additives, such as guanidinium chloride (GdmCI) (Schmid and Baldwin, 1979),that strongly decrease protein stability but do not affect prolyl peptide bond isomerization. T h e reaction is catalyzed by strong acid and it shows an activation energy of 88 kJ/ mol, as expected for prolyl isomerization (Schmid and Baldwin, 1978). 3. T h e refolding of the Us molecules involves slow steps that are limited by prolyl peptide bond isomerization. Folding steps and prolyl isomerization steps can be mutually interdependent. On the one hand the presence of incorrect isomers in the chain can decelerate crucial folding steps, and on the other hand rapid chain folding can affect

30

FRANZ X. SCHMID ET AL

the equilibrium and the kinetic properties of Xaa-Pro peptide bond isomerization. This close interrelationship between structure formation and prolyl peptide bond isomerization is a key feature of slow-folding steps and is of crucial importance for understanding the role of prolyl isomerases in these processes. Prolyl isomerization is now well established as a slow reaction in the folding of various unfolded proteins, such as pancreatic RNase (Schmid and Baldwin, 1978; Cook et al., 1979; Schmid et al., 1986; Grafl et al., 1986; Lang and Schmid, 1990), the C, fragment of the immunoglobulin light chain (Goto and Hamaguchi, 1982), thioredoxin (Kelley and Stellwagen, 1984; Kelley and Richards, 1987), yeast iso-1 and iso-2 cytochromes c (Ramdas et al., 1986; White et al., 1987; Wood et al., 1988), RNase T1 (Kiefhaber et al., 1990b,c), barnase (Matouschek et al., 1990), staphylococcal nuclease (Kuwajima et al., 1991), and the chymotrypsin inhibitor CI2 (Jackson and Fersht, 1991b). Nevertheless, other potential sources for slow interconversion reactions in unfolded protein molecules should not be disregarded. Slow ligand exchange (Tsong, 1977), loopthreading reactions of disulfide-bonded chains (Nall et al., 1978), and isomerizations of non-proline-containing peptide bonds were discussed as potential sources for Us species. Even though the correct trans state is strongly favored for peptide bonds that d o not contain proline, the large number of such bonds in a protein molecule could nevertheless lead to a significant portion of molecules with the wrong peptide bonds (Brandts et al., 1975). Experimental evidence for a role of cis peptide bonds not involving proline in protein folding is still missing.

D. Classif cation of Pralines Probably not all proline residues are important for protein folding. Evidence for “nonessential” prolines came from a comparison of several homologous pancreatic RNases (Krebs et al., 1983, 1985) and cytochromes c (Babul et al., 1978; Nall, 1990) that differ in the number of proline residues. Such prolines could be nonessential because they do not interfere with folding, or, alternatively, because they remain nativelike as regards isomeric state, after unfolding. Other evidence for different classes of proline residues has come from energy calculations (Levitt, 1981; Ihara and Ooi, 1985), in which the destabilization of the native state was calculated when one proline at a time was incorporated, in its incorrect isomeric state, into the protein. Levitt (198 1) classified these proline residues into three categories. Type I prolines destabilize the native state only to a small extent when in the incorrect isomeric state. Such prolines should barely affect folding.

PROLYL ISOMERASES

31

Incorrect isomers of type I1 prolines are intermediate, they decrease the free energy of stabilization by an amount that is smaller than the total free energy of stabilization of the protein, and they should still allow folding to proceed, although at a reduced rate. Nonnative isomers of type 111 prolines destabilize the native state entirely and therefore they are expected to block refolding. The prediction that Pro8 of bovine pancreatic trypsin inhibitor (BPTI) is such a type 111 residue was recently confirmed by Hurle et al. (1991). After the replacement of Pro8 by Gln in a two-disulfide variant of BPTI, the slowest phase of folding was no longer observable. Apparently, the incorrect proline, which destabilizes the native conformation most, also leads to the strongest deceleration of folding. The classification of proline residues into these three types is a little arbitrary, but very useful for illustrating the varying impact of incorrect isomers on folding. At strictly “nonessential” prolines the isomer distribution should not change during folding. Thermodynamic linkage relation requires that prolines that do not affect folding (e.g., by modifying the stability of intermediates) should reciprocally also not be affected in their isomerization rates and equilibria by folding events. Pro43 of calbindin may be an example for such a nonessential proline. Its cis-trans equilibrium is apparently unaffected by the folded state of the protein (Kordel et al., 1990a). Clearly, the location of an Xaa-Pro bond in the folded structure and the solvent conditions selected for folding are important determinants for the effect of an incorrect prolyl isomer on the folding mechanism. 111. PROLYL ISOMERASES

A . Discovery of Prolyl Isomerase The search for an enzymatic activity that would catalyze prolyl peptide bond isomerization began soon after the proposal of the proline hypothesis. The success came in 1984, when Fischer and co-workers discovered a peptidylprolyl czs-trans-isomerase activity in porcine kidney and other tissues by an assay that is based on the conformational specificity of chymotrypsin. This protease cleaves the 4-nitroanilide moiety from the peptide glutaryl-Ala-Ala-Pro-Phe-4-nitroanilide only when the Ala-Pro peptide bond is in the trans conformation. In aqueous solution 90% of the molecules are trans in the assay peptide and only 10% are cis. Therefore, in the presence of a high concentration of chymotrypsin, 90% of the hydrolysis reaction occurs within the dead time of manual mixing. Hydrolysis of the remaining 10% is slow, limited in rate by the cis +

32

FRANZ X. SCHMID ET AL

trans isomerization of the Ala-Pro bond of the peptide. By using this assay Fischer et al. (1984) were able to detect and purify an enzyme from porcine kidney that accelerates this isomerization very efficiently. Accordingly, the enzyme was named peptidylprolyl cis-trans-isomerase. It is a monomeric protein with a molecular weight of 17,700. The prolyl isomerase assay has been significantly improved by Kofron et al. (1991). They were able to increase the fraction of the cis isomer in the assay peptide from 10% up to 70% by dissolving it in an anhydrous mixture of trifluoroethanol and LiCl. By using this improved assay they determined the K , value for the cis peptide as 1 mM and k,,, as 13,200 sec-' (Kofron et al., 1991). The corresponding k,,,lK, value of 1.3 X lo7M - ' sec-' (at 5°C) indicates that prolyl isomerase from porcine kidney is a very effective catalyst. A surprising result emerged from the sequencing of PPI (Fischer et al., 1989; Takahashi et al., 1989). It was found to be identical with cyclophilin, the major high-affinity binding protein for the immunosuppressive drug cyclosporin A (CsA) in the cell. Incidentally, this protein was discovered in the same year as the PPI activity (Handschumacher et al., 1984). Prolyl isomerases of the cyclophilin type apparently occur in all organisms and in all subcellular compartments, notably in the cytoplasm, the mitochondria, and the endoplasmic reticulum. Binding of CsA to mammalian PPI is very tight, with a dissociation constant in the nanomolar range (Handschumacher et al., 1984). CsA inhibits PPI activity in a competitive manner with an inhibition constant of the same order of magnitude (Fischer et al., 1989; Kofron et al., 1991). A second class of prolyl isomerases, the FK506-binding proteins (FKBPs), was discovered in 1989 (Siekierka et al., 1989; Harding et al., 1989). These proteins are inhibited by the immunosuppressants FK506 and rapamycin. They do not show similarity in sequence to the cyclophilins, and they do not bind CsA. Nevertheless they catalyze prolyl isomerization in an oligopeptide (Harding et al., 1989) as well as during protein folding (Tropschug et al., 1990). Little is known about the function of FKBPs in protein folding and they are not considered further in this article. We will use the term PPI when the enzymatic activity as prolyl isomerase is concerned, and the terms cyclophilin and FKBP to discriminate the two known families of prolyl isomerases that are inhibited by CsA and by FK506, respectively. Cyclophilin and FKBP differ strongly in sequence specificity, The PPI activity of cyclophilin shows only a small dependence on the chemical nature of the amino acid Xaa that precedes proline in the assay peptide. T h e activity of FKBP, however, varies by three orders of magnitude when the same set of peptides is used in the PPI assays (Harrison and Stein, 1990; see Stein, this volume).

PROLYL ISOMERASES

33

Three-Dimensional Structure of Cyclophilin A ribbon plot of the three-dimensional structure of human cytoplasmic cyclophilin is shown in Fig. 2. It shows the overall folding topology and the binding site for the substrate peptide N-acetyl-Ala-Ala-Pro-Alaamidomethylcoumarin. The structure of the enzyme-peptide complex and the binding site for cyclosporin A were determined by a combination of X-ray crystallography and two-dimensional NMR spectroscopy (Kallen et al., 1991; Wuthrich et al., 1991). Cyclophilin has an eight-stranded antiparallel /3-barrel structure and two a helices. The substrate peptide is bound in a long groove on the surface of the /3 barrel. The Ala-Pro bond of the bound peptide is in the trans conformation. Residues that were identified to participate in CsA binding by NMR were found to cluster at the peptide-binding site, indicating that CsA binds at the PPI active site as a competitive inhibitor. Amino acids near the peptidebinding site include Cysll5 and Cys62, T r p 12 1 , and His 126. A detailed account of the structure and mechanism is given by Stein in this volume (see also Note Added in Proof). B.

C. Cyclophilin Family

After the initial discoveries of cyclophilin by virtue of its prolyl isomerase activity in porcine kidney (Fischer et al., 1984) and as a binding

FIG. 2. Ribbon plot of the overall fold of human cyclophilin. The complex with the tetrapeptide substrate N-acetyl-Ala-Ala-Pro-Ala-amidornethylcoumarin is shown. From Kallen et al. (1991). Reprinted by permission from Nature 353, 276. Copyright 0 1991 Macmillan Magazines Ltd.

34

FKANZ X. SCHMID ET AL.

protein for cyclosporin A in bovine thymocytes (Handschumacher et al., 1984) additional members of the cyclophilin family were detected in many cells and tissues. Undoubtedly, cyclophilins occur in all organisms and in various subcellular compartments, including the endoplasmic reticulum (ER) (Hasel et al., 1991; Caroni et al., 1991; Price et al., 1991) and the periplasm of Escherichia coli (Kawamukai et al., 1989; Liu and Walsh, 1990). A secreted form was found in human milk (Spik et al., 199 1). Cyclophilins are abundant proteins, constituting u p to 0.4% of the total cellular protein. The amino acid sequences of the cyclophilins remained highly conserved during evolution. This holds in particular for the proteins from eukaryotes. The cyclophilins from bovine thymus and from porcine kidney are identical in sequence (Takahashi et al., 1989), and the human and the bovine cyclophilins share 98% identical amino acids (Haendler et al., 1987). The homology between the mammalian cyclophilins and the cytosolic PPI from E . coli is about 25% (Hayano et al., 1991). The PPIs from porcine kidney and E . cola cytoplasm were used in most of the work on the function of prolyl isomerases as catalysts of protein folding that will be discussed herein. The NinaA protein of Drosophila is also related to the cyclophilins. It possesses a hydrophobic extension of about 30 residues at its carboxy terminus, which is probably used to anchor the protein in the ER membrane (Shieh et al., 1989; Schneuwly et al., 1989). The function of the NinaA protein is related to the intracellular folding and/or transport of certain classes of rhodopsin molecules in the Drosophila eye (Stamnes et al., 1991; Colley et al., 1991).

D . Catalysis of Slow-Folding Steps The initial attempts to demonstrate catalysis of folding by prolyl isomerase from porcine kidney concentrated on the folding of bovine RNase A. This protein was selected, since U, and Us species were first found for RNase A, and good, but indirect, evidence existed for the involvement of prolyl isomerization in slow steps of its unfolding and refolding. ‘The respective experiments, carried out in several laboratories, however, indicated that the slow phases of RNase A refolding are insensitive to the presence of PPI (Fischer and Bang, 1985; Lang et al., 1987; Lin et al., 1988). This negative result originated probably from a poor accessibility of one or more prolyl peptide bonds, brought about by the proximity of disulfide bonds (Pro93 is close to the 40-95 disulfide bond) and/or the rapid formation of ordered structure during folding. Such an explana-

PROLYL ISOMERASES

35

tion was supported by results on the S-protein fragment of RNase A (residues 2 1- 124). This fragment contains all proline residues and disulfide bonds of the parent protein, but it is less stable and folds much more slowly than intact RNase A. Folding of the S-protein is indeed accelerated by PPI, although the efficiency of catalysis is poor (Lang et al., 1987; cf. also Fig. 6). Subsequently, the action of porcine prolyl isomerase in folding was examined for a number of proteins (Lang et al., 1987; Lin et al., 1988). For these experiments small proteins, mostly with disulfide bonds, were selected, for which existing kinetic data had already suggested that prolyl isomerization steps were involved in slow folding. Good catalysis by prolyl isomerase was observed only for a few proteins, such as RNase T1 or the immunoglobulin light chain (see later, Figs. 5 and 6). Other proteins, such as porcine RNase, cytochrome c, o r pepsinogen, showed only moderately enhanced folding rates in the presence of PPI. Similar to bovine RNase A, slow refolding of thioredoxin was not catalyzed by PPI at all (Lang, 1988).There is evidence, however, from experiments with an engineered variant that Pro76 is involved in a late, slow step of the folding of thioredoxin (Kelley and Richards, 1987). The simplest explanation for the lack of catalysis by PPI is that, as in RNase A, rapid formation of ordered structure renders Pro76 of thioredoxin inaccessible to PPLProlyl and hydroxyprolyl isomerization is a rate-limiting step in the maturation of the collagen triple helix and it is accelerated in the presence of PPI (Bachinger, 1987).T h e folding of collagen in vitro and in vivo is discussed in detail in Section VIII, B. Of course, not all slow steps in protein folding involve prolyl isomerization. In particular, the very slow folding of large proteins can be limited in rate by other events, such as correct domain pairing or subunit association (Vaucheret et al., 1987). The in vitro unfolding and refolding of many large proteins are only partially reversible. In most cases this is caused by aggregation reactions that compete with correct folding in the cell as well as in the test tube (Jaenicke, 1987, 1991). Chaperones, such as GroEL, can inhibit aggregation by binding to exposed hydrophobic regions of folding intermediates (Goloubinoff et al., 1989; Buchner et al., 1991; Mendoza et al., 1991). We examined whether prolyl isomerase can also suppress aggregation, not by binding to the protein, but by increasing the rate of early folding steps that compete with aggregation. Attempts to find such an effect of prolyl isomerase were not successful. Neither the rate nor the yield of reactivation of two large oligomeric proteins, lactate and malate dehydrogenases, was increased in the presence of PPI (M. Kongsbak-Reim and F. X. Schmid, unpublished).

36

FRANZ X. SCHMlD ET AL.

IV. RNase T 1

AS

MODELSYSTEMTO PROBECATALYSIS OF FOLDING

RNase T 1 proved to be a good model system to investigate the function of prolyl isomerases in protein folding. Its folding kinetics are dominated by the slow trans + cis isomerizations of two prolyl residues that are in the cis conformation in the native protein. Folding and unfolding reactions can be studied in the presence and in the absence of the two disulfide bonds. Furthermore, an interesting interrelationship between the two slow events, prolyl isomerization and formation of disulfide bonds, is observed in the oxidative folding of reduced RNase T1. A. Structure and Stability of RNase TI

RNase T 1 from Aspergdlus oryzae is a small single-domain protein of 104 amino acids (Pace et al., 1991) with an extended Q helix of 4.5 turns and two antiparallel /3 sheets (Heinemann and Saenger, 1982; Koepke et al., 1989). The structure is shown schematically in Fig. 3. Two disulfide bonds form a small (2-10) and a large (6-103) covalently linked loop. RNase T 1 contains four prolyl peptide bonds; two are trans (Trp59Pro60 and Ser72-Pro73) and the other two are cis (Tyr38-Pro39 and

N

cis-Pro39

FIG. 3. Schematic drawing of the backbone conformation of RNase T1, cornplexed with guanosine 2’-phosphate. The positions of the cis-prolines (Pro39 and Pro%), as well as the trans-prolines(Pro60 and Pro73) are indicated. Drawing courtesy of Udo Heinemann, Berlin.

37

PROLYL ISOMERASES

Ser54-Pro55) in the native protein. It belongs to the family of microbial RNases and is not related to the pancreatic RNases in sequence or in three-dimensional structure. T h e unfolding transition of RNase T 1 is reversible under a wide variety of conditions (Pace, 1990; Pace et al., 1990) and is well described by the two-state approximation (Kiefhaber et al., 1990d; Pace et al., 1991). A peculiar feature of RNase T1 is the strong stabilization of the native state in the presence of NaCl (Oobatake et al., 1979; Pace and Grimsley, 1988). The two disulfide bonds are not absolutely required to maintain the protein in the folded conformation. RNase T I can fold to a nativelike, catalytically active form in the absence of the disulfide bonds under favorable conditions, such as low temperature and the presence of NaCl (Oobatake et al., 1979; Pace et al., 1988). Consequently, the folding and unfolding kinetics of this protein can be studied in the presence as well as in the absence of disulfide bonds. B . Folding Kinetics of RNase TI

Refolding of RNase T1 with intact disulfides is a complex process that consists of a minor fast and several slow processes. The slow-refolding phases originate from U,’ species that are formed after unfolding by prolyl isomerizations in the denatured protein. The two cis-prolyl peptide bonds at Pro39 and Pro55 isomerize largely to the incorrect trans state after rapid unfolding and thus create slow-refolding species (cf. Scheme I). Irrespective of the isomeric state of the prolyl residues, unfolded

13%

70%

SCHEME I. Kinetic model for the unfolding and isomerization of RNase T1.This model is valid for unfolding only. The superscript and the subscript indicate the isomeric states of Pro39 and Pro55, respectively, in the correct, nativelike cis (c) and in the incorrect, nonnative trans (t) isomeric state. As an example, U55,39tstands for an intermediate with Pro55 in the correct cis and Pro39 in the incorrect trans state. The two isomerizations are independent of each other, therefore the scheme is symmetric with identical rate constants in the horizontal and vertical directions, respectively. The given percentages for the individual unfolded species are estimates only. From Kiefhaber et al. (1990b.c).

38

FRANZ X. SCHMID Kl’ AL.

RNase T 1 molecules can rapidly form extended secondary structures in the time range of milliseconds when the protein is transferred to refolding conditions. This is indicated by the rapid regain of a nativelike circular dichroism spectrum in the amide region within the dead time of stoppedflow mixing (15 msec) (Kiefhaber et al., 1992b). Several slow reactions follow that involve prolyl isomerization. They are coupled with further folding and are monitored by changes in the absorbance and fluorescence of the aromatic amino acids. Refolding of RNase T 1 apparently occurs on a minor fast and two major slow pathways. This is revealed when an unfolding assay is used to determine the time course of formation of native molecules. The assay is based on the finding that the completely folded species, N , is separated from all other unfolded or partially folded molecules by a high activation barrier. Therefore only N molecules unfold slowly, whereas partially folded intermediates unfold rapidly. The folding kinetics obtained by this method are thus not affected by the formation of such intermediates. Of the unfolded molecules (the U, species), 3.5% refold to N within the dead time of the experiment. In addition to this rapid reaction two slow processes occur: a sequential two-step reaction (with time constants of 190 and 500 sec) and a very slow reaction with T = 3000 sec (Kiefhaber et al., 1990a,b). A simplified kinetic mechanism for refolding of RNase T 1 is shown in Scheme 11. It is based on the assumption that the czs-prolyl peptide bonds at Pro39 and Pro55 isomerize largely to trans in the unfolded protein and thus dominate the refolding kinetics. T h e results obtained for the wild-type protein and a variant with substitutions at positions 54 and 55 (see below) suggest that indeed Pro39 and Pro55 are both 80-90% trans in denatured RNase T1. The two isomerizations in the unfolded protein lead to four distinguishable species (cf. Scheme I>. About 3.5% of all molecules contain the correct isomers (U55,39c)and fold rapidly (U, + N). In addition, three slow-folding species exist (cf. Scheme I): two with one incorrect proline isomer each (U55tS9c and U55c39f) and another, dominant species with two incorrect prolines (U55t.79t).In the refolding mechanism (Scheme 11) it is proposed that all slow-folding molecules can regain rapidly most of their secondary structure and presumably part of their tertiary structure (U, + Ii steps). The subsequent slow, rate-limiting steps of folding are caused by the reisomerizations of the incorrect prolyl isomers in these partly folded species. The major unfolded species with two incorrect isomers (U55t39t) can enter two alternative folding pathways (the upper or the lower pathway in Scheme II), depending on which reisomerization occurs first. This choice is determined by the relative rates of reisomerization at the stage of the intermediate 155t39t.Though complex, the kinetic Schemes I and I1 for unfold-

PROLYL ISOMERASES

\

39

74 = 190 sec

r39c 55t

SCHEME 11. Kinetic model for the slow-refolding reactions of RNase TI under strongly native conditions. U stands for unfolded species, I for intermediates of refolding, and N is the native protein. The superscript and the subscript indicate the isomeric states of Pro39 and Pro55, respectively, in the correct, nativelike cis (c) and in the incorrect, nonnative trans stands for an intermediate with Pro55 in the correct (t) isomeric state. As an example, 15:9‘ cis and Pro39 in the incorrect trans state. The time constants given for the individual steps refer to folding conditions of 0.15 M GdmC1, 0.1 M Tris-HCI, pH 8.0, at 10°C. From Kiefhaber et al. (1990b,c).

ing and refolding of RNase T 1 nevertheless represent simplifications, since contributions from the isomerizations of the two trans-prolines (Pro60 and Pro73) are not considered. Such contributions are probably small and might be masked by the dominant contributions of the cis-prolines. According to Scheme 11, protein folding and reisomerization of prolyl peptide bonds are interrelated processes in the refolding of RNase T1. Under strongly native conditions intermediates with nativelike secondary structure are formed very rapidly and they can tolerate the presence of nonnative proline isomers. Their stability, however, is lowered by these incorrect isomers, and they are not populated under marginally native conditions. Pro39 and Pro55 of RNase T 1 could thus be classified as “type 11” prolines (cf. Section 11,D). Correct isomers are required for the final events of folding, and hence the last steps of folding are limited by reisomerization. T h e rapid formation of ordered structure affects the

40

FRANZ X. SCHMID ET AL.

isomerization kinetics. Unlike the situation in RNase A, where an acceleration of isomerization was found (Cooketal., 1979; Schmid and Blaschek, 1981), one isomerization in RNase T 1 (at Pro-39 in the Ij5c39t -+ N step) is strongly decelerated in a folding intermediate (Kiefhaber et al., 1990b). The folding mechanism in Scheme I1 is valid only under strongly native conditions, where intermediates are populated and reverse reactions are not significant.

C . Catalysis of RNase TI Folding by Prolyl Isomerases The slow refolding of RNase T1, as measured by the changes in absorbance o r fluorescence, can be approximated as a sum of two phases, an “intermediate” phase with a time constant, T , of about 400 sec, and a “very slow” phase with a time constant of about 3000 sec (at pH 8, 10°C) (Kiefhaber et al., 1990a). The intermediate phase is slightly heterogeneous, with contributions from several refolding steps (cf. Scheme 11). To a first approximation it is dominated by contributions from the reisomerization of Pro55 (in the 155t39t + 155c39tand the 155t.19C + N steps) and from the reisomerization of Pro39 in molecules that additionally contain an incorrect Pro55 (the 155t39t+ 155tS9c step). This assignment was supported by results on the Ser54GlylPro55Asn variant, where the intermediate phase was missing (Kiefhaber et al., 1990~). The very slow phase originates from the 155:Yt + N step (Scheme 11) and is limited in rate by the reisomerization of Pro39 in molecules that had already undergone trans + cis isomerization at Pro55. The slow phases are catalyzed by prolyl isomerases, albeit with a strongly different efficiency. Catalysis of Pro55 isomerization is generally good. Decent catalysis of Pro39 isomerization is only observed as long as a second incorrect isomer (at Pro55) is present. Molecules with a correct cis isomer at Pro55 and an incorrect trans isomer at Pro39 only (155:9t) can apparently form extensive structure. This rapid folding has two effects: it strongly decelerates trans -+ cis isomerization at Pro39 (in the 155rS9t-+ N step) and it renders catalysis by PPI very poor (Fig. 4).The folding reactions that constitute the intermediate phase are well catalyzed by PPI (Fig. 4). All isomerizations occur after the formation of extensive structure at the stage of largely folded intermediates. Possibly Pro39, but not Pro55, is already well shielded from the solvent at this stage of folding and is not readily accessible for prolyl isomerase. In native RNase T1, Pro55 is located at the surface; Pro39, however, is buried in the interior of the protein. Cytoplasmic prolyl isomerase from E. coli catalyzes the slowest phase of folding better than the enzyme from porcine kidney does. When increasing concentrations of the enzyme from E . coli are employed, cataly-

41

PROLYL ISOMERASES

0

180

360

3600 7200

540

Time (sec)

FIG. 4. Catalysis of the slow-refolding reactions of RNase T1 by 17.7-kDa PPI from porcine kidney. The increase in tryptophan fluorescence during folding is shown as a function of the refolding time. Folding in the absence of (W) PPI. Folding in the presence of (0)10 Klml PPI, (A) 50 Klml PPI, and (A) 1000 Klml PPI. Refolding was initiated by a 40-fold dilution of unfolded RNase T1 (8.0 M urea, 0.1 M Tris-HCI, pH 8.0) to final conditions of 2.0 p M RNase TI in 0.2 M urea, 0.1 M Tris-HCI, pH 8.0, at 10°C in the presence of the various concentrations of PPI (E. R. Schonbrunner, unpublished).

sis of RNase TI folding can become very effective (Fig. 5 ) . In the presence of 7200 K/ml prolyl isomerase (see Table I for definition of K ) the intermediate phases are complete in the dead time of mixing (2 sec) and the time constant of the very slow phase is reduced from 7 = 3000 sec (in the absence of prolyl isomerase) to 7 = 10 sec. This is equivalent to a 300-fold increase in folding rate at a prolyl isomerase concentration that is similar to the concentrations found in the cell (Schonbrunner et al., 1991).

20'

0

I

I

5 10 refolding time

I

15 (minl

I

I

60

120

I

FIG. 5. Catalysis of slow refolding of RNase T1 in the presence of increasing concentrations of cytoplasmic PPI from E. coli (0)No PPI, (0)25 Klml PPI, (A) 500 Klml PPI, and (A) 7200 Klml PPI. The refolding experiments were carried out as described in the legend to Fig. 4. From Schonbrunner et al. (1991).

42

FRANZ X. SCHMID E T AL.

D. Dependence of Catalysis on Presence of Prolines The name prolyl isomerase implies that this enzyme acts by catalyzing the isomerization of prolyl peptide bonds. This has been shown conclusively for the short synthetic peptide glutaryl-Ala-Ala-Pro-Phe-4-nitroanilide. This artificial assay was selected because it allowed exploitation of the conformational specificity of chymotrypsin for an efficient screening procedure ( Fischer et al., 1984). Only proline-containing peptides were able to compete with this substrate in the assay; proline free peptides or protein fragments had no effect. This is no proof, however, that the acceleration of slow protein folding reactions by PPI is also mediated by this prolyl isomerase function. In the initial experiments (cf. Section III,D) the correlation between the involvement of prolyl isomerization into the folding of several proteins and the respective catalysis by PPI was not very convincing. At this time it was alternatively discussed that PPI functions as a general “polypeptide-binding protein” that interacts with exposed chain segments and thereby facilitates folding. Positive evidence for Xaa-Pro sequences as targets for PPI in refolding protein chains was obtained from folding experiments with the Ser54Gly/ Pro55Asn variant of RNase T1, which lacks the cis-prolyl peptide bond between Ser54 and Pro55. T h e folding results obtained with this variant were obvious (Kiefhaber et al., 1 9 9 0 ~ )The . intermediate phase of folding was almost completely absent, and the good catalysis of folding by PPI was no longer observed. This result bears two implications. First, isomerization of Pro55 is indeed a rate-limiting step in the folding of RNase T1, and second, the good catalysis of refolding of the wild-type protein by PPI is mediated by the presence of Pro55. This suggests that Pro55 is a target for PPI in the catalysis of the intermediate phase of RNase T 1 folding. The efficient catalysis of this folding reaction supports the original assumption that prolyl isomerases accelerate slow steps in protein folding by facilitating the isomerization of incorrect prolyl peptide bonds.

V. CATALYSIS OF FOLDING I N ABSENCE OF DISULFIDE BONDS Most of the small proteins that were used initially as substrates to test the function of prolyl isomerases contained disulfide bonds, which were left intact during unfolding and refolding. These proteins were used because their unfolding is reversible under a wide variety of conditions and because good evidence existed for a number of them that prolyl isomerizations were involved as rate-limiting steps in their slow-folding reactions. A protein chain without disulfides should be a better model

43

PROLYL ISOMERASES

for the cellular folding of a newly formed chain in which the cysteines are still in the reduced form. Again, RNase T 1 was a good model to investigate in vitro the role of prolyl isomerization for the folding of the non-cross-linked protein chains and the catalysis by prolyl isomerases. The two disulfide bonds of RNase T 1 can be reduced in the unfolded state and stabilized either by the presence of a reducing agent or by covalent modification with iodoacetate or iodoacetamide (Pace and Creighton, 1986). Opening of the disulfides strongly destabilizes the protein. At low temperature, near pH 5, and in the presence of NaCl, however, it can be induced to fold to a nativelike and enzymatically active form (Oobatake et al., 1979; Pace et al., 1988). At pH 8 and 15°C the disulfide-reduced and carboxymethylated form of RNase T1 (RCMRNase T1) is less stable and is largely unfolded in the absence of NaC1. TABLE I Catalysis of Refolding of RCM-RNase TI by PPl"

0 15 40 78 150 300

1820 1133 7 13 460 243 140

1.0 1.6 2.6 4.0 7.5 13.0

" The protein was first unfolded in 80 mM TrisHC1, pH 7.8, 25°C. in the absence of NaCl. The refolding was induced by 40-fold dilution to give final conditions of 0.5 pM RCM-RNase T1 in 2.0M NaCl, l00mM Tris-HC1, pH 8.0, 15°C. PPI from E. coli cytosol was used. The PPI activity is expressed as the factor by which the prolyl isomerization of the assay peptide Suc-Ala-Ala-Pro-Phe-4-nitroanilide is accelerated relative to the uncatalyzed isomerization minus one ( T ~ , / T~ ~ I, = K). This factor is zero in the absence of PPI. The conditions for the determination of the PPI activity and for the refolding experiments were identical. Time constant of the single-phase refolding reaction of RCM-RNase T1. "Acceleration factor expressed as the ratio of the rates of folding in the presence (1 /rJ and in the absence ( 1 / ~ ~of) PPI (Miicke and Schmid, 1992).

44

FRANZ X. SCHMID ET AL.

A reversible transition to a folded form occurs when the concentration of NaCl is increased. T h e midpoint of this folding transition is near 1 M NaCI; above 2 M NaCl RCM-RNase T 1 is in the folded form. This folded form of RCM-RNase T 1 is catalytically active (Pace et al., 1988). Refolding induced by transfer of the protein from 0 to 2 M NaCl is a very slow reaction (T = 1820 sec) and it is accelerated in the presence of prolyl isomerase (Table I). This result allows two conclusions to be drawn: (1) Similar to the slow refolding of the disulfide-intact form, the slow refolding of RNase T 1 without cross-links also involves prolyl isomerization and it is limited in rate by this reaction. (2) Prolyl isomerization in chains, where the disulfides have not yet formed, is also catalyzed by prolyl isomerases. The catalysis is more efficient in the absence of the disulfide bonds than in their presence when folding is compared under identical conditions (Table 11). The simplest explanation is that the rapid formation of ordered structure early in folding affects the effectiveness of catalysis by prolyl isomerase. Such structure is most extensively and most rapidly formed when the disulfides are left intact and when refoldingis carried out under strongly native conditions, such as in the presence of 3 M NaCI. An increase in the concentration of NaCl in the refolding conditions diminishes the efficiency of catalysis for both RCM-RNase T 1 and the form with intact disulfides (Miicke and Schmid, 1992). VI. CATALYSIS OF PROLYL ISOMERIZATION DURING UNFOLDING AND REFOLDING

A . Catalysis in Unfolded Proteins Prolyl isomerases are enzymes. They catalyze the reaction in either direction and they d o not determine the isomeric states of the prolyl peptide bonds in the refolding protein substrates. The distribution of cis and/or trans isomers depends only on the stability of the folding protein under the chosen solvent conditions. Under refolding conditions the native isomers are locked in by rapid folding; under unfolding conditions PPI should catalyze the cis-trans equilibration reaction. Catalysis of prolyl isomerization in an unfolded protein is difficult to investigate because unfolding conditions would denature prolyl isomerase, and also because equilibration reactions in the unfolded state are usually “silent,” i.e., they are not correlated with a change in a spectroscopic signal. The first problem could be overcome by using RCM-RNase T 1 as a substrate. This protein is only marginally stable and can be induced to unfold by a dilution from 2 to 0.4 M NaCl at pH 8 and 15°C. Under these conditions prolyl isomerases are stable and retain their full enzymatic activity. Within

45

PROLYL ISOMERASES

TABLE I1 Calalytic Efficiency of PPI in the Refolding of RCM-RNase TI and of RNase TI" ~

Refolding; of RCM-RNase TI'

~

~

~~~~

Refolding of RNase TI''

PPI activityb (Klml)

TI1

Tq?

TIP

Tpl

(sec)

(sec)

A,!

Ap/

(sec)

(sec)

A,!

A,/

0 8 19 38 76 152

1245 964 798 615 358 192

80 65 51 50 28 22

0.94 0.93 0.92 0.90 0.87 0.84

0.06 0.07 0.08 0.10 0.13 0.16

883 911 909

119 114 119 112 98 73

0.29 0.29 0.26 0.21 0.14 0.11

0.71 0.71 0.74 0.79 0.86 0.89

1009

450 403

Efficiency of catalysisg

K values Kk KloldlKAd

RCM-T1 (1)"

RCM-TI (2)'

RNase TI (1)"

RNase T1 (2)'

Peptide'

5.4 0.036

2.8 0.018

1.3 0.009

0.6 0.004

152 1

" The protein was first unfolded in 80 mM Tris-HCI, pH 7.8, containing 8 M urea, 25°C. Refolding was induced by 40-fold dilution to give final conditions of 0.7 P M RNase T 1 and RCM-RNase TI, respectively, in 3.0 M NaC1, 0.2 M urea, 100 rnM Tris-HCI, pH 8.0, 15°C. PPI from E. coli cytosol was used. The K values are defined as described in footnote b to Table I. ' Both disulfide bonds were reduced and then carboxymethylated to prevent the reforrnation of the disulfide bonds. Both disulfide bonds remain intact during unfolding and refolding. ' Time constants of phases 1 and 2, respectively. Fractional amplitudes of phases 1 and 2, respectively; A , + A2 = 1. R The acceleration of the different phases of the refolding reactions are compared with the acceleration of the isomerization of the assay peptide. " Phase 1 of the refolding reaction of RCM-RNase TI and RNase TI, respectively. 'Phase 2 of the refolding reaction of RCM-RNase TI and RNase TI, respectively. Isomerization of the assay peptide Suc-Ala-Ala-Pro-Phe-ONa. Ir K values for the different refolding reactions in the presence of 152 Klml PPI. Ratio of the K values for the respective refolding reaction and for the catalysis of the isomerization of the assay peptide. A value of 1 shows that the reaction is catalyzed with the same efficiency as the isomerization of the assay peptide. Lower values indicated reduced catalytic efficiency. J

'

*

the transition region the rapid unfolding equilibrium (N U,) and the Us reaction [cf. Eq. (l)] subsequent slow prolyl isomerization in the U, are kinetically coupled. At equilibrium the Us species is strongly favored over U, in unfolded RNase TI and thus the slow U, $ Us reaction leads to a marked shift in the coupled N U, equilibrium. Hence, the slow Us equilibrium is coupled with further unfolding attainment of the U, of N and therefore correlated with a large decrease in fluorescence, which

*

*

46

FRANZ X. SCHMID ET AL

is easily measured. Unfolding, or, to be more precise, the equilibration of U, and Us species of RCM-RNase T1, is indeed catalyzed by prolyl isomerase (Table 111). The efficiency of PPI is the same when the catalysis is compared in unfolding and refolding experiments under identical conditions of 0.8 M NaCl near the midpoint of the unfolding transition (Mucke and Schmid, 1992).Clearly, prolyl isomerases have no directional information, and the final product of action, a cis-prolyl peptide bond in a native protein or an equilibrium mixture of cis and trans isomers, is determined only by the solvent conditions and by the stability of the substrate protein. This is comparable to the action of protein disulfideisomerase in oxidative folding. Disulfide-isomerase can catalyze the reduction, the oxidation, or the scrambling of disulfide bonds, solely depending on the redox conditions and on the stability of the substrate protein (Freedman, 1984).

B . Dependence on Substrate Concentration of Folding CatalysG In the initial demonstration of an effect of PPI on protein folding, 1.6 pM PPI (1200 K/ml) was required for a sevenfold acceleration of the folding of 2 pM immunoglobulin light chains (Lang et al., 1987). From TABLE 111 it1 Utlfolded Form of RCM-KNasr TI"

Catalysis of Prolyl 1soein.ization

Time constant (secr

Amplitude'"

PPI activity" (Klml)

71

72

Al

A,

1 15 35 70 138 275

337 223 I54 98 73 54

8 8 9 6 13 8

0.88 0.87 0.88 0.89 0.89 0.92

0.12 0.13 0.12 0.1 I 0.11 0.08

7,,h"

1.0 1.5 2.2 3.4 4.6 6.2

" T h e protein was first folded in 2.25 M NaCI, 100 m M NaAc, pH 5.0, 10°C. The unfolding was induced by 48-fold dilution to give final conditions of 0.7 pM RCM-RNase TI in 0.4 M NaCI, 100 m M Tris, pH 7.8, 15°C. The PPI was from E. coli cytosol. T h e K values are defined as described in footnote 6 to Table I. Time constants for phases 1 and 2, respectively. Fractional amplitudes of phases 1 and 2, respectively. Acceleration factor expressed as the ratio of the rates of unfolding in the presence of various concentrations of PPI (I/T,,,,) and in the absence of PPI (l/r,)).

"

47

PROLYL ISOMERASES

these results it appeared possible that PPI does not act as a catalyst in protein folding, but rather as a roughly stoichiometric polypeptidebinding protein. Such an alternative is clearly ruled out for the action of PPI on the slow phases in the folding of RNase TI. Good catalysis is observed when the PPI concentration is much smaller than the concentration of the substrate RNase TI and the extent of catalysis is independent of the concentration of RNase T I . In the absence of PPI, the slow refolding of RNase T 1 with intact disulfide bonds can be approximated as the sum of two phases with time constants of 3000 and 400 sec, respectively. The 400-sec phase (the "intermediate" phase) is heterogeneous (cf. Section IV,B). In the presence of 100 Klml E. coli PPI, slow refolding is strongly accelerated and the intermediate phase splits into two phases that differ in the extent of catalysis (cf. Section IV,C). The time constants as well as the relative amplitudes of the individual phases of slow refolding are independent of the RNase T1 concentration in the examined range from 0.6 to 30 p M RNase TI (Table IV). The catalysis depends on PPI concentration (Fig. 5), suggesting that under the employed conditions the saturation of PPI with the substrate RNase TI is low. This is not surprising regarding the small substrate concentrations that are used

TABLE

Iv

Cutalysis of RNasr T1 Folding by PPI: Variation uf Coticrtifrution uf RNusc TI"

Amplitude"

Time constant (sec)h [RNase T l ]

(PM) 12 0.6 1.8 3 6 12 18

24 30

PPI activity (Klml) 0 100 100 100 100 100 100 100 100

HA

3440 730 735 680 700 720 740 670 630

450 63 60 62 58 58 64 64 60

12 12 12 12 13 13 13 14

0.25 0.18 0.19 0.19 0.19 0.18 0.18 0.17 0.16

0.23 0.23 0.23 0.24 0.25 0.24 0.22 0.23

0.44 0.29 0.30 0.31 0.29 0.29 0.31 0.31 0.31

0.69 0.70 0.72 0.73 0.72 0.72 0.72 0.70 0.70

" Unfolding conditions were 8 M urea, 100 mM Tris-HC1, pH 7.8. Refolding was started by dilution of 25 pI unfolded RNase T I in 975 pI 100 mM Tris-HC1, pH 7.8, at 1O.O"C to the given final concentrations; the concentration of urea was 0.2 M . The concentration of cytosolic E . colz PPI equivalent to 100 Klml was 0.14 /uM. Time constants of the various phases of RNase TI refolding. In the absence of PPI, phases 2 and 3 cannot be separated. ' Amplitudes of the three phases of refolding measured by the increase in fluorescence at 320 nm. The fluorescence of the refolded protein was set as 1.0 in each experiment.

18

FKANZ X. SCHMID E T AL

in refolding experiments, and in view of the K,, value of about 1 mM obtained for the assay peptide. K , values for protein substrates are difficult to measure because of the prohibitively high protein concentrations that would be required in the folding experiments. C. Efficiency of Catalysis

The efficiency of the catalysis of slow-folding reactions by prolyl isomerases is strongly variable. It depends on the particular PPI that is used, on the substrate protein, and on the folding conditions. Virtually no acceleration of slow folding was found for bovine RNase A for chymotrypsinogen, P-lactoglobulin, and thioredoxin (Lang et al., 1987; Lin et al., 1988; Lang, 1988). For these proteins evidence existed that prolyl isomerization might be involved in slow folding. The folding reactions of cytochrome c and pepsinogen were marginally increased in the presence of high PPI concentrations (Lin et al., 1988). Table V provides a detailed review of the efficiencies of various prolyl isomerases as catalysts of the slow-folding reactions of different proteins under varying folding conditions. The catalytic efficiencies are given relative to the PPI activities measured in the standard assay with the peptide Suc-Ala-Ala-Pro-Phe4-nitroanilide. An efficiency of 1 implies that the same relative increase in rate is observed in the folding reaction and in the isomerization of the assay peptide. Figure 6 provides representative plots for selected proteins. Several trends are apparent from the data compilation in Table V: (1) The efficiency of catalysis depends mainly on the substrate protein. The catalysis of folding of the S-protein fragment of bovine RNase A by porcine PPI is more than 3000-fold less efficient than catalysis of isomerization of the assay peptide. For the slow-folding reaction of the immunoglobulin light chain, catalyzed by the same PPI, however, this ratio is reduced to a factor of 60. (2) T h e catalytic efficiencies in the folding of RNase TI of the various eukaryotic prolyl isomerases are very similar. The cytoplasmic PPI from E. coli, however, shows a markedly higher activity in all three slow-folding reactions of RNase T1. T h e strongest difference is observed for the very slow 3000-sec phase of folding. This reaction is about %fold better catalyzed by the enzyme from E. coli, relative to the eukaryotic PPIs. (3) Opening of the disulfides of RNase T1 leads to a significant improvement of the catalysis by PPI. This is probably due to the strongly reduced stability of folding intermediates in the absence of the disulfide bonds. (4) A related decrease in catalytic efficiency is observed when the structure of folding intermediates is stabilized by the addition of NaCI. NaCl strongly stabilizes folded forms of RNase T 1 in the presence as well as in the absence of disulfide bonds.

49

PROLYL ISOMERASES

Activity

of

TABLE V Various Prolyl Isomcrases as Catalysts of Protein Folding Efficiency" Kl,,lrllnRl

~il.loldrr

PPI source

Cyclophilins Cytosolic 17 kDa, porcine kidney

Folding protein RNase A

S-Protein Porcine RNase Ig light chain RNase T1

0.30 M 0.30 M 0.25 M 0.20 M

urea, urea, urea, urea,

10°C 10°C 10°C 10°C

0

I ,2

1250

3.0 M NaC1, 0.20 M urea, 15°C

0.032

4

RNase TI

880 120

3.0 M NaCl, 0.20 M urea, 15°C

0.013 0.012

4

RNase TI

3400 450 450

0.20 M urea, 10°C

0.006 0.013 0.013

5

Cytosolic, Neurospora crassa Cytosolic, yeast

0.20 M urea, 10°C

0.20 M urea, lO0C

0.20 M urea, 10°C

0.20 M urea, 10°C

0.20 M urea, 10°C

2.0 M NaC1, 15°C

0.0003 0.0007 0.0 17 0.006 0.16 0.035 0.001 0.07 0.07 0.05 0.40 0.06 0.002 0.17 0.05 0.006 0.06 0.06 0.0016 0.012 0.0058 0.043

Ref.'

RCM-RNase T1

Cytosolic, human

Neurospora crassa

50 0.25 M urea, 10°C 200

Kpep,,dc

RCM-RNase TI

Cytosolic, E. Cali

FK-binding protein Cytosolic,

Folding conditions

650 540 200 3400 450 450 3400 450 450 3400 450 450 3400 450 450 3400 450 450 3400 450 450 1820

19 kDa, porcine kidney

Cytosolic, E. coli

(sec)

1 1 1 3

3

3

3

3

3

4

" Time constant for the uncatalyzed folding reaction.

'

The efficiency K ~ , , I ~ I , , , relates ~ I K ~ the ~ , ,PPI ~ ~ activity in protein-folding reactions (Kf,,lrllog) with the activity in the isomerization of the assay peptide Suc-Ala-Ala-Pro-Phe-p-nitroanilide (Kpepude).The values are defined as described in the footnotes to Table 1. ' (I) Lang el al. (1987), ( 2 ) Lin et al. (1988). ( 3 ) Schonbrunner et al. (1991). ( 4 ) M. Miicke and F. X. Schmid (unpublished data), ( 5 ) Tropschug et al. (1990).

50

FRANZ X. SCHMID E T AL.

L

Q s C

0 .c ! I a

-a

H 0

1000

2000

3000

4000

5000

PPI activity (Wml) FIG. 6. Acceleration of refolding of different proteins as a function of PPI activity. The acceleration factor is given as the ratio klk,, of the observed rate constants for folding in the presence of PPI, k , and in the absence of PPI, k,,. The PPI activity is given as Klml. The K values are defined as described in footnote b to Table I. The following protein concentrations were used in the refolding experiments: (0)2 p M immunoglobulin light chain, (0) 11 porcine pancreatic RNase, and (A)17 p M S-protein fragment of bovine RNase A. The final conditions for refolding were 0.25 M urea (0.30 M urea for porcine RNase), 0.1 M Tris-HCI, pH 8.0, 10°C. Based on data from Lang et al. (1987).

In summary, the accessibility of the Xaa-Pro bonds in the refolding protein appears to be a major factor for the efficiency of catalysis by PPI. The extensive data for RNase T 1 indicate that catalysis is poor at a bond that is buried in the native protein and presumably also in a folding intermediate (at Pro%). Catalysis is much more efficient at a bond (at Pro55) that remains at least partially accessible throughout folding. Variations that lead to a decrease or to a destabilization of rapidly formed structure during folding, such as the removal of the disulfide bonds, o r a decrease in the NaCl concentration, also lead to an increase in the efficiency of catalysis. A correlation with the rate of the uncatalyzed refolding reaction is not evident from the data in Table V. The importance of the local amino acid sequence around the prolines cannot be evaluated yet, since the data base is too small. It should be noted, however, that bovine and porcine RNase A, RNase T1, and the immunoglobulin light chain contain Tyr-Pro peptide bonds that are cis in the respective native proteins. Generally, Tyr-Pro bonds have the highest tendency to occur in the cis conformation in folded proteins (Stewart et al., 1990).

PROLYL ISOMERASES

51

D. Native State Isomerization

The inhibitory action of the complexes of cyclophilin with CsA and FKBP with FK506 on cellular signal transduction pathways raised the intriguing question whether prolyl isomerases might also catalyze slow conformational transitions of folded proteins that were thought to involve prolyl cis-trans isomerizations, such as in concanavalin A (Brown et al., 1977) and in bovine prothrombin (Marsh et al., 1979). Lin et al. (1988), however, could not find evidence for an increase in rate of these two slow isomerizations in the presence of porcine PPI. Either these reactions are limited by molecular processes other than prolyl isomerization, o r the folded forms of prothrombin and concanavalin A are not accessible for prolyl isomerases. Two slowly interconverting folded forms were also found for calbindin (Kordel et al., 1990a). They differ in the isomeric state of the Gly42-Pro43 peptide bond. This bond is flexible and accessible to solvent and its rate of cis-trans interconversion is close to the values expected for prolyl isomerization in small peptides (cf. Grathwohl and Wuthrich, 1981). This isomerization is, however, unaffected by the addition of PPI (Kordel et al., 1990b).

VII.

OF PROLYL ISOMERASE A N D PROTEIN SIMULTANEOUS ACTION DISULFIDE-ISOMERASE AS CATALYSTS OF FOLDING

At least two major slow processes occur in the folding of disulfidecontaining proteins: the cis-trans isomerizations of Xaa-Pro peptide bonds and the formation of the correct disulfide bonds. T h e latter is catalyzed by protein disulfide-isomerase (PDI ). This enzyme occurs at high concentration in the endoplasmic reticulum (Hawkins et al., 1991) and there is good experimental evidence that PDI is required for the de novo folding of nascent secretory proteins (Bulleid and Freedman, 1988). Cyclophilins have recently also been localized in the ER (Hasel et al., 1991) and in other compartments of the secretory pathway (Caroni et al., 1991). Their biological function is not known. In i n vitro experiments prolyl isomerase accelerates the oxidative folding of reduced RNase T 1 (i.e., folding coupled with formation of the disulfide bonds) and the catalysis of disulfide bond formation by PDI is markedly improved when PPI is present simultaneously (Schonbrunner and Schmid, 1992). The oxidative folding of RNase T1 in the presence of a mixture of reduced and oxidized glutathione is a slow process and it can be followed by the increase in tryptophan fluorescence (Fig. 7). Folding is strictly linked to disulfide bond formation under the conditions

52

FRANZ X . SCHMID E T AL

100

-

'8

80 60 40

20 0

~ oA : o o o o o o o 1

0

1

1

1

1

1

3600

1

1

1

0 0 0 0 0 0 0 0 0 0 0 0 1

7200

1

1

1

1

10800

1

1

1

1

14400

1

I

I

I

18000

Time (sec) FIG.7. Oxidative refolding of reduced RNase TI. Reoxidation conditions were 0.1 M Tris-HCI, pH 7.8, 0.2 M guanidinium chloride, 4 mM reduced glutathione, 0.4 mM oxidized glutathione, 0.2 mM EDTA, and 2.5 pM RNase TI at 25°C. The kinetics of oxidative refolding were followed by the increase in tryptophan fluorescence intensity at 320 nm (0).by an unfolding assay (Kiefhaber et al., 1990b) that measures the formation of native protein molecules (A),and by the increase in the intensity of the band for native RNase T 1 in native polyacrylamide gel electrophoresis (0).Fluorescence emission in the presence of 10 mM reduced dithioerythritol to block disulfide bond formation (0).The small decrease in signal after several hours is caused by slight aggregation of the reduced and unfolded protein. (From Schonbrunner and Schmid (1992).

of Fig. 7, and the protein remains unfolded under reducing conditions that prevent disulfide bond formation. PPI accelerates the oxidative folding of RNase T 1 (Fig. 8). This facilitated formation of the disulfide bonds during folding in the presence of PPI is probably an indirect effect. Partially nativelike structure and thus the correct set of disulfides are presumably formed most rapidly from the unfolded protein molecules that contain the correct set of nativelike prolyl peptide bonds. These molecules are analogous to the rapidly folding U, species of unfolded RNase T 1 with intact disulfides. UF molecules can refold in the time range of milliseconds (Kiefhaber et al., 1990a,b). The other unfolded species that have incorrect prolyl isomers cannot form such structures easily. The interconversion of the forms with the correct and the incorrect prolyl isomers is known to be slow and it can influence the rate of disulfide bond formation. Prolyl isomerase catalyzes these isomerizations and thereby indirectly facilitates the formation of the correct disulfide bonds in oxidative folding reactions. An analogous effect of prolyl isomerase is noted when disulfide bond formation is catalyzed by disulfide isomerase. In the absence of prolyl

53

PROLYL ISOMERASES

100

8 80 u)

?? 0

2 60 LL

9

.% -

40

d

20

0

1800

3600

5400

7200

9000

Time (sec) FIG. 8. Acceleration of the oxidative refolding of RNase TI by PPI and PDI. T h e increase in fluorescence at 320 nm is shown as a function of the time of reoxidation. T h e final conditions were 2.5 p M RNase TI in 0.1 M Tris-HCI, 0.2 M GdmCI, 2 mM EDTA, 3 mM glycine, 0.4 mM oxidized glutathione, and 4 mM reduced glutathione at pH 7.8 and 25°C. Reoxidation (0)in the absence of PPI and PDI, (0)in the presence of 1.4 p M PPI, (A) in the presence of 1.6 p M PDI, and (A) in the presence of both 1.6 p M PDI and 1.4 p M PPI. In all experiments more than 90% of the observed kinetics were well approximated by single first-order processes, as indicated by the continuous lines. T h e respective time constants (7)are: (0)T = 4300 sec, (0)T = 2270 sec, (A)T = 1500 sec, (A) T = 650 sec. In all cases the initial fluorescence signal was about 10% of the final emission of the native protein. From Schonbrunner and Schmid (1992).

isomerase, addition of 1.6 p M PDI increases the rate of oxidative folding of RNase T 1 by a factor of 2.9 (Fig. 8). In the presence of 1.4 p M PPI, however, the same concentration of PDI leads to 6.6-fold acceleration of reoxidation (Fig. 8). When the concentration of PPI is further enhanced to 6.9 p M , the oxidative folding is 12-fold increased. This factor was reduced to 2.9 when PPI was omitted from the reoxidation solution (Schonbrunner and Schmid, 1992). This accelerating effect of prolyl isomerase on the oxidative folding of RNase T I in the presence or in the absence of PDI is most pronounced under conditions wherein prolyl isomerization steps and disulfide bond formation are similar in rate. In earlier experiments, no acceleration of disulfide bond formation was found during the folding of pancreatic RNase A under similar conditions. Only when the concentrations of reduced and oxidized glutathione were strongly reduced in the reoxidation experiments, a small acceleration of the initial part of the reoxidation kinetics was noted (Lin et al., 1988). The simplest explanation is that in the reoxidation of pancreatic RNase A incorrect disulfides form rapidly during folding unless the concentra-

54

FRANZ X. SCHMII) E T AL

tion of glutathione is very small. The rate-limiting event for reactivation is the slow reshuffling of the incorrect disulfide bonds and this reaction is not affected by the presence of prolyl isomerase. Clearly, the action of prolyl isomerases is not restricted to the slow folding of polypeptide chains with intact disulfides, but they also accelerate the oxidative folding of reduced proteins, which resemble more closely the nascent polypeptide chains as they occur in the endoplasmic reticulum. The simultaneous presence of' PPI markedly enhances the efficiency of PDI as a catalyst of disulfide bond formation. Both enzymes act according to their specificity and catalyze the isomerization of prolyl peptide bonds and the formation of disulfide bonds, respectively, in the folding protein chains. It remains to be demonstrated that a similar concerted action of the two enzymes can take place in the course of de novo synthesis and folding of proteins in the cell. VIII.

ROLEOF PROLYL ISOMERASE FOR CELLULAR FOLDING

The enzymatic functions of prolyl isomerases in vitro are fairly well characterized. They catalyze cis-trans isomerizations of Xaa-Pro bonds in small peptides and some proline-limited steps in the slow folding of several proteins. The efficiency of prolyl isomerases in these in vitro reactions can be very high, with k,,,lK, values as high as 10' M-' sec-' (Harrison and Stein, 1990; Kofron et al., 1991). In contrast to this good characterization of the in vitro activities, the cellular functions of this class of proteins are largely unclear. The simultaneous roles as efficient catalysts of prolyl isomerization and as high-affinity receptors for immunosuppressants are difficult to reconcile, and it is not known at present whether the inhibition of prolyl isomerase activities by immunosuppressants, such as CsA or FK506, is biologically relevant. Indirect evidence for a possible function of prolyl isomerases in cellular protein folding is provided by two experimental findings: (1) catalysis of protein folding is a conserved property of cyclophilins of strongly different origin, and (2) the in vivo maturation of two proteins is slightly retarded in the presence of the PPI inhibitor CsA. A . Prolyl Isomerase Activity Conserved in Evolution

Proteins homologous to the cytosolic cyclophilins from vertebrates or the corresponding genes are found virtually in every organism and in every subcellular compartment. All members of this family that could be obtained in purified form had a high prolyl isomerase activity and were inhibited by CsA, albeit with different effectiveness. Notably, the affinity

PROLYL ISOMERASES

55

of the cytosolic PPI from E . coli for CsA is about 1000-fold lower in comparison to the eukaryotic cyclophilins. Folding experiments with RNase T 1 as a model system indicated that a sample of six different prolyl isomerases of the cyclophilin family from vastly different eukaryotic and prokaryotic species all catalyzed the slow folding of this protein with similar efficiency (Schonbrunner et al., 1991; cf. also Table V). This evolutionary conservation of function suggests that the catalysis of prolyl peptide bond isomerization may indeed be an important function of the cyclophilins and that they could be involved in the de novo protein-folding process.

B . Effect of Cyclosponn A on Cellular Protein Maturation

A retarding effect of CsA on cellular folding, mediated possibly by the inhibition of PPI activity, was observed for two large proteins, collagen and transferrin. Both are secreted proteins that mature to their native oligomeric structure in the endoplasmic reticulum. 1. Folding of Collagen

Procollagen, the precursor of collagen, contains peptide extensions at both the amino- and the carboxy-terminal portions of the chains. The extensions at the carboxy terminus are organized in a globular folded structure in which the three constituent chains are covalently cross-linked by interchain disulfide bonds. Folding and annealing to the triple helix of the three parallel chains of collagen are thought to start at this carboxyterminal globule and to proceed in an ordered unidirectional fashion from the C to the N terminus of the triple helix (Engel and Prockop, 1991). Collagen is readily cleaved by trypsin in the unfolded state, but not in the triple-helical native conformation. Bachinger et al. (1978, 1980) used trypsin pulses applied after various times of folding to detect and follow the occurrence of intermediates that increased in size in the time course of in vitro refolding of type 111 pN collagen. This collagen fragment still contains the carboxy-terminal disulfide-bonded knot. The kinetic analysis, together with measurements of the change in circular dichroism that accompanies triple helix formation, led to the proposal of a folding model for collagen. In the model, the triple helix grows with a uniform rate in a zipperlike manner starting at the carboxy-terminal nucleus. This zero-order helix propagation reaction was suggested to be limited in rate by the cis + trans isomerization of Xaa-proline and/or Xaa-hydroxyproline bonds. Bruckner et al. ( 1981) developed a model that could explain the folding kinetics quantitatively by assuming a rate

56

FRANZ X. SCHMID ET AL.

constant for cis 4 trans isomerization of 0.015 sec-' (at 25°C) and an average number of 30 tripeptide units separating consecutive cis peptide bonds. After the discovery of prolyl isomerase, Bachinger (1987) showed that the in vitro annealing of the collagen triple helix is indeed increased by a factor of about three in the presence of 0.03 mg/ml PPI. Similar results were obtained with type IV procollagen (Davis et al., 1989). The gain of resistance toward proteolysis by trypsin can also be used to follow the maturation of collagen in intact cells. Steinmann et al. (1991) have employed this method to follow the folding of procollagen I in chicken embryo tendon fibroblasts. They found that 8.5 min are required for half-completion of the triple helix. When 5 p M CsA was diffused into the cells, this value increased to 13.5 min (Fig. 9). This retardation of folding led to an overmodification of the collagen chains and also to an increased intracellular degradation of collagen. These results are most

0

5

10 15 20 CHASE T I M E ( m i d

25

30

FIG. 9. Effect of CsA on the rate of procollagen 1 triple helix formation in suspended chicken embryo tendon cells. The time course of procollagen 1 triple helix formation was monitored in a pulse-chase experiment by separation of protease-resistant a l ( 1) and a2( I ) chains by SDS-polyacrylamide gel electrophoresis. T h e Buorograms (upper panel) show the appearance of protease-resistant and hence triple-helical collagen I in the absence (-) or in the presence (+) of 5 p M CsA. T h e kinetics are shown in the lower panel: (0)no CsA; (0)5 p M CsA. Best fits are drawn according to the model of Bruckner and Eikrnberry (1984). From Steinmann et al. (1991).

PROLYL ISOMERASES

57

easily explained by assuming that the rate-limiting prolyl and hydroxyprolyl isomerizations during the in uiuo folding of collagen are catalyzed by prolyl isomerase. Inhibition of this activity by CsA consequently leads to retarded folding and to more pronounced side reactions of the unassembled chains, such as hydroxylations and also nonspecific aggregation. 2. Maturation of Transferm'n Transferrin, a large protein with 19 disulfide bonds, is very slowly secreted from HepG2 cells. Secretion has a half-time of about 3 hr and is sensitive to CsA. When the CsA concentration is increased from lo-' to lop5M , the level of transferrin secretion drops by about threefold. Other secretory proteins, such as serum albumin or a,-antitrypsin, are transported more rapidly and their secretion is not affected by CsA (Lodish and Kong, 1991). Incompletely folded and only partially disulfide-bonded forms of transferrin migrate more slowly than the native correctly cross-linked protein in SDS gel electrophoresis when reducing agents are absent and give rise to diffuse bands on the gel. Lodish and Kong (1991) have used this property to follow the time course of transferrin maturation after 35S pulse labeling in the endoplasmic reticulum in the absence and in the presence of CsA. They note that the maturation of this protein is very slow in either case (Fig. 10). In the presence of CsA, however, intracellular folding shows an initial lag that is absent in the experiment without CsA. Other immunosuppressants, such as FK506 and rapamycin, had no effect. T h e authors conclude that this constitutes evidence for the retardation by CsA of an initial step in the folding of transferrin, and that this might be a step that is accelerated by a prolyl isomerase of the cyclophilin type, which resides in the endoplasmic reticulum. It remains unclear at present why 10-15% of the transferrin molecules adopt a fast-migrating form very rapidly in the absence as well as in the presence of CsA (Fig. 10). In both the maturation of collagen and the folding of transferrin the inhibitory effects of CsA on cellular folding were found to be fairly small and difficult to discriminate from potential effects of CsA on other cellular processes. It is important, however, to consider that prolyl isomerization proceeds during protein folding in the absence of prolyl isomerase as well, albeit at a slower rate. Additionally, even a small retardation of intracellular folding can be deleterious because proteins that are in a partially folded conformation for an extended time can be much more susceptible to nonproductive side reactions, such as aggregation, unwanted covalent modifications, or proteolytic digestion.

58

FRANZ X. SCHMID ET AL

1

0.01 0

,

20

6

40

1

60

I

80

unfolded folded

1

100

Minutes of chase FIG. 10. Effect of CsA on the folding of newly formed transferrin. (A)HepC2 cells were preincubated for 1 hr with (+) or without (-) I p M CsA,pulse labeled at 23°C for 10 min, then chased for the indicated period at 23"C, all in the presence (+) or absence (-) of CsA. Transferrin immunoprecipitates were analyzed by nonreducing SDS-polyacrylamide gel electrophoresis. The arrows denote the very slow-migrating transferrin that accumulates in the presence of CsA. (B) Quantification of the effect of 1 p M CsA on transferrin maturation, as determined by densitometry of the gel in A. In the pulse-labeled samples 12-1576 of transferrin migrated as the folded species. From Lodish and Kong (1991).

PROLYL ISOMERASES

59

IX. CONCLUSIONS A. Prolyl Isomerases as Tools in Protein Folding The characterization of the molecular nature of rate-limiting steps is a major aim in the elucidation of the folding mechanism of proteins. It is now clear that cis-trans isomerizations of prolyl peptide bonds can be such slow steps. Folding reactions that involve prolyl isomerization were traditionally identified by measuring their kinetic properties and by comparing them with the properties of prolyl isomerization in short peptides (Brandts et al., 1975; Nall et al., 1978; Schmid and Baldwin, 1978). Prolyl isomerizations have the following characteristic properties: (1) They are slow, with time constants in the 10- to 100-sec range (25°C). (2) T h e activation energy is near 20 kcal/mol. (3) T h e kinetics of isomerization are independent of the concentration of denaturants, such as urea and GdmC1. Such properties were indeed found in the U, Us reactions in several unfolded proteins. The identification of prolyl isomerization steps during refolding is severely complicated by the coupling between isomerization and the formation of folded structure, which affects the properties of the measured kinetics. Usually, the experimental activation energy is significantly smaller that 85 kJ/mol and the rates of slow folding decrease with increasing denaturant concentration. Prolyl isomerases specifically catalyze the isomerization of Xaa-Pro peptide bonds. The acceleration of a particular folding reaction observed in the presence of a PPI therefore constitutes simple and clear evidence that prolyl isomerization is involved in the rate-limiting step of this reaction. A good example is provided by the chymotrypsin inhibitor CI2. Both slow phases of the kinetics of its refolding were accelerated by PPI and could thus be easily identified as prolyl isomerizations (Jackson and Fersht, 199la,b). Unfortunately, such results are only significant when catalysis by PPI is actually found. A lack of catalysis by PPI is more difficult to interpret. Of course no catalysis will be observed when folding is limited in rate by a molecular process other than prolyl isomerization, but also when the respective prolyl peptide bonds are not accessible for PPI because ordered structure forms rapidly in folding. In the latter case other prolyl isomerases could be tried with a higher activity toward prolines with low accessibility. Cytoplasmic PPI from E. coli might be a good choice in such a case, since it shows a fairly high activity toward partially buried Xaa-Pro bonds (Schonbrunner et al., 1991). Alternatively, the rapid structure formation can be suppressed, usually by increasing the residual concentration of denaturant in the refolding solution. Unfortunately, this approach is restricted by the decrease in activity of many

60

FRANZ X. SCHMII) ET AL

PPIs with increasing concentration of denaturant. A significant inhibition is observed for the enzymes from porcine kidney and from E . coli already in the presence of less than 0.5 M urea or GdmC1. It is conceivable that the folding pathway of a protein can be influenced by prolyl isomerases. In refolding molecules with more than one incorrect isomer, a difference in the catalysis of their reisomerization by PPI could lead to changes in the rank order of isomerization and thus to a different route of refolding. Alternatively, a change in mechanism could occur when prolyl isomerization is sufficiently accelerated and another process becomes rate limiting for folding. Such a change should lead to a marked decrease in catalytic efficiency of PPI at high concentration and was observed in the folding of an immunoglobulin light chain (Lang et al., 1987).

B . Prolyl Isomerization in Cellular Folding It is clear now that prolyl isomerization is a rate-limiting step in the in vitro folding of many proteins. The folding reactions of small proteins are frequently decelerated from the time range of milliseconds to the time range of seconds and minutes when incorrect prolyl isomers are present in the protein chains. The folding of large and oligomeric proteins is often very slow and limited in rate by processes other than prolyl isomerization. T h e question whether prolyl isomerization reactions are also relevant for the de novo folding of proteins in the cellular environment cannot be answered at present, since good experimental evidence is still lacking for the in vivo folding of globular proteins. One notable exception is the cellular maturation of the collagen triple helix, which apparently follows the same proline-limited mechanism in vivo and in vitro (see Section VII1,B). The conformation of Xaa-Pro peptide bonds in the newly synthesized polypeptide chains prior to cellular folding is not known. The product of protein biosynthesis could be a uniform chain with all peptide bonds in the trans conformation. If this chain starts to fold immediately, then the trans-prolines would be in the correct conformation already, the cisprolines would be in the incorrect isomeric state, and their trans + cis isomerization would be involved in the folding of all molecules. Alternatively, if there is sufficient time available for the Xaa-Pro bonds of the nascent chains to reach a &/trans equilibrium (e.g., when folding is transiently arrested by binding to other proteins, such as heat-shock protein (HSP70), then the distribution of prolyl cis and trans isomers prior to cellular folding could be similar to the distribution found in the unfolded protein in vitro. Such a case was encountered in the maturation

PROLYL ISOMERASES

61

of collagen in the endoplasmic reticulum (Section VII1,B). In either case, prolyl isomerization should be involved in cellular folding. In particular, prolyl peptide bonds that are cis in the native protein are entirely or to a large extent in the incorrect trans state, unless the ribosome synthesizes them already as cis peptide bonds. Accepting that prolyl isomerizations do occur in the course of cellular folding, it is still unclear whether these processes influence the kinetics of folding. Prolyl isomerization in oligopeptides is already fairly rapid at 25°C (T = 10-100 sec), and can occur in the time range of a few seconds at 37°C. In addition, initial chain-folding steps can lead to a partially native structure, which could increase the rate of isomerization. Such an “intramolecular catalysis” was observed in the in uitro folding of pancreatic RNase A (Cook et al., 1979; Schmid and Blaschek, 1981). Finally, the in vitro folding of many proteins is much slower than prolyl isomerization and limited in rate presumably by other processes. Taken together, prolyl isomerizations occur almost certainly in the course of protein folding in the cell. It is not clear, however, whether these isomerizations are also rate-determining steps of folding in vivo. These considerations complicate a straightforward assessment of the necessity for enzymatic catalysis of slow steps in cellular protein folding. The restrictions that determine the allowed time window for cellular folding are not known, and they may vary significantly for different proteins. The rate of folding could be an important factor for the steadystate concentration of regulatory proteins that are in very rapid turnover. Another critical aspect may be the time that a protein spends in partially folded conformations that are sensitive to aggregation. Aggregation can be suppressed by rapid but reversible binding to other proteins, such as the chaperones. Aggregation could also be minimized by shortening the time of exposure of interactive surfaces in folding intermediates, i.e., by catalyzing critical slow-folding steps, such as prolyl isomerizations. Prolyl isomerases of the cyclophilin type show some properties that would be expected for a catalyst of cellular protein folding. Cyclophilins occur in all cellular compartments where folding reactions occur. The activity toward accessible prolyl bonds is high, and the specificity with regard to the chemical nature of residue Xaa is low. Additional experiments are clearly needed, however, to clarify the possible role of prolyl isomerases for the in uivo folding process of nascent proteins. C . Relation with Immunosuppression and Signal Transduction

The finding that both classes of prolyl isomerases, the cyclophilins and the FK506-binding proteins, are strongly inhibited by immunosuppres-

62

FRANZ X. SCHMID ET AL

sive agents is intriguing, but its implications are not understood at present. Immunosuppressants, such as CsA, are isolated from microorganisms, and they are probably not produced by these organisms to affect the immune system of mammals. I t is more likely that they interfere with more fundamental processes common to all cells. This is reflected in the ubiquitous distribution of the respective target proteins, the cyclophilins and the FK-binding proteins. Intensive efforts are being made to understand the role of prolyl isomerases and of their complexes with immunosuppressants for signal transduction pathways (Schreiber, 1991; Flanagan et al., 1991). First results indicate that these complexes, but not the unliganded enzymes, interact with calcineurin, a Ca2+-dependent protein phosphatase (Liu et al., 199 1). At present it is too early to speculate whether the primary cellular function of prolyl isomerases is correlated with processes that involve protein folding or with signal transduction pathways. Possibly, prolyl isomerases are involved in all these processes and PPI activities are important for their regulation. Whether such a multitude of tasks is accomplished by the same enzymes or rather by specialized prolyl isomerases is not known.

REFERENCES Adler, M., and Scheraga, H. A. (1990). Biochemistry 29, 8211-8216. Alexandrescu, A. T . , Ulrich, E. L., a n d Markley, J. L. (1989). Biochemistry 28, 204-21 1 . Alexandrescu, A. T., Hinck, A. P., and Markley, J. L. (1990). Biochemistry 29, 4516-4525. Babul, J., Nakagawa, A,, and Stellwagen, E. (1978).J. Mol. Biol. 126, 117-121. Bachinger, H.-P. (1987).J. Biol. Chem. 262, 17144-17148. Bachinger, H.-P., Bruckner, P., Timpl, R., and Engel, J. (1978). Eur. J . Biochem. 90, 605-613. Bachinger, H.-P., Bruckner, P., Timpl, R., Prockop, D. J., and Engel, J. (1980). Eur. J . Biochem. 106, 619-632. Brandts, J. F., Halvorson, H . R., a n d Brennan, M . (1975). Biochemistry 14, 4953-4963. Brandts, J. F., Brennan, M., and Lin, L.-N. (1977). Proc. Natl. Acad. Sri. U.S.A. 74, 41 78-4 182. Brown, R. D., 111, Brewer, C. F., and Koenig, S. H. (1977). Biochemistry 16, 3883-3896. Bruckner, P., and Eikenberry, E. F. (1984). Eur.J. Biochem. 140, 391-395. Bruckner, P., Eikenberry, E. F., and Prockop, D. J. (1981). Eur.1. Biochem. 118,607-613. Buchner, J., Schmidt, M., Fuchs, M . , Jaenicke, R., Rudolph, R., Schmid, F. X., and Kiefhaber, T. (1991). Biochemistry 30, 1586-1591. Bulleid, N. J . , and Freedman, R. B. (1988). Nature (London) 335, 649-651. Caroni, P., Rothenfluh, A,, McGlynn, E., and Schneider, C. (1991). J . Biol. Chem. 266, 10739-10742. Chazin, W. J., Kordel, J., Drakenberg, T., Thulin, E., Brodin, P., Grundstrom, T., a n d Forsen, S. (1989). Proc. Natl. Acad. Sci. U.S.A. 86, 2195-2198. Cheng, H. N., and Bovey, F. A. (1977). Biopolymers 16, 1465-1472. Colley, N. J., Baker, E. K., Stamnes, M. A,, and Zuker, C. S. (1991). Cell (Cambridge, Muss.) 67,255-263.

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Cook, K. H., Schmid, F. X., and Baldwin, R. L. (1979). Proc. Natl. Acad. Sci. U.S.A. 76, 6157-6 161. Davis, J. M., Boswell, B. A,, and Bachinger, H.-P. (1989).J. Bid. Chem. 264, 8956-8962. Engel, J.. and Prockop, D. J. (1991). Annu. Rev. Biophys. Biophys. Chem. 20, 137-152. Evans, P. A., Dobson, C. M., Kautz, R. A., Hatfull, G., and Fox, R. 0. (1987). Nature (London) 329, 266-268. Fischer, G., and Bang, H . (1985). Biochim. Biophys. Acta 828, 39-42. Fischer, G., Bang, H., and Mech, C. (1984). Biomed. Biochim. Acta 10, 1101-1 111. Fischer, G., Wittmann-Liebold, B., Lang, K., Kiefhaber, T., and Schmid, F. X. (1989). Nature (London) 337, 476-478. Flanagan, W. M., Corthesy, B., Bram, R. J., and Crabtree, G. R. (1991). Nature (London) 352,803-807. Freedman, R. B. (1984). Trends Biochem. Sci. 9, 438-441. Garel, J . R., and Baldwin, R. L. (1973). Proc. Natl. Acad. Sci. U.S.A. 70, 3347-3351. Goloubinoff, P., Christeller, J. T., Gatenby, A. A,, and Lorimer, G. H. (1989). Nature (London) 342, 884-889. Goto, Y., and Hamaguchi, K. (1982).J. Mol. Biol. 156, 891-910. Grafl, R., Lang, K., Wrba, A,, and Schmid, F. X. (1986).J. Mol. Biol. 191, 281-293. Grathwahl, C., and Wuthrich, K. (1976a). Biopolymers 15, 2025-2041. Grathwahl, C., and Wuthrich, K. (197613).Biopolymers 15, 2043-2057. Grathwahl, C., and Wuthrich, K. (1981). Biopolymers 20, 2623-2633. Haendler, B., Hofer-Warbinek, R., and Hofer, E. (1987). E M B O J . 6, 947-950. Hagerman, P. J. (1977). Biopolymers 16, 731-747. Handschumacher, R. E., Harding, M. W., Rice, J., Drugge, R. J., and Speicher, D. W. (1984). Science 226, 544-547. Harding, M. W., Galat, A., Ueling, D. E., and Schreiber, S. L. (1989). Nature (London) 341, 758-760. Harrison, R. K., and Stein, R. L. (1990). Biochemistry 29, 3813-3816. Hasel, K. W., Glass, J. R., Godbout, M., and Sutcliffe, J. G. (1991). MoL. Cell. Biol. 11, 3484-349 1. Hawkins, H. C., Blackburn, E. C., and Freedman, R. B. (1991). Biochem. J. 275,349-353. Hayano, T., Takahashi, N., Kato, N., Maki, N., and Suzuki, M. (1991). Biochemistry 30, 3041-3048. Heinemann, U., and Saenger, W. (1982). Nature (London) 299, 27-32. Herning, T., Yutani, K., Taniyama, Y., and Kikuchi, M. (1991).Biochemistry 30,9882-9891. Higgins, K. A., Craik, D. J., Hall, J. G., and Andrews, P. R. (1988). Drug Des. Delivery 3, 159- 170. Hurle, M. R., Anderson, S., and Kuntz, I. D. (1991). Protein Eng. 4, 451-455. Ihara, S., and Ooi, T. (1985). Biochim. Biophys. Acta 830, 109-112. Jackson, S., and Fersht, A. R. (1991a). Biochemistry 30, 10428-10435. Jackson, S., and Fersht, A. R. (1991b). Biochemistry 30, 10436-10443. Jaenicke, R. (1987). Prog. Biophy. Mol. Biol. 49, 117-237. Jaenicke, R. (1991). Biochemistry 30, 3147-3161. Jorgensen, W. L., and Gao, J. (1988).J. Am. Chem. Soc. 110, 4214-4216. Kallen, J., Spitzfaden, C., Widmer, H., Wuthrich, K., and Walkinshaw, M. D. (1991).Nature (London) 353, 276-279. Kato,S.,Okamura, M., Shimamoto, N., and Utiyama, H. (1981).Biochemistry20,1080-1085. Kato, S., Shimamoto, N., and Utiyama, H. (1982). Biochemistry 21, 38-43. Kawamukai, M., Matsuda, H., Fujii, W., Utsumi, R., and Komano, T . (1989).J. Bacteriol. 171,4525-4529.

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Kelley, R. F., and Richards, F. M. (1987). Biochemistry 26, 6765-6774. Kelley, R. F., and Stellwagen, E. (1984). Biochemistry 23, 5095-5102. Kelley, R. F., Wilson, J . , Bryant, C., and Stellwagen, E. (1986). Bzochemi.qtry 25, 728-732. Kiefhaber, T., and Schmid, F. X. (1992).J. Mol. Bid. 224, 231-240. Kiefhaber, T., Quaas, R., Hahn, U., and Schmid, F. X. (1990a). Biochemistry 29,3053-3060. Kiefhaber, T., Quaas, R., Hahn, U., and Schmid, F. X. (1990b).Biochemistry 29,3061-3070. Kiefhaber, T., Grunert, H.-P., Hahn, U., and Schmid, F. X. (1990~).Biochemistry 29, 6475-6479. Kiefhaber, T., Schmid, F. X., Renner, M., Hinz, H.-J., Hahn, U., and Quaas, R. (199Od). Biochemistry 29, 8250-825 7. Kiefhaber, T., Kohler, H.-H., and Schmid, F. X. (1992a).J.Mol. B i d . 224, 217-229. Kiefhaber, T., Chaffotte, A , , Engelborghs, Y., and Schmid, F. X. (1992b). Proteins Struct. Funct. Genet. 12, 171-179. Kim, P. S., and Baldwin, R. L. (1982). Annu. Rev. Biochem. 51, 459-489. Kim, P. S., and Baldwin, R. L. (1990). Annu. Rev. Biochem. 59, 631-660. Koepke, J., Maslowska, M., Heinemann, U., and Saenger, W. (1989). J . Mol. Biol. 206, 475-488. Kofron, J . L., Kuzmic, P., Kishore, V . , Colon-Bonilla, E., and Rich, D. (1991). Biochemistry 30,6127-6134. Kordel, J., Forsen, S., Drakenberg, T., and Chazin, W. J. (1990a). Biochemistry 29, 4400-4409. Kordel, J., Drakenberg, T., ForsCn, S., and Thulin, E. (1990b). FEBS Lett. 263, 27-30. Krebs, H., Schmid, F. X., and Jaenicke, R. (1983).J. Mol. Bid. 169, 619-633. Krebs, H., Schmid, F. X., and Jaenicke, R. (1985). Biochemistry 24, 3846-3852. Kuwajima, K., Okayama, N., Yamamoto, K., Ishihara, T., and Sugai, S. (1991).FEES Lett. 290, 1357138. Lang, K. (1988). Ph.D. Thesis, University of Regensburg, West Germany. Lang, K., and Schmid, F. X. (1990).J. Mol. Bid. 212, 185-196. Lang, K., Schmid, F. X., and Fischer, G. (1987). Nature (London) 329, 268-270. Levitt, M. (1981).J. Mol. Bid. 145, 251-263. Lin, L.-N., and Brandts, J. F. (1978). Biochemistry 17, 4102-4110. Lin, L.-N., Hasumi, H., and Brandts, J. F. (1988). Biochim. Biophys. Actu 956, 256-266. Liu, J., and Walsh, C. T. (1990). Proc. Natl. Acad. Sci. U.S.A. 87, 4028-4032. Liu, J., Farmer, J. D., Jr., Lane, W. S., Friedman, J., Weissman, I., and Schreiber, S. S. (1991). Cell (Cambridge, Mass.)66, 807-815. Lodish, H. F., and Kong, N. (1991).J. Biol. Chem. 266, 14835-14838. MacArthur, M. W., and Thornton, J . M. (1991).J. Mol. B i d . 218, 397-412. Marsh, H. C., Hiskey, R. G., and Kochler, K. A. (1979). Biochem.j. 183, 513-517. Matouschek, A . , Kellis, J. T., Serrano, L., Bycroft, M., and Fersht, A. R. (1990). Nature (London) 346, 440-445. Mendoza, J. A., Lorimer, G. H., and Horowitz, P. M . (1991). ,J. Bid. Chem. 266, 16973-16976. Mucke, M., and Schmid, F. X. (1992). Biochemistry 31, 7848-7854. Nall, B. T. (1990). In “Protein Folding” L. M. Gierasch and J . King, eds.), pp. 198-207. AAAS Press, Washington, DC. Nall, B. T., Garel, J.-R., and Baldwin, R. L. (1978).J. Mol. B i d . 118, 317-330. Oobatake, M., Takahashi, S., and Ooi, T. (1979).J. Biochem. (Tokyo) 86, 65-70. Pace, C. N. (1990). Trends Biocliem. Sci. 15, 14-17. Pace, C. N., and Creighton, T. E. (1986).J. Mol. Bid. 188, 477-486. Pace, C. N., and Grimsley, G. R. (1988). Biochemistry 27, 324‘2-3246.

PROLYL ISOMERASES

65

Pace, C. N., Grimsley, G. R., Thomson, J. A , , and Barnett, B. J. (1988).J. Biol. Chem. 263, 11820-1 1825. Pace, C. N., I.aurmts, U . V., and Thornson.J. A. (1990). Wiorlarmistq 29, 2‘51i,l-2572. Pace, C. N., Heinemann, U., Hahn, U., and Saenger, W. (lYYl). Angew. Chem. Inl. Ed. Engl. 30, 343-360. Price, E. R., Zydowsky, L. D., Mingjie, J., Baker, C. H., McKeon, F. D., and Walsh, C. T . (1991). Proc. Natl. Acad. Sci. U.S.A. 88, 1903-1907. Ramdas, L., Sherman, F., and Nall, B. T. (1986). Biochemistry 25, 6952-6958. Ridge, J. A,, Baldwin, R. L., and Labhardt, A. M. (1981). Biochemistry 20, 1622-1630. Schmid, F. X. (1982). Eur. J . Biochem. 128, 77-80. Schmid, F. X. (1992).I n “Protein Folding” (T. E. Creighton, ed.), pp. 197-241. Freeman, New York. Schmid, F. X., and Baldwin, R. L. (1978). Proc. Natl. Acad. Sci. U.S.A. 75, 4764-4768. Schmid, F. X., and Baldwin, R. L. (1979).J. Mol. Biol. 133, 285-287. Schmid, F. X., and Blaschek, H. (1981). Eur.J. Biochem. 114, 11 1-1 17. Schmid, F. X., Grafl, R., Wrba, A,, and Beintema, J. J. (1986). Proc. Natl. Acud. Sci. U.S.A. 83, 872-876. Schneuwly, S., Shortridge, R. D., Laarrivee, D. C., Ono, T., Ozaki, M., and Pak, W. L. (1989). Proc. Natl. Acad. Sci. U.S.A. 86, 5390-5394. Schonbrunner, E. R., Mayer, S., Tropschug, M., Fischer, G., Takahashi, N., and Schmid, F. X. (1991).J . Biol. Chem. 266, 3630-3635. Schonbrunner, E. R., and Schmid, F. X. (1992).Proc. Natl. Acad. Sci. U.S.A. 89,4510-4513. Schreiber, S. S. (1991). Science 251, 283-287. Shieh, B.-H., Stamnes, M. A., Seavello, S., Harris, S. L., and Zuker, C. S. (1989). Nature (London) 338,67-70. Siekierka, J. J., Hung, S. H. Y., Poe, M., Lin, C. S., and Sigal, N. H. (1989).Nature (London) 341,755-757. Spik, G., Haendler, B., Delmas, O., Mariller, C., Chamoux, M., Maes, P., Tartar, A , , Montreuil, J., Stedman, K., Kocher, H. P., Keller, R., Hiestand, P. C., and Movva, R. (1991).J. Biol. Chem. 266, 10735-10738. Stamnes, M. A., Shieh, B.-H., Chuman, L., and Zuker, C. S. (1991). Cell (Cambridge, Mass.) 65, 219-227. Steinmann, B., Bruckner, P., and Supertifurga, A. (1991).J. Biol. Chem. 266, 1299-1303. Stewart, D. E., Sarkar, A,,and Wampler, J. E. (1990).J. Mol. Biol. 214, 253-260. Takahashi, N., Hayano, T., and Suzuki, M. (1989). Nature (London) 337, 473-475. Tropschug, M., Wachter, E., Mayer, S., Schonbrunner, E. R., and Schmid, F. X. (1990). Nuture (London) 346, 674-677. Tsong, T. Y . (1977)./. B d . Chem. 252, 8778-8780. Vaucheret, H., Signon, L., Le Bras, G., and Garel, J.-R. (1987). Biochemistry 26, 2785. White, T . B., Berget, P. B., and Nall, B. T. (1987). Biochemistry 26, 4358-4366. Wood, L. C., Muthukrishnan, K., White, T . B., Ramdas, L., and Nall, B. T . (1988). Biochemistry 27, 8554-8561. Wuthrich, K., Spitzfaden, C., Memmert, K., Widmer, H., and Wider, G. (1991).FEES Lett. 285,237-247.

NOTEADDEDIN PROOF.The structure of free prolyl isomerases of the cyclophilin type was refined at 1.63 8, (Ke, 1992). Further work on the complex of human cyclophilin with a tetrapeptide showed that the peptide N-acetyl-Ala-Ala-Pro-Ala-amidomethylcoumarin

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(cf. Fig. 2) is actually bound with a cis Ala-Pro bond (Kallen and Walkinshaw, 1992). The structure of the complex of human cyclophilin with CsA was solved by x-ray crystallography (Pflugl et al., 1993) and by modelling based on a combination of x-ray and NMR data (Spitzfaden et al., 1992). Additional members of the cyclophilin family were discovered with molecular weights in the 22-kDa range (Thalhammer et al., 1992) and 40-kDa range (Kieffer et al., 1992). Similarly, homologs of FKBP of higher molecular weight, notably in the 25- and 55-kDa region, were found in various tissues (Galat et al., 1992; Jin et al., 1992; Callebaut et al., 1992; Peattie et al., 1992; Tai et al., 1992; Yem et al., 1992). Prolyl isomerase activities also occur in various plant organelles (Breiman et al., 1992). T h e evidence is good now that the immunosuppressive effects of the cyclophilin/ cyclosporin and FKPB/FK 506 complexes in uivo are mediated by the inhibition of calcineurin activity (Clipstone and Crabtree, 1992; Fruman et al., 1992). A new, continuous assay for prolyl isomerases was developed by Garcia-Echeverria et al. (1992). Breiman, A,, Fawcett, T . W., Ghirardi, M. L., and Mattoo, A. K. (1992).J. B i d . Chem. 267, 21293-2 1296. Callebaut, I., Renoir, J.-M., Lebeau, M.-C., Massol, N., Burny, A., Baulieu, E.-E., and Mornon, J.-P. (1992). Proc. Natl. Acad. Scz. U.S.A. 89, 6270-6274. Clipstone, N. A,, and Crabtree, G. R. (1992). Nature (London) 357, 695-697. Fruman, D. A,, Klee, C. B., Bierer, B. E., and Burakoff, S. J. (1992). Proc. Natl. Acad. Scz. U.S.A. 89, 3686-3690. Galat, A., Lane, W. S., Standaert, R. F., and Schreiber, S. L. (1992). Biochemistry 31, 2427-2434. Garcia-Echeverria, C., Kofron, J. L., Kuzmic, Kishore, V., and Rich, D. H. (1992).J. Am. Chem. Soc. 114, 2758-2759. Jin, Y.-J., Burakoff, S. J., and Bierer, B. E. (1992).J. Bid. Chem. 267, 10942-10945. Kallen, J., and Walkinshaw, M. D. (1992). FEES Lett. 300, 286-290. Ke, H. (1992).J. Mol. B i d . 228, 539-550. Kieffer, L. J., Thalhammer, T., and Handschumacher, R. E. (1992).J . Biol. Chem. 267, 5503-5507. Peattie, D. A., Harding, M. W., Fleming, M. A,, DeConzo, M. T., Lippke, J. A., Livingston, D. J., and Benasutti, M. (1992). Proc. Natl. Acad. Sci. U.S.A. 89, 10974-10978. Pflugl, G., Kallen, J., Schirmer, T., Jansonius, J., Zurini, M. G. M., and Walkinshaw, M. D. (1993). Nature (London) 361, 91-94. Spitzfaden, C., Weber, H.-P., Braun, W., Kallen, J., Wider, G., Widmer, H., Walkinshaw, M. D., and Wuthrich, K. (1992). FEBS Lett. 300, 291-300. Tai, P.-K. K., Albers, M. W., Chang, H., Faber, L. E., and Schreiber, S. L. (1992). Science 256, 1315-1318. Thalhammer, T., Kieffer, L. J., Jiang, T.-R., and Handschumacher, R. E. (1992). Eur. J . Biochem. 206, 31-37. Yem, A. W., Tomasselli, A. G., Heinrikson, R. L., Zurcher-Neely, H., Ruff, V. A,, Johnson, R. A., and Deibel, M. R., Jr. (1992).J. Biol. Chem. 267, 2868-2871.

STRUCTURE AND MECHANISM OF 70-kDa HEAT-SHOCK-RELATED PROTEINS By DAVID 0. McKAY Beckman Laboratories for Structural Biology, Department of Cell Biology, Stanford University School of Medicine, Stanford, California 94305

I. Overview of Stress-70 Proteins

. . . . . . . . . . . . . . .. . .. . . . . . . . . . . . . . . . . . .. .

11. Biochemical Activities of Stress-70 Proteins . . . . . . . . . . . . . . . .

B. Interactions with Polypeptides. . . . . . . . . . C. Renaturation Activities . . . . . . . . . . . . . . . D. Common Mechanism for All Activities.. 111. Structure of Stress-70 Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Primary Structure . . . . . . . . . B. Tertiary Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . C. Temperature-Dependent Structural Transitions IV. Enzymatic Mechanism of Stress-70 Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. ATPase Mechanism ....................... B. Peptide Recognition . . . C. Coupling of Peptide-Binding and ATPase Activities . . . . . . . . . . . . . . . . . D. Clathrin Uncoating Reaction . . . . . V. Modulators of Stress-70 Protein Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Accessory Proteins B. Posttranslational Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.... . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

67 69 69 70 71 72 73 73 75 79 80 80 83 85 86 89 89 91 92 93

I. OVERVIEW OF STRESS-70 PROTEINS T h e family of 70-kDa heat-shock-related proteins is a widespread family of proteins that are essential for normal cell viability and thermotolerance, but whose specific in vivo biochemical functions are not yet well understood. T h e members of this family that were identified initially had levels of expression substantially enhanced in response to heat shock; for example, the seminal observation of heat-inducible 70-kDa proteins in Drosophilu was reported by Tissieres and colleagues in 1974. Only later did it become apparent that cells had other homologues, closely related in sequence to the inducible representatives, that were expressed constitutively or in a less stringently regulated fashion. As a consequence, the nomenclature by which members of this family have been referred to, i.e., “heat-shock” proteins (HSPs) or “heat-shock-related’’ proteins, has historically inflicted some confusion as to the functions of the proteins. 67

Cop! i-iglit 0 1993 In Ac;idemic Press. Inc. All rights ol reproduction i i i an! loriii reserved.

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It is now realized that members of this family of proteins are present in cells under normal conditions, and that their presence is not strictly stress related. Induction of expression of some members in response to heat shock or other forms of cell stress is only a manifestation of an additional facet of the collective functions of the larger family of proteins. However, for simplicity in nomenclature, I will follow the convention adopted in another recent review and refer to the family of 70-kDa heat-shockrelated proteins as the “stress-70”protein family (Gething and Sambrook, 1992). Typically, bacteria have been found to have only a single stress-70 protein, whereas eukaryotic cells have several different representatives (Pelham, 1986). T h e Escherichia coli representative is the dnaK protein; other bacteria have been found to have dnaK homologues (Bardwell and Craig, 1984; Birkelund et al., 1990; Danilition et al., 1990; Garsia et al., 1989; Gomez et al., 1990; Hearne and Ellar, 1989; Kornak et al., 1991; Sussman and Setlow, 1987). In eukaryotic cells, the HSP70-related proteins can be broadly segregated into three groups, based on their compartmentalization within the cell. The first group consists of stress-70 proteins that are found in all mitochondria, and additionally in chloroplasts in the case of plant cells (Amir-Shapira et al., 1990; Craig et al., 1989; Engman el al., 1989; Leustek et al., 1989; Marshall 1990; Mizzen et al., 1989). T h e second group is composed of the inducible heat-shock proteins (HSP70s), whose synthesis is induced by various cellular stresses, and their constitutive counterparats, often referred to as heat-shock cognates (HSC70s), which are found in the cell cytoplasm under normal physiological conditions. Generally these proteins appear to localize in the nucleolus after a cell is stressed. The discrimination between inducible versus constitutive representatives is not distinct. For example, in yeast there are eight genes that segregate into four complementation groups for HSP70-related proteins. One particular group, expressing HSP70and HSC70-like proteins, has four genes (SSAl -SSA4) whose expression is interdependent. SSAl and SSA2 are constitutive in wild-type cells, although SSAl mRNA increases severalfold after heat shock. SSA3 is normally inducible, but is expressed constitutively in cells in which SSAl and SSA2 genes are functionally deleted; SSA4 is strictly inducible (Werner-Washburne et al., 1987). A stress-70 protein is also localized in the endoplasmic reticulum (Munro and Pelham, 1986). It is referred to as the binding protein, or BiP, since it has been observed to bind immunoglobulin heavy chains during their synthesis and assembly in lymphocytes (Haas and Wabl, 1983),as well as other proteins in the endoplasmic reticulum. Historically, it has also been referred to as the glucose-regulated protein (GRP78),

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because its level in cells increases in response to glucose starvation, a, physiological response whose functional significance is still nebulous (Shiu and Pastan, 1979). Other reviews have summarized many of the apparent biological functions of the stress-70 proteins (Craig et al., 1990, Gething and Sambrook, 1992; Haas, 1991; Winfield and Jarjour, 1991). This review focuses primarily on the protein chemistry and mechanistic enzymology of this protein family. 11. BIOCHEMICAL ACTIVITIES OF STRESS-70 PROTEINS

Although an exhaustive review of the literature on stress-70 protein function is beyond the scope of this discussion, it is informative to highlight the diverse types of activities with which these proteins have been associated. These can be generally grouped into (1) participation in assembly or disassembly of macromolecular complexes, (2) interactions with polypeptides, including short (<30 residues) oligopeptides as well as full-length proteins, and (3) renaturation of denatured proteins. In surveying these varied activities, one is left with an intriguing question: How does a single protein, or small group of closely related proteins, manage to accomplish all these tasks? A . Participation in Disassembly of Specific Oligomeric Complexes One of the activities first identified for a stress-70 protein was the participation of the E . coli dnaK protein in the initiation of A phage replication. A mutant was isolated that failed to support A replication (Georgopoulos, 1977) and subsequent mapping of the gene and identification of the gene product indicted the dnaK protein as a participant in the initiation of A replication (Georgopoulos et al., 1979). It is now known that the dnaK protein, in concert with the E. coli proteins dnaJ and grpE, facilitates the dissociation of the A P protein from a replication preinitiation complex, in an ATP-dependent reaction (Mensa-Wilmot et al., 1989; Yamamoto et al., 1987). A mutant that fails to promote this activity, classically referred to as dnaK756, has been isolated; in many subsequent biochemical studies, activities of the dnaK756 protein have been examined in parallel with wild-type dnaK protein. Wickner and colleagues have demonstrated a role for the E. coli dnaK protein in replication of bacteriophage P1 (Wickner, 1990). In P1 replication, the phage repA protein binds specifically to the P1 origin of replication; it appears to be a monomeric repA that binds to the origin with high affinity. Dimers of repA, in a 2 : 2 subunit complex with the E . coli

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dnaJ protein, can be dissociated into monomers by dnaK in an ATPrequiring reaction, thereby activating the repA protein and allowing high-affinity binding to the origin (Wickner et al., 1991a,b). Another activity associated with a stress-70 protein is the in vitro disassembly of clathrin cages into triskelions (see Section IV,D). Rothman and colleagues isolated and characterized a protein from bovine brain that facilitated the disassembly of clathrin cages in a reaction that required hydrolysis of ATP; they referred to the protein as a “clathrin-uncoating ATPase” (Schlossman et al., 1984). Subsequently, it was realized that this protein was a constitutively expressed member of the stress-70 family (HSC70) (Chappell et al., 1986). Hence, several in uitro activities have demonstrated a participation of stress-70 proteins in the disassembly of macromolecular complexes. T h e participation of dnaK in initiation of replication for A and P1 phages appears to be dependent on ancillary proteins, specifically dnaJ and grpE. This raises the possibility that substrate specificity, in these cases, may be intrinsic to the ancillary proteins rather than residing solely in the dnaK protein. In the case of in vitro dissassembly of clathrin cages, the HSC70 protein can accomplish the reaction without accessory proteins. In all the above cases, ATP hydrolysis is essential for the activities. B . Interactions with Polypeptides

There is also evidence that the stress-70 proteins interact with extended, or “unfolded,” polypeptides. It has been demonstrated that a mitochondrial stress-70 protein is essential for import of proteins into mitochondria (Kang et al., 1990; Ostermann 1990). More specifically, polypeptides bind to, and can be cross-linked to, the mitochondrial stress70 protein during import (Scherer et al., 1990). It has further been demonstrated that cytoplasmic stress-70 proteins are essential for efficient transmembrane translocation of proteins into mitochondria or microsomes (Chirico et al., 1988; Deshaies et al., 1988); this has led to the suggestion that the cytoplasmic stress-70 proteins may bind to and stabilize extended, unfolded conformations of polypeptide prior to their transmembrane translocation. Additionally, Welch and colleagues have demonstrated by immunoprecipitation with monoclonal antibodies against cytoplasmic stress-70 proteins that these proteins appear to interact with a plethora of polypeptides in the cell, possibly recognizing them as nascent, unfolded polypeptides during translation (Beckmann et al., 1990). In yeast it has been shown that the BiP protein is essential for the

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translocation of proteins into the endoplasmic reticulum, as evidenced by the fact that loss of function of temperature-sensitive mutant BiP at the nonpermissive temperature results in loss of secretion of several proteins, as well as protease sensitivity of the protein precursors on the cytoplasmic side of the endoplasmic reticulum membrane (Nguyen et al., 1991; Vogel et al., 1990). Although direct interaction of a stress-70 protein with a presumably unfolded protein has been demonstrated only in the case of a mitochondrial stress-70 protein that could be cross-linked to a “stuck,” partially translocated polypeptide, there is substantial indirect and circumstantial evidence that supports a general model in which the stress-70 proteins “meet” extended polypeptides before they fold into a compact tertiary structure, be it cytoplasmic HSC70 binding to nascent polypeptides during translocation, or BiP (in the endoplasmic reticulum) or the mitochondrial stress-70 proteins binding extended polypeptides during the transmembrane translocation process. ATP binding and hydrolysis appear to be required for peptide release. These observations have given rise to the notion that one of the functions of stress-70 proteins may be to assist in the folding, or at a minimum, to prevent the aggregation and misfolding, of polypeptides. C . Renaturation Activities

Under certain conditions, the stress-70 proteins can participate in the renaturation of denatured or inactivated proteins. The renaturation capabilities of E. coli dnaK protein have been most extensively documented. It has been shown that in vitro, dnaK can protect E. coli RNA polymerase from aggregation when the polymerase is incubated at elevated temperatures that would normally result in loss of activity, and, further, that dnaK can disaggregate and reactivate polymerase, once it has been inactivated by heat denaturation (Skowyra et al., 1990). These activities are absolutely dependent on ATP hydrolysis. T h e mutant dnaK756 protein is effective in protecting active RNA polymerase against heat inactivation, but is incapable of disaggregating and reactivating polymerase, once it has been heat inactivated. A second demonstration of participation in renaturation of heatinactivated proteins by dnaK is provided by experiments with a temperature-sensitive repressor protein of bacteriophage A , Ad857 protein (Gaitanaris et al., 1990). Activities of the AcI wild-type and mutant proteins were measured in an in vitro operator DNA binding assay and by in vivo expression from a hcl-regulated operon fusion of hPRORand the

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E . coli gal operon. It was shown that the activity of hcI857 protein that was heat denatured in E . coli at a nonpermissive temperature (42°C) for 30 min was restored in strains expressing wild-type dnaK, as measured by both in vivo repression of a P-galactosidase expression and in vitro A operator DNA binding by protein from cell lysates. However, the activity of hcI857 repressor was not restored efficiently in strains with mutations in the dnaK gene (dnaK756, as well as dnaK25, a recently isolated mutant that fails to grow at 37°C). Further, recovery of AcI857 repressor activity was also inefficient in E. coli strains harboring mutations in the dnaJ (dnaJ259) and grpE (grpE280) genes. By inference, it appears that the presence of wild-type dnaK correlates with efficient renaturation of a heat-inactivated temperature-sensitive hcI repressor protein, and that the presence of wild-type dnaJ and grpE correlates with the augmented efficiency of the process.

D. Common Mechanism for All Activities In summary, the stress-70 proteins have been demonstrated to participate in diverse and seemingly unrelated activities: disassembly of specific macromolecular aggregates, interation with extended and presumably unfolded polypeptides, and renaturation or reactivation of certain proteins under some conditions. All of these activities are dependent on ATP hydrolysis. This suggests that the stress-70 proteins have a common mechanism for transducing free energy of nucleotide hydrolysis into interactions with the protein/polypeptide substrates. The specific nature of this mechanism is not understood at this time. One apparent dilemma that makes it difficult to reconcile all of these activities into a common mechanism is how a single protein (such as dnaK in E . coli or BiP in the endoplasmic retidulum) or group of closely related proteins (in cases where several homologues are expressed simultaneously) can recognize, with a substantial degree of discrimination, such a broad diversity of polypeptide and protein substrates. Based on available evidence, it can be suggested that binding of relatively unstructured segments of polypeptides by the stress-70 proteins might be a common feature of their interaction with unfolded polypeptides, partially denatured proteins, and some macromolecular complexes such as clathrin light chains (discussed below). However, such a simplistic view will require substantial modification and augmentation based on further experimental evidence before a satisfactory understanding of the specificity of polypeptide substrate recognition by this family of proteins is achieved.

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STRUCTURE OF STRESS-70 PROTEINS A . Primary Structure

More than 40 complete nucleic acid sequences have been reported for genes or cDNAs of stress-70 proteins (GenBank release 70.0; December, 1991).A general consensus picture for the derived amino acid sequences, which may evolve further as more sequences become available, has emerged. (Fig. 1). Generally, the eukaryotic HSP70 and HSC70 proteins, as well as the mammalian BiPs, have about a 50-55% amino acid sequence identity with the bacterial dnaK proteins. Mammalian BiPs show typically 65-7076 sequence identity with the HSP70 and HSC70 proteins. As a group, the HSP70 and HSC70 proteins have -75- 100% identity between individual members. Some mammalian stress-70 proteins show a dramatically stringent level of sequence conservation; for example, the three mammalian BiPs that have been sequenced (human, hamster, and rat) are 97-100% identical (Munro and Pelham, 1986; Ting and Lee, 1988; Ting et al., 1987). These data support an evolutionary scheme in which the ancestral precursor of HSP70, HSC70, and BiP first diverged from the dnaK-like proteins; subsequently, BiP diverged and developed independently from the HSP70 and HSC70 proteins. The level of sequence conservation is nonuniform within the primary structure. The aminoterminal (70-80% of the primary structure) is more highly conserved than the carboxy-terminal remainder. Many of the most highly conserved regions of the sequence are segments that form the nucleotide-binding environment of the ATPase region of the molecules (discussed in more detail below). T h e greatest variation in sequence occurs in the last -30-50 amino acids of the carboxy terminus; different subgroups of the stress-70 proteins have different characteristic “tails.” T h e HSP70 and HSC70 proteins have a stretch of -10-30 uncharged amino acids that is rich in glycine, alanine, proline, and hydrophobic residues; in higher animals, an unusual approximate GGMP, o r more generally, approximate GG(hydrophobic)P, motif is repeated several times. For example, bovine HSC70 has the sequence GGMP-GGMP-GGMP-GGLP-GGGAPPS . . . which is followed by a more polar, predominantly acidic termination, GP(K/T)(I/ V)EEVD. T h e particular function of this sequence motif, if there is one, has not yet been identified. T h e BiP proteins have a 10- to 15-residue G,A,S,P-rich region followed by a short acidic region and an endoplasmic reticulum retention signal-(K/H/D)DEL--KDEL-in sequences from mammals, HDEL in the yeast Saccharomyces cerevisiae (Normington et al.,

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ATPase Fragment-highly conserved in sequence

Leadermitochondrial,

Peptide Binding Fragment-more variable

C

a

C -650

N

-385

1

Relatively highly conserved in sequence

V -385

V -550

Carboxy terminus:

Highly variable

I in sequence -600

E--[

C -650

]

G,A,P,hydrophobic-rich; --G P(K/T)(i/V)EEVD in higher animals, approximate (GGMP) repeat

forHSC7O & HSP7O

--[G,A,S,P-rich region]-[short acidic region]-(K/H/D)DEL for BiPs

-[G,A,Q-rich region]-[charged, primarily acidic region] for bacterial dnaK homologs

t = major site of proteolysis (t)= secondary site of proteolysis in mammalian BiP and HSC7O

-= A

site of deletion in Bacillus dnaKs

FIG. I . Schematic drawing of the functional organization of stress-70 proteins within their primary structure. Numbers correlate with the amino acid sequence of bovine HSC70.

1989; Rose et al., 1989) as well as in the plant Nicotiana tabacum (Denecke et al., 1991), and DDEL in Kluyveromyces lactis (Lewis and Pelham, 1990).

The dnaK proteins have a 10- to 20-residue G,A,Q-rich segment, which leads into a highly charged, predominantly acidic termination. In this case, no specialized function has been ascribed to the carboxy-terminal sequence motif. An early indication of functional organization within the primary structure of stress-70 proteins was suggested by the work of Rothman and colleagues with bovine HSC70 and its clathrin-uncoating activity. Proteolysis of HSC7O with chymotrypsin yielded an amino-terminal fragment of -44 kDa that bound ATP-agarose and retained ATPase activity, but did not bind clathrin (Chappel et al., 1987). This demonstrated that

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the ATP-binding activity resided in the amino-terminal portion of the molecule, and further suggested that the peptide-binding function, which was lost on proteolysis, might reside in the carboxy-terminal portion of the molecule. Evidence consistent with this scheme was presented by Milarski and Morimoto (1989), who demonstrated a segregation of the ATP-binding and nucleolar localization functions of human HSP70 to the amino- and carboxy-terminal regions of the protein, respectively. Additional evidence has accumulated to demonstrate unequivocally that the ATPase function of stress-70 proteins is encompassed within the first -380-390 residues of the primary structure (-350-360 residues for the foreshortened members, such as those of Bacillus subtilis and Bacillus rnegateriurn)(Flaherty et al., 1990). It is presumed that the peptide-binding activity of the molecule lies within the remainder of the primary structure, although the precise boundaries of this activity have not been delineated. B.

Tertiary Structure

The three-dimensional structure of an intact stress-70 protein has not yet been solved. A structure is currently available only for the ATPase fragment of the bovine HSC70 protein; its X-ray crystallographic structure has been determined to 2.2 8, resolution (Flaherty et al., 1990). The ATPase fragment has two lobes with a cleft between them; each lobe can be further subdivided into two structural domains. In the view shown in Fig. 2, the two structural lobes are on the right and left; each lobe has an upper and lower domain. (For simplicity, throughout the remainder of this discussion the domains will be designated by their relative spatial position in this view of the molecule; e.g., “lower right,” “lower left,” etc.) The two lower domains each have a five-strand /3 sheet; the two p sheets wall the base of the cleft. The nucleotide is bound at the base of the cleft, with the phosphates bound between the central p strands. The overall topology of the ATPase fragment is shown in Fig. 3, with the topological drawings of the domains in the same relative orientation as the structural domains in Fig. 2. Notably, the two lower domains have identical folding topology: a five-strand p sheet and three a helices, with identical connectivity in the two domains. Surprisingly, the ATPase fragment, by itself, shows a substantial structural similarity to two other proteins, actin (Kabsch et al., 1990) and hexokinase (Fletterick 1975). T h e similarity is most easily visualized in the topology diagrams of the proteins (Fig. 3). Actin and the HSC70 ATPase fragment have nearly identical topology, the major difference being that essentially half of the upper right domain of the ATPase fragment is replaced by a methylhistidine-containing loop in actin. Inter-

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A

FIG.2. Stereo views of the structure of the bovine HSC70 AI'Pase fragment with bound MgADP and Pi. (A) C , carbon atom backbone. (B) Schematic drawing; cylinders represent a and 3,,) helices; arrows represent p strands.

HSC70 ATPase Fragment

Actin N

Hexokinase FIG. 3. Topology of HSC70 ATPase fragment, actin, and hexokinase. Cylinders represent a and 3,,,helices; arrows represent p strands. Domains that are identical in folding in all three proteins are shaded.

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estingly, sequence alignments within the HSP70-related protein family suggest that this same region of the upper right domain would be replaced with a foreshortened loop in the B. subtilis and B. megaterium dnaK proteins (Hearne and Ellar, 1989; Sussman and Setlow, 1987). The structural similarity of actin and the HSC70 ATPase fragment with hexokinase is less extensive, being limited to the lower domains, i.e., to the nucleotide-binding domains of the molecules. The structural similarity of these proteins was not anticipated from amino acid sequence data; indeed, when the structures of actin and the HSC70 ATPase fragment are superimposed, the resulting alignment yields only a 10% identity between the two amino acid sequences (Flaherty el al., 1991). However, now that it is apparent that these proteins belong to the same structural superfamily, sequence “fingerprints” characteristic of this family have been recognized. The strongest consensus fingerprint is derived from the first pair of /3 strands of the lower right domain, which encompass the highly conserved (V/I)CIDLGTT(Y /N)SC sequence of the stress-70 proteins. This motif of the stress-70 proteins binds the /3- and h-phosphates of the nucleotide. Alignment with actins and hexokinases suggests a consensus, (I/L/V)X(I/L/V/C)DXC(G/S/T)(G/S/T)XX(R/K/ C), where X represents an arbitrary amino acid residue. In this consensus, the aspartic acid at position 4 is essential for stabilizing the divalent metal ion of a bound metal-nucleotide complex, the glycine in position 6 is the only residue allowed because of steric interactions with the nucleotide phosphates, and a basic residue at position 11 can form a salt bridge to the phosphates of the nucleotide. This consensus sequence suggests that other kinases, such as glycerol kinase and glucokinase, belong to this superfamily of proteins. Although the structural similarity between these proteins might at first appear fortuitous, it is likely a consequence of functional imperatives. The HSP7O-related proteins, actins, and hexokinases are all phosphotransferases that bind ATP/ADP; comparison of the structure of the HSC70 ATPase domain and actin shows that the nucleotide-binding sites are very similar; many of the amino acids that are identical between the two proteins make specific contacts with the nucleotide. Bound ADP, observed crystallographically, is essentially superimposable between actin and the HSC7O ATPase fragment. This strongly suggests a parallel in the mechanism of ATP hydrolysis and product release between the two proteins. However, this suggestion has not been explored at this time. Two groups have independently suggested that the peptide-binding domain of the stress-70 proteins may be similar in structure to the peptide-binding domain of the histocompatibility antigen proteins (HLAs) (Flajnik et al., 1991; Rippmann et al., 1991). This suggestion is

-

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based on a modest sequence identity of 24% between the derived amino acid sequence of the peptide-binding region of Xenopus class I HLA and human HSC70, as well as on a similarity in the patterns of hydropathy and predicted secondary structure of the peptide-binding domain of HLA molecule and a region of the stress-70 proteins that could potentially encompass the peptide-binding activity of the molecules. By comparison with HLAs, the predicted structure of the peptide-binding domain of stress-70 proteins would encompass 180 amino acids, spanning residues -380-560 of a typical sequence (where the sequence numbering is referenced to bovine HSC70). Interestingly, in this model the peptidebinding domain would begin immediately following the apparent termination of the ATPase domain, would span a region of relatively high sequence conversation in the carboxy-terminal portion of the molecule, and would terminate prior to a region of relatively high sequence variability that extends to the carboxy terminus (Fig. 1). Consistent with the possibility of a domain boundary in the neighborhood of residue -560 of the stress-70 proteins is the observation that both bovine HSC70 and canine BiP are protease sensitive in this region, suggestive of a possible structural domain boundary (Chappell et al., 1987; Kassenbrock and Kelly, 1989). This prediction for the structure of the peptide-binding domain is provocative in light of a possible functional parallel between HLAs and the stress-70 proteins in the binding of extended (po1y)peptides. It will be of interest to see whether structural data confirm or controvert this prediction.

-

C . Temperature-DependentStructural Transitions Some intriguing observations have been made by Fink and colleagues on the tempertaure-dependent structural transitions in the stress-70 proteins (Palleros et al., 1991, 1992). Specifically, both tryptophan fluorescence and circular dichroism have been monitored as a function of temperature for both E. coli dnaK and bovine HSC70 proteins. For dnaK, two transitions are observed: in the absence of nucleotide, a cooperative transition is observed with a midpoint of 41-4Z°C, and a second transition is observed at -71°C. In the presence of MgATP o r MgADP, the first transition is shifted to 59-60°C. T h e first transition appears to induce a monomeric “molten globule” state in the protein-a state with a loss of compact tertiary structure, but with a retention of substantial welldefined secondary structure. Gel filtration indicates the apparent molten globule state of dnaK to have 13% increase in Stokes radius from the native protein (42 versus 38 A). T h e transition is reversible; heating dnaK to temperatures above the first transition, followed by incubation below

-

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DAVID B. MCKAY

the transition temperature, results in apparent conversion of the molten globule back to the native state on a time scale of minutes. Below the molten globule transition temperature, dnaK binds reduced, carboxymethylated a-lactalbumin (RCMLA, a representative denatured protein); however, above the transition temperature, the RCMLA-binding activity is lost. A different behavior is observed with the bovine HSC7O protein. Although parallel experiments reveal two similar structural transitions for HSC70 at temperatures close to those observed for dnaK, the HSC70 protein forms large, heterogeneous aggregates at temperatures above the first thermal transition, i.e., under conditions where it would be suggested, by analogy to dnaK, to form a molten globule. T h e significance of these observations of temperature-dependent properties of representative stress-70 proteins, in the context of the response of cells to thermal stress, is not yet apparent.

Iv. ENZYMATIC MECHANISMOF STRESS-70 PROTEINS A substantial number of observations have demonstrated that the stress-70 proteins bind denatured proteins, as well as some short peptides, and that ATP hydrolysis (possibly with concomitant release of Pi or ADP) results in the release of bound peptides (de Silva et al., 1990; Hendershot, 1990; Hurtley et al., 1989; Kassenbrock et al., 1988; Palleros et al., 1991).Conversely, binding of peptides to stress-70 proteins induces ATPase activity above basal levels; peptide-induced ATPase activity is often used as a facile in vitro assay for stress-70 protein activity. Schematically, this activity can be subdivided into (1) ATP binding and hydrolysis, followed by product release (Pi and ADP), (2) peptide binding and release, and (3) a mechanism of coupling peptide binding/release and nucleotide hydrolysis/product release. A . ATPase Mechanism The turnover rate of ATP by stress-70 proteins is relatively slow, and is enhanced severalfold by the binding of peptides or by the action of accessory proteins. In the absence of peptides, the E. coli dnaK protein has been reported to have basal ATPase rates in the range of 0.15 (mol ATP/mol dnaK min; measured at 37"C, pH 8.1) (McCarty and Walker, 1991) to 0.21 (mol ATP/mol dnaK min; measured at 30"C, pH 8.8) (Liberek et al., 1991a). T h e basal ATPase rate of dnaK rises steeply as a function of temperature, increasing 70-fold from 20 to 53°C. Above 53"C, the ATPase activity drops precipitously, possibly due to the onset

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of a structural transition to the molten globule state observed at -59°C by Fink and colleagues. The ATPase rate of dnaK can also be enhanced -50-fold above its basal level by the concerted effect of the grpE and dnaJ proteins. The peptide stimulation of the ATPase activity of bovine BiP protein has been studied (Flynn et al., 1989). The basal ATPase rate of BiP is reported to be 0.026 (mol ATP/mol BiP min; 37"C, pH 7.0). Stimulation with a variety of peptides 9- 15 residues in length resulted in a three- to sixfold increase in ATPase rate, to 0.08-0.16 (mol ATP/mol BiP min). Similar experiments with a subset of the same peptides on bovine HSC70 gave values of 0.23-0.24 (mol ATP/mol HSC70 min) for the peptidestimulated ATPase activity. The stimulation of HSC70 ATPase activity by clathrin light chain peptides has also been examined (DeLuca-Flaherty et al., 1990). The peptide found to be most effective in stimulating nucleotide hydrolysis induced a maximal ATPase rate of -1.0 (mol ATP/mol HSC70 min; 37"C, pH 7.0). It is instructive to compare this to the activity of the 44-kDa ATPase fragment of HSC70 under identical assay conditions: a peptide-independent rate of 0.8 (mol ATP/mol HSC70 min) is observed-approximately equal to the maximum rate observed for the intact HSC70 protein. Apparently, in the absence of peptide, the ATPase activity of the intact HSC70 protein is substantially attenuated; binding of peptide enhances the rate to a maximal level that is approximately equal to that of the ATPase domain alone. Hence, although the specific numerical values reported for ATPase rates of stress-70 proteins show some variation, possibly attributable to differences in specific protein preparation and assay procedures utilized by different individuals, the consensus scheme that these data show is that in the absence of substrate peptide, the stress-70 proteins have a low basal ATPase rate, typically found to be -0.01-0.03 (mol ATP/mol stress-70 protein min). This can be enhanced severalfold either by binding of peptides or denatured proteins, or (as demonstrated by the effect of grpE and dnaJ proteins on dnaK) by the action of ancillary proteins. T o the extent that the observations on HSC70 can be generalized to other members of the stress-70 protein family, peptide binding appears to relieve the attenuation of ATPase activity and allow it to proceed at the rate characteristic of the ATPase fragment of the protein alone. The structure of the wild-type ATPase fragment of HSC70 with MgADP + Pi bound, as well as inferences drawn from comparisons with the structures of actin with both CaATP and CaADP bound, reveal the environment of the nucleotide and suggest several residues that might participate in the ATP hydrolysis reaction (Fig. 4; numbering of amino

a2

DAVID B. MCKAY

FIG. 4. Environment of the bound MgADP and Pi in bovine HSC70 ATPase fragment. Glycines ( 0 )are highlighted at their C, carbon atoiii positions; H 2 0 molecules (+) are also shown. For clarity, only C, carbon atoms of the peptide backbone plus selected side chains of the protein are shown.

acid residues in Fig. 4 and in the discussion below is referenced to bovine HSC70) (Flaherty et al., 1990). It is apparent that the MgADP complex is stabilized by several hydrogen bonds contributed by both side chains and polypeptide backbone atoms of the protein, most notably from Thr12 and Thr-13, as well as the backbone of the antiparallel /3 strands in this region. The divalent Mg” ion is also stabilized by the carboxyls of Asp-10 and Asp-199, a pair of residues that is strictly conserved in all stress-70 proteins and whose structural equivalents are also conserved aspartic acid residues in actin. Additionally, the amino group of Lys-7 1 forms a salt bridge to the bound Pi. Two alternative mechanisms of ATP hydrolysis that appear both sterically and chemically reasonable can be suggested. The first possibility would be a direct in-line attack by a H,O molecule on the y-phosphate of ATP, with Asp-206 acting as a proton acceptor. The plausibility of this alternative was initially suggested by the comparison of the actin and HSC70 ATPase fragment structures, and more specifically by superposition of a CaATP nucleotide, with the conformation observed in actin, on the ATPase fragment model (Flaherty et al., 1991). An H,O molecule appears to intercalate between the carboxyl of Asp-206 and the hypothet-

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83

ical position of the y--phosphate of ATP; with minor adjustments to the model, distances and geometries suitable for an in-line attack by the H,O molecule, with Asp-206 acting as a proton acceptor, are achieved. An alternative possibility would be a nucleophilic attack on the yphosphate of ATP by the hydroxyl of Thr-204, resulting in the formation of a phosphoprotein ester intermediate; hydrolysis of the ester would then complete the reaction. In such a scheme, a proton acceptor would extract a proton from the hydroxyl of Thr-204. Model building suggests that with modest rotation of the Thr-204 side chain in the presence of ATP, Asp-206 would be well positioned to act as a proton acceptor. In the second step of the reaction, the hydrolysis of the phosphate ester, Glu-175, could act to extract a proton from an H,O molecule, allowing an in-line attack of an H,O positioned between the phosphate on Thr204 and the carboxyl of Glu-175. Of interest in the context of this alternative for the ATP hydrolysis mechanism is the observation that in the presence of CaATP (although not with MgATP), some members of the stress-70 protein family autophosphorylate in vitro. McCarty and Walker (1991) have identified the site of in vtiro autophosphorylation on dnaK to be Thr-199 (equivalent to Thr-204 in HSC70). They have further shown that mutagenesis of Thr- 199 substantially reduces the basal ATPase activity; most dramatically, mutation to valine reduces the ATP turnover rate by at least two orders of magnitude, to <0.001 (mol ATP/ mol dnaK min), attesting to a crucial role for the threonine in the reaction. However, at this time, there are no data that distinguish between the two alternative mechanisms: direct attack by H,O on the y-phosphate of ATP versus a phosphoprotein intermediate. B . Peptide Recognition

The stress-70 proteins interact with a broad spectrum of polypeptide substrates, but they have some degree of specificity in their interactions. In several instances, it has been shown that a stress-70 protein can bind to proteins [e.g., bovine pancreatic trypsin inhibitor (BPTI), alactalbumin] that have been stabilized in a nonnative, or denatured, form by reduction and carboxymethylation of the cysteines that would normally form disulfides; at the same time, they will not bind to the native forms of the same proteins (Liberek et al., 1991b;; Palleros et al., 1991, 1992). This suggests that the peptide-binding activity of the stress70 proteins discriminates in favor of polypeptides in a denatured, and possibly extended, conformation over those in a compact secondary and tertiary structure. NMR experiments demonstrating that the E . coli dnaK

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protein interacts with a peptide that is in an extended conformation strongly support this view (Landry et al., 1992). Beyond binding to polypeptides that are in an extended conformation preferentially over those that are folded into a compact structure, the stress-70 proteins manifest further discrimination based on amino acid sequence of the substrate peptides. Rothman and colleagues have characterized the peptide-binding site of bovine BiP with respect to both the size of the binding site and the preference for types of amino acids (Flynn et al., 1991). Specifically, they synthesized random N-mers-peptides of length N with random amino acids incorporated at each position-and characterized the affinity of BiP for the peptides as a function of length N , and subsequently, for an apparent optimal length, the preferences for each amino acid at each position. Their results indicate that the minimum peptide length required for optimal peptide binding is seven amino acids. Further, for a 7-mer of random sequence, they demonstrate that the binding preference is for amino acids with nonpolar aliphatic side chains (Leu, Ile, Met, and Val), with a notable exception occurring in position 7, which appears to have substantial affinity for positively charged side chains (Lys and Arg). The magnitudes of the discrimination, measured as a ratio of the amount of a given amino acid residue in the N-th position in the bound peptide, divided by the amount of the same residue in the Nth position in the input peptide, varied over a -30-fold dynamic range. For example, in positions 3-7, the Leu content of the bound peptide was typically enriched 3- to 4-fold over that of input peptide, while the Asn content in these positions was typically only -0.1-0.2 that of the input peptide. These “enrichment factors” were translated into a free energy of partitioning, described quantitatively as R T In (A,/A,), where A, and A , are the relative abundance of a given amino acid at the N-th position in the bound and input peptide, respectively. When the values of this parameter for amino acids at positions 3-6 of the 7-mer peptide are compared with the hydrophobicity of the residue side chain, an approximately linear relationship is found. This binding energy “fingerprint” demonstrates that the internal core of the peptide-binding site of BiP discriminates in favor of hydrophobic residues. Among hydrophobic residues, it was found that BiP shows a preference for unbranched aliphatic side chains over those with aromatic or @branched aliphatic side chains. Comparison of their results with studies on the hydrophobic stabilization of residues on the interior of proteins (Matsumura et al., 1988) led Rothman and colleagues to conlude that the “energy fingerprint of a pocket that binds an aliphatic residue to the interior of a folded protein is qualitatively indistinguishable from the fingerprint of the core of the peptide-binding site of BiP.”

HEAT-SHOCK-RELATED PROTEINS

C . Coupling of Peptide-Binding and ATPase Activities Evidence on the coupling of nucleotide hydrolysis and peptide bindinglrelease in the stress-70 proteins is consistent with, in the simplest approximation, a two-state model for the proteins. Evidence from several sources indicates that stress-70 proteins have at least two distinct conformations: one has a relatively high affinity for peptides and the other has a substantially lower affinity. T h e transition from the high-peptideaffinity state to the low-peptide-affinity state is induced by binding and hydrolysis of MgATP. Two different sets of experiments provide evidence for a conformational change in dnaK. Fink and colleagues (Palleros et al., 1991) have monitored the intrinsic tryptophan fluorescence of dnaK, and have found both a change in intensity and a shift in the wavelength of peak intensity between dnaK with MgADP bound (A,,, = 333 nm; greater peak fluorescence intensity) and dnaK with MgATP bound (A,,, = 327 nm; lesser peak intensity). T h e dnaK complexed with the nonhydrolyzable MgATPyS showed a spectrum indistinguishable from that of dnaK with MgADP, suggesting that it is hydrolysis of the nucleoside triphosphate, rather than nucleotide binding, that is required to shift the protein into an “ATP-bound” conformation. Schematically, similar conclusions were reached by Georgopoulos and colleagues (Liberek et al., 1991b), using protease digestion patterns as a probe of conformation for dnaK. Distinctly different digestion patterns were observed for dnaK in the presence of MgADP or MgATP. Consistent with the fluorescence experiments of Fink and colleagues, MgATP hydrolysis appeared necessary to shift dnaK into the ATP-bound conformation; proteolytic digestion in the presence of MgAMPPNP, a nonhydrolyzable analog of MgATP, resulted in a digestion pattern similar to that observed in the presence of MgADP. They further demonstrated that the ADP-bound conformation of dnaK had a relatively high affinity for denatured proteins (specifically, for RCM-BPTI), and that addition of MgATP to dnaK in the ADP-bound conformation complexed with RCM-BPTI resulted in both a release of RCM-BPTI and a shift to the ATP-bound conformation. Interestingly, the mutant dnaK756 protein appears to be locked in the ATP-bound conformation; it also has a substantially lower affinity for RCM-BPTI than the wild-type dnaK protein. Taken together, these results indicate that ATP hydrolysis can trigger a conformational change from a high-peptide-affinity conformation to a low-peptide-affinity conformation in the protein, in either the presence or absence of bound peptide. This relatively simple two-state model for dnaK, which can be offered as a pardigm that may be generalized for other stress-70 proteins, is consistent with numerous observations

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recorded in the literature demonstrating that ATP hydrolysis releases proteins that are bound to stress-70 proteins. T h e two-state model can also be utilized to suggest a relatively simple scheme for the interaction of stress-70 proteins with polypeptides during translation and transmembrane translocation, as well as for their “renaturation” activities. If a protein were partially or fully unfolded, a stress70 protein could bind to extended stretches of polypeptide, binding preferentially to stretches that were more hydrophobic in sequence. Binding by stress-70 proteins would interdict intermolecular aggregation of polypeptides that were not condensed into a native tertiary structure. Shifting the stress-70 protein to a low-peptide-affinity state using ATP hydrolysis would result in release of the peptide, allowing it the opportunity to fold into its native tertiary conformation in the protein. If it failed to do so, the cycle could be repeated, allowing, in essence, multiple attempts for the peptide segment to fold into a correct tertiary structure that a stress-70 protein would be unable to bind, with input of multiple cycles of ATP hydrolysis if necessary.

D. Clathrin Uncoating Reaction In discussing the mechanistic enzymology of the stress-70 proteins, one is brought back to the intriguing question of how HSC7O catalyzes the disassembly of clathrin cages. The clathrin uncoating reaction was explored extensively by Rothman and colleagues; much of the characterization was completed prior to the realization that the uncoating protein was, in fact, a member of the stress-70 protein family (Schlossman et al., 1984; Schmid and Rothman, 1985a,b; Schmid et al., 1984, 1985). It was also done before it became apparent that denatured proteins and relatively unstructured peptides are popular substrates for the stress-70 proteins. Greene and Eisenberg (1990) have extended the work on the clathrin uncoating activity of HSC70. However, it still remains an intriguing challenge to attempt to fit the clathrin uncoating activity of HSC70 into a general mechanistic scheme for stress-70 proteins. The “building block” of clathrin cages is a “triskelion,” a trimer of clathrin protomers with extended “legs”; it consists of three clathrin heavy chains (HCs; -180 kDa per protomer) and three light chains (LCs; -35 kDa) (Crowther and Pearse, 1981). Mammalian cells have two different light chains (denoted LC,, and LC,); typically, the two light chains are present in a molar ratio of - 1 : 2, LC,: LC,, although the ratio is dependent on cell type and has been shown to range from 5: 1 to 0.33:l (Acton and Brodsky, 1990). The spontaneous in vitro assembly and disassembly of clathrin cages from purified triskelions is strongly

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dependent on pH. Triskelions will assemble into stable clathrin cages at pH 6.0-6.5; conversely, cages will disassemble into triskelions at pH >7.5. T h e uncoating activity of HSC70 is optimal at pH 7.0 and minimal at pH values lower than -6.5; thus, HSC70 catalyzes the disassembly of calthrin cages under conditions where they are relatively unstable. Based on extensive data (Schmid and Rothman, 1985a, Schmid et al., 1984, 1985), Schmid and Rothman have proposed a mechanism for the uncoating reaction, which can be summarized as follows: (1) HSC70 first binds a site on clathrin that requires the presence of light chains; (2) ATP hydrolysis by HSC7O then results in exposure of a site on the triskelion that was not previously accessible; they refer to this step of the activity as a “displacement”; (3) then, in a step they call “capture,” HSC70 binds the newly exposed site on a triskelion, presumably blocking reassembly of that leg of the triskelion into the clathrin lattice. Approximately three molecules of ATP are hydrolyzed per triskelion released in the uncoating reaction. T h e amount of HSC70 bound per product triskelion-approximately 0.32 : 1.0, weight ratio of HSC70:triskelion, consistent with -3 X 70 kDa of HSC7O per triskelion of -645 kDa-is consistent with one HSC70 protomer binding for each leg of the triskelion. This suggests that removal of a triskelion is accomplished by three molecules of HSC70, each hydrolyzing one molecule of ATP during the reaction. In addition to the hydrolytic ATP-binding site on HSC70, Schmid and Rothman have suggested a second, “catalytic” ATP-binding site, where binding of ATP o r nonhydrolyzable analogs would enhance the rate of triskelion capture, without hydrolysis of the nucleotide. Subsequent work by other investigators on the kinetics of clathrin uncoating has demonstrated that the reaction displays an initial “burst” phase, in which HSC70 rapidly uncoats a stoichiometric amount of clathrin (approximately one triskelion per three HSC70 molecules)-suggesting that HSC7O can be initally “primed” for uncoating-followed by a slower steady-state rate of uncoating (Greene and Eisenberg, 1990). T h e initial burst can be inhibited substantially by the presence of ADP Pi. The steady-state rate of uncoating is observed to be in the range of -0.005-0.006 (mol triskelion released/mol HSC70 min), approximately two orders of magnitude slower that the maximum rate of ATP turnover attainable by HSC70. Thus, the displacement and/ or capture steps appear to be rate limiting for the clathrin uncoating reaction; the ATP hydrolysis by HSC70 is rapid by comparison. In the context of the initial interaction of HSC70 with clathrin requiring the presence of light chains, a site of interaction of HSC70 with light chains, and hence by inference a candidate for the initial binding site on clathrin, has been characterized (DeLuca-Flaherty et al., 1990). The

+

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candidate site is a glycine and proline-rich segment of LC,, within the stretch of amino acids 47-7 1 of the bovine light chain sequence. Peptides derived from this region of LC, are effective in stimulating the ATPase activity of HSC70; peptides from other regions of LC, are not. Peptides derived from the structurally equivalent region of LC, are also ineffective in stimulating HSC70 ATPase activity. This particular region of LC, is conformationally labile in the intact triskelion; under conditions in which clathrin cages are stable (e.g., pH 6.5), it is inaccessible to a polyclonal antipeptide antibody raised against its sequence, but under conditions used for the uncoating reaction (pH 7.0) or in the presence of -0.11.0 mM Ca2+, it is accessible to antibody. Additionally, LC, inhibits clathrin uncoating, whereas LC,, does not. The accessibility of this segment of LC, under the conditions by which HSC70 uncoats clathrin, combined with the ability of a peptide derived from this region to mimic a substrate by stimulating the ATPase activity of HSC70, suggests that the initial site of interaction of HSC7O with clathrin may be this conformationally labile, glycine- and proline-rich segment of LC,. The data raise the intriguing possibility that in vivo, the clathrin uncoating activity may be controlled by a conformational “switch”on LC, that can cycle the initial site of HSC70 binding on clathrin between accessible and inaccessible conformations. Heuser and colleagues have utilized electron microscopy to characterize a species in the uncoating reaction, as well as in the interaction of HSC70 with isolated triskelions, that they interpret as a triskelion with three HSC7O molecules bound at the vertex (Heuser and Steer, 1989). They propose that a trimer of HSC70 bound at a triskelion vertex may be an intermediate in the uncoating reaction. This contrasts with the proposal of Rothman and Schmid that HSC70 binds the terminal domains of triskelions during the capture reaction, but would be consistent with models in which HSC7O binding to triskelion vertices would block the assembly of terminal domains of neighboring triskelions into a stable clathrin lattice. In summary, although the question of whether HSC70 uncoats clathrin cages in vivo is still a point of debate, the in vitro uncoating has provided an intriguing activity for study. There appears to be general agreement that the uncoating reaction requires three molecules of HSC70 per clathrin triskelion, and that three molecules of ATP are hydrolyzed for each triskelion released. Some points that await further clarification include (1) whether HSC70 binds to the terminal domains of clathrin triskelions or at the vertex during the capture step of the reaction, and (2) whether there is a second, catalytic ATP-binding site on HSC70 that binds but does not hydrolyze ATP during the capture step.

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MODULATORSOF STRESS-70 PROTEIN ACTIVITY

A . Accessory Proteins It is well established that in some activities, the E. coli dnaK protein works in concert with accessory heat-shock proteins. The dnaK protein was first recognized as a gene product that was essential for bacteriophage A DNA replication in E. coli, as evidenced by a mutation in the dnaK gene (dnaK756) that gives rise to a strain unable to support A replication (Georgopoulos, 1977). Two other proteins, dnaJ and grpE, which act in concert with dnaK in A replication, were originally identified as products of genes in which mutations also resulted in failure to support A replication in vivo (Saito and Uchida, 1977; Yochem et al., 1978). T h e purified dnaJ protein dimer, with each half being 42 kDa (Bardwell et al., 1986; Ohki et al., 1986), is an essential component in assays for both in vitro A replication initiation (Liberek et al., 1988; Mensa-Wilmot et al., 1989) and in vitro activation of the phage P1 repA protein (Wickner, 1990; Wickner et al., 1991a,b). The purified grpE protein is a monomer of 24 kDa. It is not absolutely essential for in vitro A replication initiation; however, addition of grpE to the assay system substantially reduces the amount of dnaK required for initiation (Lipinska et al., 1988; Zylicz et al., 1988). A more extensive discussion of the participation of dnaK, dnaJ, and grpE in the initation of A replication can be found elsewhere (Georgopoulos et al., 1990). The specific effects of purified dnaJ and grpE proteins on the dnaK protein have been characterized (Liberek et al., 1991a). T h e dnaJ and grpE proteins, acting in concert, can stimulate the basal ATPase activity of dnaK -50-fold; dnaJ appears to enhance the rate of nucleotide hydrolysis by dnaK, but does not substantially affect nucleotide release. The grpE protein appears to enhance the rate of release of nucleotide bound to dnaK. Consequently, neither protein alone exerts a dramatic effect on the overall rate of ATP turnover by dnaK; however, the effect of the two proteins combined is to accelerate both the rate of ATP hydrolysis and the rate of nucleotide release, resulting in enhanced ATP turnover. The concentrations of dnaJ and grpE required for half-maximal effect are both -0.1-0.2 /AM. The specific roles of dnaJ and grpE in modulating the interaction of dnaK with other protein substrates are not clear. Possible functions include roles as specificity factors and capture factors. Since dnaJ is known to bind specific target proteins, such as the bacteriophage P1 repA protein and the A P protein, in addition to binding dnaK, it could conceivably function as a specificity factor, targeting dnaK to particular oligomeric

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assemblies as substrates for disassembly. Additionally, it has been hypothesized that grpE may capture A P protein after it is released from a replication initiation complex, to prevent its immediate reassociation with the complex (Georgopoulos et al., 1990). It is conceivable that the participation of accessory proteins such as dnaJ and grpE in some functions of dnaK will have parallels in the functions of other stress-70 proteins. Several dnaJ homologues have been documented in yeast, although as a group they d o not show the stringent level of sequence conservation found in the stress-70 proteins. In particular, the primary structure of dnaJ appears to be modular, and can be described as (1) an N-terminal region of -80- 100 amino acid residues, (2) a glycine-rich region of variable length, typically -30-50 residues, (3) a cysteine-rich region, with four -CXXC- motifs, suggestive of metalbinding sites in which pairs of cysteines would coordinate metal ions, and (4) a C-terminal region of 150-200 residues. YDJ l p is a yeast homologue of dnaJ for which mutations in the gene give rise to a slowgrowth phenotype and defects in mitochondria1 import (Atencio and Yaffe, 1992; Caplan and Douglas, 1991). It appears to be localized in the cytoplasm and nuclear envelope. It has a sequence similar to E. coli dnaJ over its entire primary structure, with an overall sequence identity of 32% between the two proteins. Another yeast homologue, SCJ l p , also shows sequence similarity over its entire primary structure; it is 37% identical to E. coli dnaJ (Blumberg and Silver, 1991). Overproduction of SCJ l p in yeast on a high-copy-number plasmid results in altered protein sorting. SCJ 1 has a signal sequence, suggesting that it may be sequestered in mitochondria or the endoplasmic reticulum. SISlp is the product of a gene that can suppress a slow-growth phenotype of a yeast strain with mutations in the SIT4 gene, which encodes a serinethreonine phosphatase (Luke et al., 1991). It shows similarity to dnaJ in the N- and Cterminal regions, but has a glycine- and proline-rich region, rather than the -CXXC- motifs, in the central region. Sec63p, which is important for protein assembly in the endoplasmic reticulum, has sequence similarity to dnaJ only over a limited segment of -80 residues in the N-terminal region (38% identity), aligning with residues 120-200 of Sec63p (Sadler et al., 1989). Hence, several proteins that have been identified in yeast show similarity to at least some regions of dnaJ. Their functional roles remain to be elucidated. Additionally, a sequence for a human homologue that shows significant sequence similarity to dnaJ in the Nand C-terminal regions has been reported. To date, genes coding for grpE homologues have been reported in B. subtilis and Chlamydia trachomatis (Engel et al., 1990; Wetzstein and Schumann 1990). The derived amino acid sequences have -40-50%

-

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sequence identity with the E . coli grpE protein, again showing substantially more sequence variation than is found in the stress-70 protein family. Eukaryotic homologues have not yet been reported.

B . Posttranslational Modification One of the earliest observations of modification of stress-70 proteins in vivo was the report in 1982 that a significant fraction of two stress-70 proteins of Dictyostelium were phosphorylated, primarily (-70-90%) on threonine residues, and to a lesser extent (-10-30%) on serine residues (Loomis et al., 1982). In 1983, it was observed that in vivo labeling of chick embryo fibroblasts and rat embryo cell lines with [“Hladenosine, as well as in nitro labeling of fibroblast whole cell lysates with [“PINAD’, resulted in labeling of an 83-kDa protein, thought to be the BiP protein, suggesting a possible ADP-ribosylation of BiP (Carlsson and Lazarides, 1983).T h e labeling decreased under condition of heat shock and glucose starvation, i.e., under conditions that are known to increase the synthesis of BiP in cells. Since these early observation on the in vivo modification of stress-70 proteins, there has been further work documenting phosphorylation of several members of the stress-70 family, both in vitro and in v i m , and further exploration of the labeling of BiP by [SH]adenosine in vivo. However, it is not yet clear what the significance of posttranslational modification of stress-70 proteins may be for regulating their activities. In vitro, with CaATP as a substrate, E . coli dnaK autophosphorylates exclusively at Thr-199 (McCarty and Walker, 1991). It does not autophosphorylate when MgATP is used as a substrate. In vivo, dnaK is found to be phosphorylated on serine as well as on threonine residues (Rieul et al., 1987). Under normal growth conditions, phosphorylation is primarily on serine; when E. coli is infected with bacteriophage M 13, the phosphorylation shifts predominantly to threonine, with minor phosphorylation of serine. The specific target residues of in v i m phosphorylation of dnaK have not yet been determined. T h e disparity between the in vitro results (autophosphorylation exclusively at Thr- 199; an absolute requirement for CaATP) and the in vivo results (phosphorylation on both serine and threonine residues, under conditions in which MgATP would be presumed to be the available intracellular substrate nucleotide) raises the questions of (1) whether the specific autophosphorylation of Thr- 199 observed in vitro also occurs in v i m , or whether it may be an artifactual side reaction when the larger Ca” ion is substituted for Mg” at the active site of the protein, and (2) whether there is a serinelthreonine protein kinase that specifically phosphorylates dnaK in vivo.

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Observations on the phosphorylation of mammalian BiP show a parallel pattern. In vitro, purified BiP protein autophosphorylates on threonine residue(s) in the presence of CaATP; the reaction is inhibited by other divalent cations, such as Mg’+, Mn2+,and Zn2+(Leustek et al., 1991). It has not yet been determined which specific threonine residue(s) is modified. In vivo, BiP is observed to be phosphorylated under conditions of normal cell growth (37”C), but not under conditions that increase production of BiP, namely, glucose starvation or heat shock (40.5”C) (Hendershot et al., 1988). Both serine and threonine residues are phosphorylated in vivo. It has also been shown that mitochondiral stress-70 proteins autophosphorylate in vitro in the presence of CaATP (but not MgATP), which is consistent with the observations on dnaK and BiP (Mizzen et al., 1991). The labeling of BiP in mouse myeloma B cells by [’Hladenosine, suggestive of ADP-ribosylation, has been shown to be a modification found on BiP free of immunoglobulin heavy chains, but not on BiP bound to heavy chains (Hendershot et al., 1988). Additionally, the [’Hladenosine labeling parallels BiP phosphorylation in vivo; conditions that increase BiP production decrease BiP phosphorylation and [‘Hladenosine incorporation, suggesting that these two posttranslational modifications might modulate BiP activity in concert in some manner. In summary, there is evidence that suggests that the activities of some stress-70 proteins may be modulated in vivo by protein phosphorylation or ADP-ribosylation. However, at this time, there is no case in which a modifying enzyme has been purified and demonstrated to modify a target stress-70 protein in vitro. Hence, the question of whether there is functionally significant posttranslational modification of stress-70 proteins remains an open question at this time. VI. EPILOGUE T h e pervasiveness of the stress-70 proteins and their near-indispensible roles in maintaining the viability of cells, combined with the nebulous state of our understanding of their biochemical functions, has made them an intriguing system for current study. Members of the stress-70 protein family were first appreciated for their participation in disassembly of specific macromolecular complexes, and only more recently for their potentially more widespread role as “molecular chaperones” that actively inhibit misfolding and/or aggregation of proteins. A problem that has not yet been adequately confronted in defining this possible chaperone function has been the difficulty of designing explicit in vitro assays of activity that can be correlated unequivocally with in vivo activities. In surveying the status of what is currently well-established about the struc-

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ture and mechanism of stress-70 proteins, we can recognize that several key questions remain to be addressed at the biophysical and biochemical level. What is the polypeptide substrate specificity of the stress-70 proteins, and how does it relate to the diverse biological functions of the proteins? What is the biochemical mechanism that couples ATP hydrolysis to peptide bindingirelease, and how does this result in a “chaperone” activity? What are the roles of accessory proteins and possible posttranslational regulatory modifications in stress-70 protein activity? ACKNOWLEDGMENTS I would like to thank the members of my laboratory group whose discussions have contributed to the comments in this review. This work has been supported by Grant GM-39928 from the National Institutes of Health and by the resources of the Beckman Laboratories for Structural Biology of the Stanford University Medical Center.

REFERENCES Acton, S. L., and Brodsky, F. M. (1990). Predominance of clathrin light chain LCbcorrelates with the presence of a regulated secretory pathway.J. Cell B i d . 111, 1419-1426. Amir-Shapira, D., Leustek, T., Dalie, B., Weissbach, H., and Brot, N. (1990). Hsp70 proteins, similar to Esclien‘cliia coli DnaK. in chloroplasts and mitochondria of Euglena gracib. Proc. Natl. Acad. Sci. U.S.A. 87, 749-752. Atencio. D. P., and Yaffe, M. P. (1992). MAS5. a yeast homolog of DnaJ involved in mitochondria1 protein import. Mol. Cell. B i d . 12, 283-291. Bardwell, J. C., and Craig, E. A. (1984). Major heat shock gene of Vrosop/iila and the Escherichia coli heat-inducible dnaK gene are homologous. Proc. Natl. Acad. Sci. U.S.A. 81,848-852. Bardwell, J. C., Tilly, K., Craig. E., King, J., Zylicz, M., and Georgopoulos, C. (1986). The nucleotide sequence of the Eschen‘chia coli K12 dnaJ+ gene. A gene that encodes a heat shock pr0tein.J. B i d . Chem. 261, 1782-1785. Beckmann, R. P.. Mizzen, L. A., and Welch, W. J. (1990). Interaction of Hsp 70 with newly synthesized proteins: Implications for protein folding and assembly. Science 248, 850-854. Birkelund, S., Lundemose, A. G., and Christiansen, G. (1990). The 75-kilodalton cytoplasmic Chlamydia trachomalis L2 polypeptide is a DnaK-like protein. Infect. Immuu. 58, 2098-2 104. Blumberg, H., and Silver, P. A. (1991). A homologue of the bacterial heat-shock gene DnaJ that alters protein sorting in yeast. Nature (Londori) 349, 627-630. Caplan, A. J., and Douglas, M. G. (1991). Characterization of YDJ 1: A yeast homologue of the bacterial dnaJ protein. J . Cell B i d . 114, 609-62 1. Carlsson, L., and Lazarides. E. (1983). ADP-ribosylation of the M,. 83,000 stress-inducible and glucose-regulated protein in avian and mammalian cells: Modulation by heat shock and glucose starvation. Proc. Natl. Acad. Sci. U.S.A. 80, 4664-4668. Chappell, T. G., Welch, W. J., Schlossman, D. M., Palter, K. B., Schlesinger, M. J., and Rothrnan, J. E. (1986). Uncoating ATPase is a member of the 70 kilodalton family of stress proteins. Cell (Cambridge, Mass.) 45, 3-13. Chappell, T. G., Konforti, B. B., Schmid, S. L., and Rothman, 1. E. (1987). The ATPase core of a clathrin uncoating pr0tein.J. B i d . Chem. 262, 746-751.

94

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Chirico, W. J., Waters, M. G., and Blobel, G. (1988). 70K heat shock related proteins stimulate protein translocation into microsomes. Nafure (Lotidoir) 332, 805-8 10. Craig, E. A., Kramer, J., Shilling, J., Werner-Washburne, M., Holmes. S.. Kosic-Smithers, J.. and Nicolet, C. M. (1989). SSCI, an essential member of the yeast HSP70 multigene family, encodes a mitochondrial protein. Mol. Cell. B i d . 9, 3000-3008. Craig, E. A., Kang, P. J., and Boorstein, W. (1990). A review of the role of 70 kDa heat shock proteins in protein translocation across membranes. Aiifonie w t i Lreuureu/ioek 58, 137- 146. Crowther, R. A., and Pease, B. M. F. (1981). Assembly and packing ot'clathrin into coats. J. Cell. B i d . 91, 790-797. Danilition. S. L., Maclean, I. W., Peeling, R.. Winston, S., and Brunham, R. C. (1990). The 75-kilodalton protein of Clilatnydia tradoiiiatis: A member of the heat shock protein 70 family? fnfrcf.ftnniuii. 58, 189-196. DeLuca-Flaherty, C.. McKay, D. B., Parhani, P., and Hill, B. L. (1990). Uncoating protein (hsc70) binds a conformationally labile domain of clathrin light chain LC, to stimulate ATP hydrolysis. Cell (Cambridge, Mass.) 62, 875-887. Denecke, J., Goldman, M. H., Demolder, J., Seurinck, J., and Botterman, J. (1991). The tobacco luminal binding protein is encoded by a multigene family. Plant Cell 3, 1025-1035. Deshaies,T.J.. Koch, B. D., Werner-Washburne, M., Craig, E. A., and Scheknian, R. (3988). A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides. Nature (London) 332, 800-805. de Silva, A. M., Balch, W. E., and Helenius, A. (1990). Quality control in the endoplasmic reticulum: Folding and misfolding of vesicular stomatitis virus G protein in cells and in vitro. J. Cell B i d . 111, 857-866. Enge1.J. N., Pollack,.J., Perara, E., and Ganem, D. (1990). Heat shock response of murine Chlamydia trarliornafis.J. Bacteriol. 172, 6959-6972. Engman, D. M., Kirchhoff, L. V., and Done1son.J. E. (1989). Molecular cloning of mtp70, a mitochondrial member of the hsp70 family. Mol. Cell. B i d . 9, 5163-5168. Flaherty, K. M., DeLuca-Flaherty, C., and McKay, D. B. (1990). Three-dimensional structure of the ATPase fragment of a 70 kilodalton heat shock cognate protein. Nuture (London) 346, 623-6'28. Flaherty. K. M., McKay. D. B., Kabsch, W., and Holnies, K. C. (1991). Similarity of the three-dimensional structures of actin and the ATPase fragment o f a 70-kDa heat shock cognate protein. Proc. Natl. Acad. Sci. U.S.A. 88, 504 1-5045. Flajnik, M. F., Canel, C., Kramer, J . , and Kasahara, M. (1991). Which came first, MHC class I or class II? finmunogetitfirs 33, 295-300. Fletterick, R. J.. Bates, D. J., and Steitz, T. A. (1975). The structure of a yeast hexokinase monomer and its complexes with substrates at 2.7 A resolution. Proc. Nafl. Arad. Sci. U.S.A. 72, 38-42. Flynn, G. C., Chappell, T. G., and Rothman. J. E. (1989). Peptide binding and release by proteins implicated as catalysts of protein assembly. Science 245, 385-390. Flynn, G. C., Poh1.J.. Flocco. M. T., and Rothman.1. E. (1991). Peptide-binding specificity of the molecular chaperone BiP. Nature (London) 353, 726-730. Gaitanaris. G. A., Papavassiliou, A. G., Rubock, P., Silverstein, S.,]..and Gottesman, M. E. (1990). Renaturation of denatured lambda repressor requires heat shock proteins. Cell ( C a m b d g e . Mass.) 61, 1013-1020. Garsia, R. J., Hellqvist, L., Booth, R. J., Kadford. A. I . , Britton, W. J., Astbury, L., Trent, R. J., and Basten, A. (1989). Homology of the 70-kilodalton antigens from Mycobacferium leprae and Mycobacteriuin boiiis with the Mycobacterium tuberculosi.~7 I-kilodalton

HEAT-SHOCK-RELATED PROTEINS

95

antigen and with the conserved heat shock protein 70 of eucaryotes. Infect, Immun. 57, 204-2 12. Georgopoulos, C . P. (1977). A new bacterial gene (groPC) which affects A DNA replication. Mol. Gen. Genet. 151, 35-39. Georgopoulos, C. P., Lam, B., Lundquist-Heil, A,, Rudolph, C. F., Yochem, J . , and Feiss, M. (1979). Identification of the E . coli dnaK (groPC756) gene product. Mol Gen. Genet. 172, 143-149. Georgopoulos, C., Ang, D., Liberek, K., and Zylicz, M. (1990). Properties of theEscherichia coli heat shock proteins and their role in bacteriophage A growth. In “Stress Proteins in Biology and Medicine” (R. 1. Morimoto, A. Tissieres, and C. Georgopoulos, eds.), pp. 191-221. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Gething, M. J . , and Sambrook, J. (1992). Protein folding in the cell. Nature (London) 355, 33-45. Gomez, S. L.. G0ber.J. W., and Shapiro, L. (1990). Expression ofthe Caulobactur heat shock gene dnaK is developmentally controlled during growth at normal temperatures. J. B a c t e d 172, 305 1-3059. Greene, L. E., and Eisenberg, E. (1990). Dissociation ofclathrin from coated vesicles by the uncoating ATPase. J. B i d . Cliem. 265, 6682-6687. Haas, 1. G. (1991). BiP-A heat shock protein involved in immunoglobulin chain assembly. Cum. Top. Microbiol. Immunol. 167, 71-82. Haas. I . G., and Wabl, M. (1983). Immunoglobulin heavy chain binding protein. Nature (London) 306,387-389. Hearne, C. M., and Ellar. D. J. (1989). Nucleotide sequence of a Bacillus subtilis gene homologous to the dnaK gene of Escfirriclzia coli. Nucleic Acids Res. 17, 8373. Hendershot, L. M. (1990). Immunoglobulin heavy chain and binding protein complexes are dissociated in uivo by light chain addition. J. Cell Biol. 111, 829-837. Hendershot, L. M., Ting,J., and Lee, A. S. (1988). Identity ofthe immunoglobulin heavychain-binding protein with the 78,000-dalton glucose-regulated protein and the role of posttranslational modifications in its binding function. Mol. Cell. Biol. 8,4250-4256. Heuser, J., and Steer, C .J . (1989). Trimeric binding ofthe 70-kD uncoating ATPase to the vertices of clathrin triskelia: A candidate intermediate in the vesicle uncoating reaction. J. Cell Biol. 109, 1457-1466. Hurtley, S. M., Bole, D. G., Hoover-Litty, H., Helenius, A,. and Copeland, C . S. (1989). Interactions of misfolded influenza virus hemagglutinin with binding protein (BiP). J . Cell Biol. 108, 2117-2126. Kabsch. W., Mannherz, H. G., Suck, D., Pai, E. F., and Holnies, K. C . (1990). Atomic structure of the actin:DNase 1 complex. Nature (London) 347, 37-44. Kang, P. J.. Ostermann, J., Shilling, J.. Neupert, W., Craig, E. A,, and Pfanner, N. (1990). Requirement for hsp70 in the mitochondria1 matrix for translocation and folding of precursor proteins. Nature (London) 348, 137- 143. Kassenbrock, C. K., and Kelly, R. B. (1989). Interaction of heavy chain binding protein (BiPKRP78) with adenine nucleotides. EMBO J. 8, 146 I - 1467. Kassenbrock, C. K., Garcia, P. D., Walter, P., and Kelly, R. B. (1988). Heavy-chain binding protein recognizes aberrant polypeptides translocated in vitro. Nature (London) 333, 90-93. Kornak, J., Kuo, C., and Campbell, L. (1991).Sequence analysis of the gene encoding the Chlamydia pneumoniae DnaK protein honiolog. Irgict. Irnmun. 59, 72 1-725. Landry, S. J., Jordan, R.. McMacken. R., and Gierasch, L. M. (1992). Different conformations for the same polypeptide bound t o chaperones DnaK and GroEL. Nature (London) 355,455-457.

96

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Leustek, T., Dalie, B., Amir-Shapira, D., Brot, N., and Weissbach, H. (1989). A member of the Hsp70 family is localized in mitochondria and resembles Escherichia coli DnaK. Proc. Natl. Acad. Sci. U.S.A. 86, 7805-7808. Leustek, T., Toledo, H., Brot. N., and Weissbach, H. (1991). Calcium-dependent autophosphorylation of the glucose-regulated protein, Grp78. Arch. Biochem. Biophys. 289, 256-26 1. Lewis, M. J.. and Pelham, H. R. B. (1990). The sequence of the Kluyeromyces lactis BiP gene. Nucleic Acids Res. 18, 6438. Liberek, K., Georgopoulos, C., and Zylicz, M. (1988). Role of the Escherichia coli DnaK and DnaJ heat shock proteins in the initiation of bacteriophage lambda DNA replication. Proc. Natl. Acad. Sci. U.S.A. 85, 6632-6636. Liberek, K., Marszalek, J., Ang, D., Georgopoulos, C., and Zylicz, M. (1991a). Escherichza coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK. Proc. Natl. Acad. Sci. U.S.A. 88, 2874-2878. Liberek, K., Skowyra, D., Zylicz, M., Johnson, C., and Georgopoulos, C. (1991b). The Escherichia coli DnaK chaperone, the 70-kDa heat shock protein eukaryotic equivalent, changes conformation upon ATP hydrolysis, thus triggering its dissociation from a bound target protein. J . Biol. Chem. 266, 14491-14496. Lipinska, B., King, J., Ang, D., and Georgopoulos, C. (1988). Sequence analysis and transcriptional regulation of the Escherichia coli grpE gene, encoding a heat shock protein. Nucleic Acids Res. 16, 7545-7562. Loomis, W., Wheeler, S., and Schmidt, J. (1982). Phosphorylation of the major heat shock protein of Dictvoslelium discoideum. Mol. Cell. B i d . 2, 484-489. Luke, M. M., Sutton, A., and Arndt, K. T. (1991). Characterization of SISl, a Saccharomyces cerevisiae homologue of bacterial dnaJ proteins. J . Cell Biol. 114, 623-638. Marshall, J. S., DeRocher, A. E., Keegstra, K., and Vierling, E. (1990). Identification of heat shock protein hsp70 homologues in chloroplasts. Proc. Natl. Acad. Sci. U.S.A. 87, 374-378. Matsumura, M., Becktel, W. J., and Matthews, B. W. (1988). Hydrophobic stabilization in T4 lysozyme determined directly by multiple substitutions of Ile 3. Nature (London) 334,406-410. McCarty, J. S., and Walker, G. C. (1991). DnaK as a thermometer: Threonine-199 is site of autophosphorylation and is critical for ATPase activity. Proc. Natl. Acad. Sci. U.S.A. 88,9513-9517. Mensa-Wilmot, K., Seaby, R., Alfano, C., Wold, M. C., Gomes, B., and McMacken, R. (1989). Reconstitution of a nine-protein system that initiates bacteriophage lambda DNA replication. J . B i d . Chem. 264, 2853-2861. Milarski, K. L., and Morimoto, R. I. (1989). Mutational analysis of the human HSP70 protein: Distinct domains for nucleolar localization and adenosine triphosphate binding. J. Cell B i d . 109, 1947-1962. Mizzen, L. A., Chang, C., Carrels, J. 1.. and Welch, W. J. (1989). Identification, characterization, and purification of two mammalian stress proteins present in mitochondria, grp 75, a member of the hsp 70 family and hsp 58, a homolog of the bacterial groEL protein. J . B i d . Chem. 264, 20664-20675. Mizzen, L. A., Kabiling, A. N., and Welch, W. J. (1991). The two mammalian mitochondria1 stress proteins, grp 75 and hsp 58, transiently interact with newly synthesized mitochondrial proteins. Cell Regul. 2, 165-179. Munro, S., and Pelham, H. R. (1986). An Hsp70-like protein in the ER: Identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell (Cambridge, Mass.) 46, 291-300.

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Nguyen, T. H., Law, D. T., and Williams, D. B. (1991). Binding protein BiP is required for translocation of secretory proteins into the endoplasmic reticulum in Saccharomyces cereuisiae. Proc. Natl. Acad. Sci. U.S.A. 88, 1565-1569. Normington, K., Kohno, K., Kozutsumi, Y., Gething, M. J., and Sambrook, J. (1989). S. cereuisiae encodes an essential protein homologous in sequence and function to mammalian BiP. Cell (Cambridge, Mass.) 57, 1223-1236. Ohki, M., Tamura, F., Nishimura, S., and Uchida, H. (1986). Nucleotide sequence of the Escherichia coli dnaJ gene and purification of the gene product. J. Biol. Chem. 261, 1778-1781. Ostermann, J., Voos, W., Kang, P. J.. Craig, E. A., Neupert, W., and Pfanner, N. (1990). Precursor proteins in transit through mitochondrial contact sites interact with hsp70 in the matrix. FEBS Lett. 277, 281-284. Palleros, D. R., Welch, W. J., and Fink, A. L. (1991). Interaction of hsp70 with unfolded proteins: Effects of temperature and nucleotides on the kinetics of binding. Proc. Natl. Acad. Sci. U.S.A. 88, 5719-5723. Palleros, D. R., Reid, K. L., McCarty, J. S., Walker, G . C., and Fink, A. L. (1992). DnaK, hsp73 and their molten globules: Two different ways heat shock proteins respond to heat. J . Biol. Chem. 267, 5279-5285. Pelham, H. R. B. (1986). Speculations on the functions of the major heat shock and glucoseregulated proteins. Cell (Cambridge, Mass.) 46, 959-96 1. Rieul, C., Cortay, J. C., Bleicher, F., and Cozzone, A. J. (1987). Effect of bacteriophage M 13 infection on phosphorylation of dnaK protein and other Escherichia coli proteins. Eur. J . Biochem. 168,621-627. Rippmann, F., Taylor, W. R., Rothbard, J. B., and Green, N. M. (1991). A hypothetical model for the peptide binding domain of hsp70 based on the peptide binding domain of HLA. EMBO J. 10, 1053-1059. Rose, M. D., Misra, L. M., and Vogel, J. P. (1989). KAR2, a karyogamy gene, is the yeast homolog of the mammalian BiP/GRP78 gene. Cell (Cambridge, Mass.) 57, 12111221. Sadler, I., Chiang, A., Kurihara, T., Rothblatt, J., Way, J., and Silver, P. (1989). A yeast gene important for protein assembly into the endoplasmic reticulum and the nucleus has homology to DnaJ, an Escherichia coli heat shock protein. J. Cell Biol. 109, 2665-2675. Saito, H., and Uchida, H. (1977). Initiation of the DNA replication of bacteriophage lambda in Escherichia coli K12. J. Mol. Biol. 113, 1-25. Scherer, P. E., Krieg, U. C., Hwang, S. T., Vestweber, D., and Schatz, G. (1990). A precursor protein partly translocated into yeast mitochondria is bound to a 70 kd mitochondrial stress protein. EMBO J . 9, 4315-4322. Schlossman, D. M., Schmid, S. L., Braell, W. A., and Rothman, J. E. (1984). An enzyme that removes clathrin coats: Purification of an uncoating ATPase. J . Cell Biol. 99, 723-733. Schmid, S. L., and Rothman, J. E. (1985a). Enzymatic dissociation of clathrin cages in a two-stage process. J. B i d . Chem. 260, 10044-10049. Schmid, S. L., and Rothman, J. E. (1985b). T w o classes of binding sites for uncoating protein in clathrin triskelions. J. B i d . Chem. 260, 10050-10056. Schmid, S. L., Braell, W. A., Schlossman, D. M., and Rothman, J. E. (1984). A role for clathrin light chains in the recognition of clathrin cages by uncoating ATPase. Nature (London) 311,228-231. Schmid. S. L., Braell, W. A., and Rothman, J. E. (1985). ATP catalyzes the sequestration of clathrin during enzymatic uncoating. J. Biol. Chem. 260, 10057-10062.

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Shiu, R. P. C., and Pastan, I. H. (1979). Properties and purification o f a glucose-regulated protein from chick embryo fibroblasts. Biochim. Siuphy. Acla 576, I4 1-150. Skowyra, D., Georgopoulos, C., and Zylicz, M. (1990). T h e E . coli dnaK gene product, the hsp70 homolog, can reactivate heat-inactivated RNA polymerase in an ATP hydrolysisdependent manner. Cell (Cambridge, Muss.) 62, 939-944. Sussman, M. D., and Setlow, P. (1987). Nucleotide sequence of a Bucillus mcguterium gene homologous to the dnaK gene of Escherichia coli. Nuclric Acids Rrs. 15, 3923. Ting, J., and Lee, A. S. (1988).Human gene encoding the 78,000-dalton glucose-regulated protein and its pseudogene: Structure, conservation. and regulation. DNA 7,275-286. Ting, J., Wooden, S. K., Kriz. R., Kelleher, K., Kaufman, R. 1.. and Lee, A. S. (1987). T h e nucleotide sequence encoding the hamster 78-kDa glucose-regulated protein (GRP78) and its conservation between hamster and rat. Gene 55, 147-152. Tissieres, A., Mitchell, H. K., and Tracy, U. M. (1974). Protein synthesis in salivary glands of Drusuphilu melunogaster: Relation to chromosome puffs. J . Mol. Biol. 84, 389-398. Vogel, J. P., Misra, L. M., and Rose, M. D. (1990). Loss of BiP/GRP78 function blocks translocation of secretory proteins in yeast. J. Cell Bid. 110, 1885-1895. Werner-Washburne, M.. Stone, D. E., and Craig, E. A. (1987). Complex interactions among members of an essential subfamily of hsp70 genes in Saccarorryrs crrenisiuc. Mol. Cell. Bid. 7, 2568-2577. Wetzstein. M., and Schumann, W. (1990). Nucleotide sequence of a Bacillus subtilis gene homologous to the grpE gene of E. coli located immediately upstream of the dnaK gene. Nucleic Acids Res. 18, 1289. Wickner, S. H. (1990).ThreeEscherichiu coli heat shock proteins are required for PI plasmid DNA replication: Formation of an active complex between E . roli DnaJ protein and the PI initiator protein. Pruc. Null. Acad. Sci. U.S.A. 87, 2690-2694. Wickner, S., Hoskins, J., and McKenney, K. (1991a). Function of DnaJ and DnaK as chaperones in origin-specific DNA binding by RepA. Nature (London) 350, 165-167. Wickner, S., Hoskins, J., and McKenney, K. (1991b). Monomerization of RepA dimers by heat shock proteins activates binding to DNA replication origin. Proc. Natl. Acad. Sci. U.S.A. 88, 7903-7907. Winfie1d.J. B., andjarjour, W. N. (1991). Stress proteins, autoimmunity, and autoimmune disease. C u n . Tup. Micrubiul. Immunul. 167, 161- 189. Yarnamoto, T., McIntyre, J., Sell, S. M., Georgopoulos, C., Skowyra, D., and Zylicz, M. (1987). Enzymology of the pre-priming steps in lambda dv DNA replication in viiro. J . Biol. Chem. 262, 7996-7999. Yochem, J., Uchida, H., Sunshine, M., Saito, H., Georgopoulos, C., and Feiss, M. (1978). Genetic analysis of two genes, dnuJ and dnuK, necessary for Escherichia coli and bacteriophage lambda DNA replication. Mol. Gen. Genet. 164, 9-14. Zylicz, M., Ang, D., Liberek, K., Yamamoto, T., and Georgopoulos, C. (1988). Initiation of lambda DNA replication reconstituted with purified lambda and Escherichia coli replication proteins. Biochim. Biophys. Acta 951, 344-350.

PapD AND SUPERFAMILY OF PERIPLASMIC IMMUNOGLOBULIN-LIKE PlLUS CHAPERONES By SCOTT J. HULTGREN,* FRANCOISE JACOB-DUBUISSON,* C. HAL JONES,* and CARL-IVAR BRANDENt * Department of Molecular Biology, Washington University Medical School, St. Louis, Missouri 63110

t ESRF, BP 220, F-38043 Grenoble, France I. General Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

99

.......... .......... ...............................

100

Composite Pilus Structure . . . IV. Postsecretional Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......... A. Immunoglobulin-like Three-Dimensional Structure o ......... B. Conserved Structural Features in Periplasmic Pilus Chaperone Superfamily . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Molecular Recognition Events Utilizing P-Barrel Motifs: A Common T h e m e . . . . . . . . . . . . . . D. PapD Pilus Protein Recognition Site E. Conformation of Chaperone-Bound ...................... F. PapD Blocks Incorrect Interacti G. Ushering of Pilus Subunits into V. Summary . . . . . . . . . . . . . . . . . . . . . . . . . .......... References ................................. ............... Note Added in P r o o f . . . . . . . . . . . . . . .

101 102 104 104 104

111 1 I6 116 117 118 120 121 123

I . GENERAL PERSPECTIVE T h e assembly of surface structures in gram-negative bacteria requires specialized chaperone systems localized in the periplasmic space to ensure that nascently translocated protein subunits are targeted to outer membrane assembly sites in the correct conformation. Detailed structural and functional analyses have revealed that members of the family of periplasmic chaperones that modulate pili biogenesis in gram-negative bacteria have similar structures consistent with the overall topology of an immunoglobulin fold. These proteins have a function that is part of a general strategy used by bacteria to cap and partition interactive subunits imported into the periplasmic space into assembly-competent complexes that are targeted to the outer membrane for the ordered ushering of the subunits into pilus fibers.

ADVANCES IN

PROTEIN CHEMISTRY. Vol. 44

99

Copyright 0 1883 by Academic Press. Inr. All rights of reprodunion in any form reserved.

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SCOTT J . HULTGREN E T AL

11.

INTRODUCTION

Microbial attachment to surfaces is a key event in colonization and infection and results mainly from a stereochemical fit between microbial adhesins, which are assembled on the surface of bacteria, and complementary receptors on host cells or other bacteria. Gram-negative bacterial adhesins that mediate extracellular colonization are usually assembled into polymeric surface fibers called pili or fimbriae (Hultgren et al., 1991). Often a bacterial adhesin is only a minor component of a pilus and is located at the tip of the fiber. The investigation of P pili biogenesis encoded by the pap operon of uropathogenic Escherichia coli will reveal basic principles in molecular biology of how protein subunits fold into domains that serve as modules for building up large assemblies after their secretion across the cytoplasmic membrane. T h e folding of protein subunits and their assembly into adhesive surface fibers in bacteria involve sequential domain-domain interactions within and between the proteins after their secretion across the cytoplasmic membrane. In all gram-negative bacteria this process may be in competition with premature interactions of complementary protein surfaces that lead to biologically nonproductive aggregates. In the periplasm, the formation of domains that serve as assembly modules requires the function of a periplasmic chaperone. Moreover, experimental data suggest that the periplasmic chaperone also participates in the secretion of the pilus subunits across the cytoplasmic membrane, probably by binding to the subunits while they are translocated (H. Jones and S. Hultgren, unpublished, 1993).Interactive siteson protein subunits must be protected by the periplasmic chaperone and targeted to the outer membrane in an assembly-capped manner to prevent nonproductive aggregation. However, at outer membrane assembly sites, chemical o r conformational changes of the subunit-chaperone complexes presumably occur to favor the release of the chaperone, allowing the correct subunit-subunit interactions to occur, driving the assembly of the ordered structure. Studies on the P pilus system have provided insight into the general structure and function of the family of specialized periplasmic chaperones and outer membrane assembly proteins that guide protein subunits down biologically productive assembly pathways after their secretion across the cytoplasmic membrane. We will describe how genetics, biochemistry, and X-ray crystallography have been used to understand how the PapD chaperone regulates the protein-protein interactions of the protein protomers destined to be assembled into highly ordered supramolecular fibers called pili.

PapD AND PILUS CHAPERONES

111.

101

pap GENECLUSTER

Escherichia coli is one of the most intensely studied free-living organisms. It is the most frequent cause of many common bacterial infections in man, including urinary tract infections, bacteremia, and bacterialrelated travelers’ diarrhea (Eisenstein, 1990; Hultgren et al., 1985; Klein and Marcy, 1976; Levine et al., 1983). It is also a leading cause of neonatal meningitis (Klein and Marcy, 1976) and a variety of other clinical manifestations, including pneumonia (Eisenstein, 1990). The initiation of many of these infections is thought to require the presentation of adhesins on the surface of the microbe in accessible configurations, promoting binding events that dictate whether extracellular colonization, internalization, or other cellular responses will occur. Adhesins are often components of long, thin filamentous protein appendages known as pili or fimbriae that are usually 5-10 nm in diameter and up to 2 pm in length (Brinton, 1959; Duguid et al., 1955, 1979; Duguid and Old, 1980; Hultgren and Normark, 1991). Historically, pili expressed by E. coli have been classified based on their sensitivities in hemagglutination reactions to specific sugar inhibitors (Duguid and Old, 1980). Most gram-negative bacteria assemble adhesive pili that generally adopt one of two basic morphologies: rodlike fibers with a diameter of approximately 7 nm, or flexible, thin fibrillae with a diameter of 2-5 nm (De Graaf and Mooi, 1986). Type 1 pili are rodlike fibers and X-ray crystallographic studies of the type 1 pilus have revealed that the subunits are arranged in a right-handed helix with 3; subunits per turn and an axial hole of approximately 2 nm (Brinton, 1965). P, S, 987P, CFAl, CS1, CS2, and F17 pili of E. coli, and type 3 pili of Klebsiella pneumoniae are also rodlike structures (De Graaf and Mooi, 1986) that seem to have an architecture similar to that of the closely related type 1 pilus rod. In contrast, K88, K99, F41, and CS3 pili (fibrillae) seem to form an open helical structure without an axial hole and consequently are much thinner and less rigid structures (De Graaf and Mooi, 1986; De Graaf et al., 1981). Interestingly, each distinct type of pilus in the Enterobacteriaceae family requires a specific member of the periplasmic chaperone superfamily for assembly. Most uropathogenic E. coli isolated from humans express P pili, which mediate binding to Gala(1-4)Gal of the globo series of glycolipids. At least 11 genes are involved in the biosynthesis and expression of functional P pili (Hultgren et al., 1991; Normark et al., 1986). These genes are found in clusters that have been identified at different sites in the E. coli chromosome (Hull et al., 1984; Lund et al., 1988; Plos et al., 1990). Thepap gene clusters from the human urinary tract E. coli isolate J96 have

102

SCOTT J. H U L K K E N E T AI.

been cloned (Hull el al., 1981) and extensively characterized (Lindberg et ul., 1984, 1986, 1987, 1989; Lund et al., 1985; Norgren et al., 1984; Normark et al., 1983, 1986; Hultgren et al., 1989, 1991; Kuehn et al., 1991, 1992). The DNA sequence of the entire pap gene cluster has been determined and was shown to encode 1 1 genes (see Fig. 1). The stalk of the pilus fiber is composed of repeating monomers of pilin encoded by the papA gene (Kuehn et al., 1992; Baga et al., 1984), which is located at the promoter proximal end of the pap operon. Inactivation of papA abolishes pilus expression; however, the receptor-binding and hemagglutinating ability of the bacterium are still retained (Lindberg et al., 1986; Normark et al., 1986; Uhlin et al., 1985). This observation was the first indication that the adhesive property of P pili was not encoded by the structural subunit gene. Subsequently, it was shown that the Gala(1-4)Gal-binding property of P pili was dependent on the expression of the pupG gene, which is located at the distal end of the pap operon (Hultgren et al., 1989; Lindberg et al., 1984, 1986, 1987; Lund et al., 1987; Norgren et al., 1984). It was also shown that PapC, together with the products of three other genes in the pap operon, papE, papF, and papK, were minor components of the P pilus fiber (Lindberg et al., 1986; Jacob-Dubuisson et al., 1993) located exclusively at the tip (Lindberg et a1.,1987). PapE, PapF, and PapK are pilinlike proteins that are related in primary structure to PapA (Lindberg et al., 1986; Marklund et al., 1992). These tip proteins form architecturally distinct thin fibrillar fibers that are joined end to end to the pilus rod (Kuehn et al., 1992).

Composite Pilus Structure Freeze-etch electron microscopy of P pili revealed that they are composite fibers consisting of two distinct structures (Kuehn et al., 1992) (see later, Fig. 8). The stalk of the pilus is composed of repeating monomers

Pilus Assembly Machinery

-

Major Regulation Pilus Subunit

{IHBHA H

H

Pilus Shalt Pilus Protein Anchor

-

Pilus Tip Fibrillurn Subunits I

H Outer Membrane C H D H J H K H MajorE HLinksF HGalo(1-4) G I Usher

Periplasmic Chaperone

Tip Tip Adhesin Length to Regulation Component Fibrillurn

Gal-binding Adhesin

FIG. 1. Schematic representation of the pap gene cluster listing the functions ot' the various gene products.

PapD AND I’ILUS CHAPEKONES

103

of the 18-kDa PapA protein packed into a right-handed helical rod that is 6.5 nm in diameter (Gong et al., 1992). At the distal end of the PapA shaft is a thin, tip fibrillum that is approximately one-third the diameter of the pilus shaft. Tip fibrillae were found to be composed mostly of repeating PapE subunits arranged in linear polymers. The PapG adhesin has been localized to the tips of fibrillae that seemingly are flexible since they often appear to be bent in various orientations in electron micrographs. The major component of the tip fibrillum, PapE, may facilitate interactions with extracellular matrix proteins such as fibronectin, which would make the entire fibrillar structure a multifunctional virulence determinant (Westerlund et al., 1991). The distal location of PapG in the tip fibrillum probably maximizes its ability to recognize glycolipid receptors on eukaryotic cells. The polymeric rodlike pilus fiber may serve to present the adhesive tip fibrillum away from the negatively charged cell surface and beyond the steric interference of lipopolysaccharides. The products of two genes,papC and pupD, are required for the translocation and assembly of pilus subunits into pili; genetic inactivation of either of these two genes has been found to abolish piliation (Hultgren et al., 1989; Lindberg et al., 1989; Norgren et al., 1984, 1987; Normark et al., 1986). PapC is an 88-kDa outer membrane protein that forms the assembly platform where chaperone-subunit complexes are targeted and dissociated, and subunits are ushered into the pili in a distinct order (Dodson et al., 1993; Jacob-Dubuisson et al., 1993). PapC belongs to a family of proteins called molecular ushers (Dodson et al., 1993). The papD gene product encodes the 28.5-kDa periplasmic chaperone protein (Kuehn et al., 1991; Lindberg et al., 1989). A genetic lesion in papD results in a rapid proteolytic degradation of the major and minor pilus subunits (Lindberg et al., 1989) as well as limited degradation of PapC (Hultgren el al., 1989). This finding suggests that PapD interacts with the different subunits of the P pilus in the periplasm, stabilizing them in an assemblycompetent form. It is now known that PapD modulates pilus assembly by preventing nonproductive subunit-subunit interactions and facilitates the targeting of subunits to assembly sites (Kuehn et al., 1991). The polymerization of pilus subunits into the growing pilus is thought to be driven in part by the dissociation of PapD. The requirement of PapD in orchestrating pilus assembly without being a component of the final structures demonstrates its function as a periplasmic chaperone and emphasizes the general principles involved in postsecretional assembly reactions. Our studies suggest that P pili are highly ordered structures that may have evolved from two genetic sources, one encoding long, right-handed helical rods and another encoding open helical fibrillar adhesive poly-

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mers. These distinct structures may have been joined end to end by the introduction of adaptor proteins that allowed polymerization of the fibrillum to the stalk structure (Jacob-Dubuisson et al., 1993). Interestingly, the ordered assembly of the entire composite fiber requires the same periplasmic PapD chaperone. T h e molecular details of the structure and mechanism of action of PapD will be discussed in the remaining sections of this article. IV. POSTSECRETIONAL ASSEMBLY Molecular chaperones are found in the cytoplasm of bacteria and in various cellular compartments in eukaryotes; they maintain proteins in nonnative conformations that permit their secretion across membranes or assembly into oligomeric structures, as exemplified elsewhere in this volume. Virtually nothing, however, has been reported about a similar requirement for molecular chaperones in the periplasm of gram-negative bacteria. The pilus biogenesis pathway provides an excellent model to understand the biological principles involved in postsecretional folding and assembly pathways. The exposure of interactive surfaces of protein protomers at the wrong time during intermediate stages of postsecretional assembly could cause biologically nonproductive interactions that lead to kinetically dead-end pathways and aggregation. However, when these surfaces are protected by a chaperone, the subunits are stabilized in assembly-competent states. We will discuss PapD as the prototype member of the class of chaperones required for pilus assembly. A. Immunoglobulin-like Three-Dimensional Structure of PapD

T h e three-dimensional structure of the PapD periplasmic chaperone that forms transient complexes with pilus subunit proteins has been solved by Holmgren and Branden (1989). PapD consists of two globular domains oriented in the shape of a boomerang (Fig. 2). Each domain is a P-barrel structure formed by two antiparallel P-pleated sheets that have a topology similar to an immunoglobulin fold. The relationship between PapD and other immunoglobulin-like proteins is discussed in Section 1V.C.

B . Conserved Structural Features in Periplasmic Pilus Chaperone Superfamily Pilus assembly in gram-negative bacteria requires that pilus subunit proteins correctly fold into domains that can serve as modules for building up large supramolecular structures. T h e correct domain-domain

PapD AND PILUS CHAPERONES

105

FIG. 2. Ribbon model of the three-dimensional structure of the PapD chaperone. T h e consensus sequence for twelve members of the family was superimposed on the tertiary structure of PapD. The position of' the invariant amino acid residues is shown in black, and that of the residues conserved in at least eight of the sequences, in gray.

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SCOTT J. HULTGREN E T AL.

interactions required for folding and assembly of the pilus proteins must occur after their secretion across the cytoplasmic membrane. In gramnegative bacteria, the correct cascade of protein-protein interactions leading to the assembly of a specific pilus requires a distinct member of the periplasmic chaperone superfamily of proteins. The 10 periplasmic pilus chaperones required for the assembly of 10 different pilus structures in five different gram-negative organisms are listed in Table I. All 10 proteins were found to be 30-600/0 identical in sequence (Table 11), suggesting that they have an immunoglobulin fold similar to that of PapD. Analysis of an alignment of the chaperone sequences has revealed invariant and highly conserved residues that form part of a chaperone consensus sequence (Holmgren et al., 1992) (Fig. 3). T h e consensus sequence was superimposed onto the three-dimensional structure of PapD to investigate the structural and functional significances of the conserved residues (Fig. 2). Most of the amino acids conserved throughout the chaperone superfamily were found concentrated in p strands in the cleft region between the domains and most likely are important structurally to maintain the overall fold of the proteins. However, some of the quasiinvariant residues occupy critical points in loops or are involved in intramolecular interactions that serve to orient some of the loops. Thus, N24, N39, N 145, and N 195 form hydrogen bonds to critical main-chain atoms to position the loops between strands B and C, and C and D, of the first domain and strands B and C, and F and G , of the second domain, respectively. In addition, the loop between strands B and C of the second domain contains two conserved residues, T147 and P148. T h e reverse

TABLE I P i h Cliuprrone ~

Chaperone

Length (amino acids)

PapD F17D FanE FaeE SfaE PilB MrkB HifB SefB Caf I M

218 222 209 225 206 205 215 214 246 238

‘l’vpe ot pilus

Organism

E . roll E . roll E . roll E. roli E . rob E . roll Klebsiella pneumoniae Hauniopliilii.\ iiilluutizcir type b Salinonrlla eiitrriditis

Yeniiiin pestis

P F17 K88 KYY S

.I‘vpe I Type 3 b Fimbrin F, envelope antigen

107

PapD AND PlLUS CHAPEKONES

PapD F17D FanE FaeE SfaE MrkB

PilB HifB Cafl M SefB

PapD

F17D

FanE

FaeE

SfaE

MrkB

*

32/56

31/59 32/50

29/54 27/54 39/63

35/62 40163 34/52 31/56

37/60 43/62 34/60 34/61 35/65

*

*

*

*

*

PilB

Hill3

Cafl M

SefB

33/58 36/58 36/61 45/64 33/52 3 1 I54 35/58 34/56 68/82 38/58 35/57 40161 * 36/59

26/54 3 1 155 3015 1 28/50

26/55 24/52 27/48 26/52 28/55 28/51 32/57 26/50 35/61

*

40159 31/56 37/58 30155

*

*

turn between F and G in the second domain is also an invariant feature that requires G198. The shifting of p strands A1 and D1 from one sheet to another was also found to be a conserved feature among the chaperone family, most likely due to the invariant T7 and P54 residues that are positioned at the respective bends. Finally, the P117 is also an invariant residue located at the elbow bend between the domains. All of these features are highlighted in black on the ribbon model in Fig. 2 and are described in more detail in Holmgren et al. (1992). A second invariant feature of the chaperone superfamily is an internal salt bridge formed by D196 and R116 in association with E83. This invariant interaction probably serves to orient the two domains toward one another to create the cleft region between the domains, which may form the interactive surface for subunit binding (see Holmgren et al., 1992). The interactive surfaces of pilus chaperones necessary for binding to pilus subunit proteins may be composed in part of residues that are conserved throughout the chaperone superfamily. Interestingly, there exists a group of conserved surface-exposed residues that have no apparent structural function, since their side chains are oriented toward the solvent without making any specific interactions with other side chains (Fig. 4). The side chains of three of these residues (T7, R8, and K112) point into the empty cleft between the domains. Three other residues (193, P94, and W36) comprise part of a hydrophobic patch on the surface of the first domain (Holmgren et al., 1992). We suggest that these conserved surface-exposed residues may be important in chaperone function. Recent studies have shown that the recognition of pilus subunit proteins by PapD involves the conserved cleft of the chaperone (Slonim et

Consensus

-

40

30

10

Inl> n=i->

I

c1 100

70

60

180 I

190

m

200 I

50

110

210

I

7)

FIG. 3. Periplasmic pilus chaperone consensus sequence. Amino acid sequence of the PapD chaperone (top line) and consensus sequence derived from the comparison of twelve chaperones (second line). Amino acids are indicated using the one-letter code. In the consensus sequence, a letter shows a residue that is present in at least eight out of twelve sequences, an asterisk designates an invariant residue, and a box shows a position with a hydrophobic residue in all twelve periplasmic chaperones. The arrows underneath the sequence represent the p strands found in the PapD structure.

PapD AND PILL'S CHAPERONES

109

al., 1992). Site-directed mutations in the invariant arginine-8 (R8) cleft residue (see Fig. 4) abolished PapD €unction.The most likely interpretation of these studies was that R8 formed part of an interactive surface in the PapD cleft that contributed to subunit recognition and binding. An alternative explanation was that the glycine, alanine, or methionine muta-

FIG.4. (A) Space-filling model of the PapD chaperone. The solvent-exposed conserved amino acid residues are the same as in (B) and are shown in light gray and the rest of the molecule is dark gray. It should be noted that in order to show clearly the important amino acid residues, the PapD molecule is oriented in this figure at 180" from the view in Fig. 2. Therefore, domain 1 is on the right and domain 2 is on the left. Note the binding cleft of the protein and the hydrophobic patch in domain 1. (B) Ribbon model, showing the amino acid residues that are conserved throughout the periplasmic chaperone family for no obvious structural reason. The side chains of these residues are solvent exposed and have been highlighted on the C, backbone of PapD. Most invariant or quasi-invariant residues are located in domain 1. The orientations of the side chains in the conserved binding cleft (Thr-7, Arg-8, Lys-112, Met-172) and in the hydrophobic patch (Ala-106, Ile-93, Pro-94, Trp-36) are shown, as well as those of Arg-68 and Asp-81.

110

SCOTT

1. HULTGKEN E'I AL

FIG.4. (continued)

tions constructed at this residue altered the overall conformation of PapD but this was much less likely for the following reasons. T h e mutated R8 residue is situated in a bulge where p strand A of the first domain switches from one p sheet to another (see Fig. 4). T h e side chain of this residue points straight into the solvent in the empty cleft region and does not make any specific interactions with other side chains of PapD (Fig. 4). A site-directed mutation in the invariant R8 cleft residue was thus expected to change only the surface properties of this restricted region and not cause any other structural alterations. This was corroborated by the fact that the R8 mutants localized correctly to the periplasmic space and maintained their ability to bind PapC, albeit with a weaker affinity (Slonim et al., 1992). T h e folding of pilus subunit proteins into domains that serve as modules for pilus assembly required an interaction with PapD that was

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strongly dependent on the invariant R8 cleft residue. The binding efficiency of PapD to PapG, PapK, and PapA increased as the steric volume of the mutated residue 8 approached that of the wild-type arginine, from glycine to alanine to methionine (Slonim et al., 1992). These results argued that PapD binds to similar motifs that must be present on each pilus subunit type. Most gram-negative pilus proteins that require a PapD-like chaperone for assembly share extensive sequence homology in the carboxy terminus, which has previously been shown to be important for PapD binding to PapG (Hultgren et al., 1989). The binding of PapD to a subunit is most likely dependent on several of the subunit side chains fitting into a subset of the available pockets present in the PapD cleft and forming a critical interaction with R8. Site-directed mutations in another invariant cleft residue (K112; see Fig. 4)of PapD also abolished its ability to bind subunits and modulate pilus assembly (L. Slonim and S. Hultgren, unpublished results, 1993). The cleft is highly conserved among the entire chaperone superfamily and may have a universal function in subunit binding in all the members of the family. The pilus chaperone superfamily of proteins is required for the assembly of adhesive pili in gram-negative bacteria, a process that is often critical in pathogenesis. This ideal model system exposes general biological problems that are probably encountered by most secreted proteins that have periplasmic preassembly intermediates.

C. Molecular Recognition Events Utilizing P-Barrel Motifs: A Common Theme

T w o higher eukaryote protein superfamilies-the immunoglobulin superfamily (Williams and Barclay, 1988), which includes antibodies, cell surface adhesion molecules, and T cell receptors, and the cytokine receptor superfamily (Cosman et al., 1990; Bazan, 1990), to which the growth hormone receptor is highly related (De Vos et al., 1992)-have made recurrent use of the immunoglobulin fold for molecular recognition processes. Although mainly encountered on cell surfaces, immunoglobulin-like domain proteins are not exclusively anchored to the cell membrane, as demonstrated by secreted antibody molecules. T h e basic structure of an immunoglobulin domain is best described as two antiparallel /3 sheets packed tightly against each other to form a hydrophobic core (Fig. 5 ) (Williams and Barclay, 1988).An immunoglobulin constant domain (C) contains seven /3 strands arranged in two 0 sheets (A, B, E, D and G, F, C ; see Fig. 6, Ig C ) pinned together by a disulfide bond bridging strands B and F. T h e constant domains of the Fab molecule are associated through the outer face of their four-strand

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FIG.5 . Schematic representation ofap-barrel, which forms the skeleton of the immunoglobulin fold domain. Note that the fl strands run antiparallel and are connected by loops that cluster at the ends of the barrel.

/3 sheets and the interface is tightly packed and mainly hydrophobic (Williams and Barclay, 1988). All heavy and light chain constant domains have the same structure (Branden and Tooze, 1991). An immunoglobulin variable domain (Fig. 6, Ig V ) contains two additional strands (C’ and C ) as well as a different order of the strands in the sheets (D, E, B, a1 and a2, G, F, C, C’, C ) . T h e two additional strands, C’ and C”, arejoined by a loop region, which makes up a hypervariable or complementaritydetermining region (CDR2). In addition, each variable domain contains two other CDRs. CDRl connects p strands B and C while CDR3 connects /3 strands F and G. The association of the V domains (heavy and light chains) occurs via their five-strand p sheets, which run almost parallel. This interaction brings the three hypervariable regions of each domain together, forming at one end of the p barrel a large, flat surface: the antibody combining site (Amit et al., 1986). Residues from all six CDRs contribute to the antibody combining site, the conformation of which is determined by the amino acid sequence of the CDRs. In the case of antibodies, the immunoglobulin fold provides a structural framework to support specific recognition surfaces formed by the CDR loops, which cluster at one end of the /3 barrel (Fig. 7, Fab’). T h e recently reported structure of the human growth hormonebinding protein (hGHbp) complexed with its ligand has shown that it is a member of the cytokine receptor superfamily and makes use of the immunoglobulin fold for molecular recognition (De Vos et al., 1992).

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Pap D Domain 1

A

CD4:2

Pap D Domain 2*

FIG.6. Topology diagrams of four immunoglobulin-like domains. Shown are domain 1 of PapD. avariabledomain ofan immunoglobulin, aconstant domain of an immunoglobulin, and the second domain of the CD4 molecule. The p strands are represented as arrows and the two p sheets in each domain are separated by a space. Note that strands A and D in the first domain of PapD are shared by the two /3 sheets (strand switching), and similarly strand A in the variable domain of the immunoglobulin passes from one sheet to the other. CDRl, CDR2, and CDR3 represent the complementarity-determining (or hypervariable) regions of the variable domain of the immunoglobulin. *, PapD domain 2 has an additional H strand (see text).

Specifically, the two domains of hCHbp are variations of the immunoglobulin fold and are identical to the second domain of CD4 (Fig. 6, CD4:2), as well as the second domain of PapD. Important residues for interaction of hCHbp and human growth hormone are localized, for the most part, in loop residues from both domains that form a binding surface near the hinge region between the two domains. Residues from loops in both domains are involved in ligand binding as well as one residue in a p strand which precedes the linker junction between the two domains [Fig. 7-(hGHbp),]. T h e relative orientation of the two @barrel domains in hGHbp is strikingly different from the orientation between the constant and variable domains of immunoglobulin molecules, and a salt bridge between the amino- and carboxy-terminal domains seems to

sco-rr J .

114

Pap D

EIULTGKEN E I AL

Fab’

Ig Molecule FIG. 7. Three different binding sites used hv the imniunoglobulin-like proteins: the PapD chaperone, the antigen-binding fragment of an inimunoglol~i~liii (Fab’), and the domain is human growth hornlone-binding protein (hGHbp). Each iiii~iiunoglob~~lin-like represented as an oval (white, Ig V-like; hatched, 0 4 - l i k e ; shaded, Ig C-like). T h e binding sites of Fah’ and hCHbp for antigen A and growth hormone H, respectively, are known to he those shown. I n the case of the chaperone, the binding cleft for the subunit S is hypothesized to he formed by the two linked domains, on the basis of site-directed mutagenesis experiments (see text).

be important in stabilizing the structure. A disulfide bond cross-links the two /3 sheets of the amino-terminal domain of hGHbp (between strands C’ and E) and, in contrast to an immunoglobulin constant domain, two additional disulfide bonds link neighboring strands of the domain. T h e carboxy-terminal domain of hGHbp does not contain any intersheet disulfide cross-links. Only minor structural variations have been noted among the members of the immunoglobulin superfamily in spite of rather weak sequence similarities. The variations include the absence of the disulfide bond typically bridging the two /3 sheets as well as several modulations in the topology of the /3 strands and in the connectivity between them (Wang et al., 1990; Ryu el al., 1990; Williams and Barclay, 1988). The immunoglobulin fold domain structure provides a stable platform for the display of specific recognition surfaces either formed by the loops connecting the /3 strands or by regions located on the outer faces of the p sheets (Williams and Barclay, 1988; Lesk and Chothia, 1982). The immunoglobulin-like domains of these classes of proteins seem to have distinct roles, integrating recognition and effector functions in the same molecule (Ryu et al., 1990; Williams, 1982). In some cases, the immunoglobulin module forms a noncovalent, lateral association with a similar module from another polypeptide chain, as is seen in antibodies

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and T cell receptors, in order to create the ligand-binding site (Chothia et al., 1988; Amit et al., 1986; David and Bjorkman, 1988). For the receptor proteins, transient lateral interactions are suggested to be part of the signal transduction mechanism, as exemplified by the growth hormone receptor (De Vos et al., 1992). In the case of hGHbp, the binding of the ligand triggers dimerization via the carboxy-terminal domains (Cosman et al., 1990). In contrast, ligand binding by an antibody may elicit other effector molecules such as complement. We suggest that the periplasmic chaperone PapD, which is the prototype of the only known prokaryotic protein family composed of immunoglobulin-like domains, is a variation on the same theme (Fig. 7, PapD). It differs from the classic immunoglobulin fold due to strand switching at the edges of the sheets. The C-terminal domain, domain 2, has structural features analogous to domain 2 of the HIV receptor, CD4, which is a variation of the classic immunoglobulin fold (Ryu et al., 1990, Fig. 6, CD4:2). T h e upper sheet is composed of strands E, B, and A and the lower sheet of strands is composed of G, F, C, and D. In addition, the C-terminal domain of PapD also has in the lower sheet a short strand, H, which is disulfide bonded to strand G. PapD lacks the intersheet disulfide bonds normally seen in constant domains (Branden and Tooze, 1991). Domain 1 of PapD is most similar to the variable domain of an immunoglobulin. In the classical immunoglobulin fold of a constant domain, the strand order is D, E, B, A for the upper sheet and G , F, C for the lower sheet. The variable domain of an immunoglobulin shares strand A between the sheets so that the strand order is D, E, B, a l , and a2, G, F, C, C ’ ,C”. In domain 1 of PapD, both strands D and A are shared between the two sheets, giving the strand order d l , E, B, a1 and a2, G, F, C , d2, making it more similar to variable domains than to constant domains (Fig. 6, PapD domain 1). We hypothesize that the two immunoglobulin-like domains of the chaperone form a different binding paradigm from that used by antibodies or the growth hormone receptor. This suggests that immunoglobulinlike domains make various uses of their surfaces to recognize ligands and partner proteins. Specifically, it seems that PapD utilizes the immunoglobulin fold in two linked domains that are oriented to form a binding cleft. In this model PapD binds pilin subunits through interactions with conserved residues in the cleft (Fig. 7, PapD). Our current model suggests that the P-barrel structure also stabilizes variable loop regions that surround the cleft and impart specificity to the chaperone. The parallel between PapD and the members of the other two families can be extended to the “effector functions” of these molecules, as the binding of a pilin subunit to the chaperone is necessary for the subsequent targeting of the

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subunit protein to the PapC assembly platform, as discussed below. It is easy to envision conformational changes taking place in the binding and/ or release steps. The stability in the external medium (Williams and Barclay, 1988) and the modular character of the immunoglobulin domain, as well as its potential to display versatile binding surfaces, make the immunoglobulin fold ideally suited for a chaperone in the Pap pilus system.

D . PapD Pilus Protein Recognition Site PapD probably binds to similar motifs on each of the pilus subunits as they emerge from the cytoplasmic membrane. By using galabiose-sepharose in affinity chromatography, the PapC adhesin was isolated in a preassembly complex with PapD from the periplasmic space (Hultgren et al., 1989). PapD binds to a domain of PapC in a way that does not sterically interfere with the receptor-binding domain of PapC since PapG mediates specific binding to Gala( 1-4)Gal, even when in a complex with PapD. The receptor-binding domain of PapG has been mapped to the amino-terminal half of the protein while the carboxy terminus of the adhesin was suggested to form a domain recognized by PapD. Specific sequence homologies among pilus proteins in the amino and carboxyl termini suggest that PapD may recognize a common motif on each of the pilus subunit proteins. Binding of PapD to PapG is required for the incorporation of the adhesin into the pilus and was found to protect PapG from proteolytic cleavages (Hultgren et al., 1989). The sensitivity of the pilus proteins to proteolytic cleavages in the absence of PapD might indicate that the PapD-subunit interactions assist in the correct folding of the polypeptides. Native polyacrylamide gel electrophoresis (PAGE) and amino acid composition analyses demonstrated that the PapD-PapG complex was a discrete bimolecular moiety that consisted of an equimolar ratio of PapD (28.5 kDA), and PapC (35 kDa). The isoelectric point (pl) of the PapD-PapC complex was found to be 7.4, which is intermediate between the pl values of PapD (pl 9.4) and PapG (pl 5.0) (Hoschuetzky et al., 1989). The opposite charge of these proteins could suggest that charged amino acids are important in the interaction. E . Conformation of Chaperone-Bound Adhesin

PapG seems to maintain the same receptor-binding specificity, whether it is bound to PapD or incorporated into the tip of the pilus, by recognizing the same polar edge of galabiose via hydrogen bonding to hydroxyl

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groups HO-6, OH-2’, OH-3’, OH-4’, and OH-6’ (Hultgren et al., 1989; Kuehn et al., 1991; Kihlberg et al., 1989). The binding specificity of PapC seems likely to be a function of its tertiary structure, as is the case for most lectins (Weis et al., 1988), arguing that PapC exists in a nativelike state when bound to PapD. In contrast, cytoplasmic chaperones such as SecB, trigger factor, and GroEL have been shown to bind to and maintain polypeptides in a nonnative state (Hardy and Randall, 1991; Lecker et al., 1989, 1990). Thus, in contrast to cytoplasmic chaperones, the role of periplasmic chaperones such as PapD may be to maintain the bound pilus subunits in nativelike conformations that are assembly competent. Supporting this hypothesis was the detection of a typical @sheet spectrum of PapC within the complex as determined by circular dichroism (Kuehn et al., 1991). In addition, the four cysteine residues in PapC are most likely involved in disulfide bridge formation in PapC that is bound to PapD. F. PapD Blocks Incorrect Interactions to Prevent Misassembly

T h e stability of the PapD-PapC complex has been examined in the presence of urea. At 2 M urea in the presence of dithiothreitol (DTT), 80% of the complex was destroyed. In an attempt to reform the native complex from the reduced, denatured preparation, the sample was diluted to conditions in which the complex was previously shown to be stable. In this analysis, the proteins formed large aggregates that prevented reformation of the complex. These results argue that the site recognized by PapD is part of an interactive surface on PapC. T h e reducing and denaturing conditions may essentially “uncap” this interactive surface. In the absence of PapD, the uncapped interactive surface may participate in incorrect interactions that are kinetically favorable, driving the proteins down a nonproductive pathway of aggregation. The ability of purified PapD to bind and cap an interactive surface of PapC and maintain it in a soluble, distinct bimolecular complex was tested in an in vitro assay. A denatured PapD-PapC preparation, where the interactive surface of PapC was uncapped, was diluted in the presence of increasing concentrations of purified PapD. The distinct bimolecular PapD-PapG complex was reformed in proportion to the concentration of PapD in the renaturing solution (Kuehn et al., 1991). Native PapD present in the diluent bound to PapC to reform the native soluble complex and block the nonproductive interactions. The pilus is thought to grow from the base, as has been shown for the closely related type 1 pilus (Lowe et al., 1987). T h e surface on the pilus protein recognized by PapD may also be part of the same surface that

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interacts with nascently incorporated subunits in the growing pilus. Thus, PapD may function as a reversible capping protein that modulates polymerization by capping and uncapping interactive surfaces of pilus subunits that are imported into the periplasmic space. When PapD is bound to the subunits, incorrect interactions are blocked and the release of PapD at the assembly site uncaps the assembly surface and allows the subunit to be polymerized into the pilus rod. T h e binding and release of PapD are orchestrated to occur at distinct sites within the cell, in order to guide the protein protomers along a productive assembly pathway. T h e targeting of chaperone complexes to the outer membrane assembly site seems to involve the recognition of the pilus protein protomer in the context of PapD. G . Ushering of Pilus Subunits into Pili from Chaperone Complexes

T h e production of adhesive P pili requires PapC, which is an 88-kDa outer membrane protein. Genetic lesions in PapC result in a block in the assembly pathway, leading to an accumulation of chaperone-pilus protein complexes in the periplasm (Norgren et al., 1987). Thus, in the absence of PapC it seems that the chaperone preassembly complexes are no longer targeted to outer membrane assembly sites. As a consequence, the periplasmic chaperone remains bound to the subunits, preventing their assembly into pili. T h e various complexes that PapD forms with each ofthe pilus subunit proteins were recently used in an in vitro assay to test their ability to recognize partially purified PapC. PapD alone was unable to recognize PapC in vitro. In contrast, PapD-PapG, PapD-PapE, and PapD-PapF complexes all specifically bound to PapC (Dodson et al., 1993). These exciting results suggested that PapD has an effector function of targeting pilus subunits to PapC after binding to them. Remarkably, PapD-PapA complexes did not bind to PapC in vitro. We interpret these results by suggesting that in vivo, PapD-PapA complexes are not targeted to empty PapC sites, but instead only recognize PapC when in the context of a growing tip fibrillum. Supporting this hypothesis was the in uivo finding that in the absence of tip fibrillar proteins PapA is unable to bind to PapC and be polymerized into pilus rods (Jacob-Dubuisson et al., 1993).These results suggested a mechanism that ensures that every pilus rod is joined end to end to an adhesive tip fibrillum (Fig. 8). PapD and PapC appear to act as molecular escorts, regulating the interactions of each pilus protein. As a molecular chaperone, PapD prevents nonproductive interactions of the subunits and allows the subunits to fold properly. We propose that PapC is a member of a new class of proteins that we have named molecular ushers. T h e PapC usher acts as

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-

A Chaperone Subunit Interaction

D

Pilus Rod Assembly

M on

each subunit type

B Targeting of Chaperone - Subunit Complexes

OUfM

---.

Membrane,

Ic

I

Tip Fibrillum Assembly

-Membrane outet --Periplasm

Pap D IS released and ts palymerlzed

&&

FIG. 8. Model for the ordered assembly of the P pilus. (A) PapD binds t o each subunit as it is translocated into the periplasinic space, niaintaining them in assembly-competent conformations that are able to recognize and bind to PapC. The subunits are then targeted

to PapC. In the initial stage, the PapD-PapG complex has the highest relative affinity tor PapC and binds to PapC first (B); subsequently PapC is localized to the distal ends of tip fibrillae. T h e other subunits that compose the tip fibrillum (PapF, PapE, and PdpK) are successively assembled (C). PapA-PapD complexes are unable t o bind to PapC alone, but in the presence of tip fibrillae they are added to the growing structure (D). The inability of PapD-PapA to bind to PapC initially ensures that pilus rods are not made in the absence of' tip fibrillae. + , Receptor binding site.

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a molecular doorkeeper, modulating the ordered targeting of chaperone-subunit complexes to outer membrane assembly sites, which results in the formation of a composite structure. T h e ordered assembly of each pilus subunit type seems to be controlled by molecular recognition events that involve stereochemical fits between complementary surfaces on each subunit type (Jacob-Dubuisson et al., 1993). The ability of the PapD chaperone to bind transiently to pilus subunits and cap interactive surfaces allows the subunits to pass through the periplasm without aggregation. The uncapping of PapD at the outer membrane seems to expose polymerization sites and drive assembly. T h e mechanism of chaperone uncapping is unknown but is seemingly ATP independent and may involve PapC. PapC is a representative member of a family of outer membrane ushers, including FanD, FaeD, FimD, and MrkC (Mooi et al., 1986; Klemm and Christiansen, 1990; Allen et al., 1991), which are required for pilus assembly in gram-negative bacteria. It is likely that all of these proteins have similar ushering functions, acting in concert with their respective chaperone partners (Holmgren el al., 1992) to assure the correct interactions necessary for the production of ordered adhesive structures, which are important in colonization and infection of susceptible hosts.

SUMMARY The formation of a P pilus requires a molecular chaperone in the periplasm and a molecular usher in the outer membrane. Each pilus is composed of six different types of proteins that are assembled into a composite fiber in a defined order. The correct folding of subunits into domains that can serve as assembly modules requires an association with the periplasmic chaperone. PapD is the prototype member of the family of bacterial pilus chaperones that have a three-dimensional structure consistent with an immunoglobulin fold. In general, proteins with an immunoglobulin fold structure have molecular recognition functions in eukaryotic cells that are often integrated with effector functions. PapD has also a recognition function, binding nascently translocated pilus subunits and maintaining them in assembly-competent conformations. The association of the chaperone with the subunit triggers the targeting of the latter to an outer membrane usher. T h e usher serves as a molecular gatekeeper, allowing the ordered incorporation of the pilus subunits into the pilus structure from the periplasmic chaperone complexes. The two immunoglobulin-like domains of PapD are oriented to form a cleft that contains the subunit binding site. This is a different binding paradigm from that used by either antibodies or the growth hormone receptor. T h e V.

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blend of genetics, biochemistry, X-ray crystallography, and carbohydrate chemistry in the study of pili biogenesis will continue to give insight into some of the most basic intellectual challenges in molecular biology concerning how proteins fold into domains that serve as modules for the formation of larger assemblies, and relating these processes to microbial pathogenesis.

ACKNOWLEDGMENTS We wish to thank Karen Dodson and Lynn Slonim for sharing unpublished results. F.J.-D. is the recipient of a European Molecular Biology Organization Long-Term Postdoctoral Fellowship; H.J. is a recipient of a Fellowship from the Keck Foundation of Washington University. This work was supported by grants to S.J.H. from the Lucille Markey Charitable Trust, Washington University/Monsanto Biomedical Research Contract, National Institutes of Health (Support Grant IROlAI29549), Institutional Biomedical Research (Support Grant 2-S07-RR-5389), and the American Cancer Society (Grant IN-36).

REFERENCES Allen, B. L., Gerlach, G.-F., and Clegg, S. (1991).J. Bacteriol. 173, 916-920. Amit, A. G . , Marriuzza, R. A., Phillips, S. E.. and Poljak, R.J. (1986). Science 233,747-753. Baga, M., Normark, S.. Hardy. J., OHanley, P., Lark, D., Olsson, 0..Schoolnik, G., and Falkow, S. (1984).J. Bacten’ol. 157, 330-333. Bazan, J. F. (1990). Proc. Natl. Acad. Sci. U.S.A. 87, 6934-6938. BrandCn, C.-I., and Tooze, J. (1991). “Introduction to Protein Structure.” Garland, New York. Brinton, C. C., Jr. (1959). Nature (London) 183, 782-786. Brinton, C. C., Jr. (1965). Trans. N.Y. Acad. Sci. 27, 1003-1165. Chothia, C., Boswell, D. R.,and Lesk, A. M. (1988). E M B O J . 7, 3745-3755. Cosman, D., Lyman, S. D., Idzerda, R. L., Beckmann, M. P., Park, L. S., Goodwin, R. G . , and March, C. J. (1990). Trends Biochern. Sci. 15, 265-270. Davis, M. M., and Bjorkman, P. J. (1988). Nature (London) 334, 395-401. De Graaf, F. K., and Mooi, F. R. (1986). Adv. Microb. Phyiol. 28, 65-143. De Graaf, F. K., Klemm, P., and Gaastra, W. (1981). Infect. Immun. 33, 877-883. De Vos, A., Ultsch, M., and Kossiakoff, A. (1992). Science 255, 306-312. Dodson, K., Jacob-Dubuisson, F., Striker, R., and Hultgren, S. (1993). Proc. Natl. Acad. Sci. U.S.A. (in press). Duguid, J . P., and Old, D. C. (1980). In “Bacterial Adherence Receptors and Recognition” (E. H. Beachy, ed.), pp. 186-217. Chapman & Hall, London. Duguid, J. P., Smith, 1. W., Dempster, G., and Edmunds, P. N. (1955).J. Pathol. Bacteriol. 70,335-348. Duguid, J. P., Clegg, S., and Wilson, M. 1. (1979).J. Med. Microbiol. 12, 213-227. Eisenstein, B. J. (1990). In “Enterobacteriacae in Principles and Practice of Infectious Diseases” (G. L. Mandel, R. G. Danglers, and J. E. Bennet, eds.), Chapter 195, pp. 1658- 1673. Churchill-Livingstone, London. Gong, M., and Makowsky, J . (1992).J. Mol. B i d . 224, 735-742. Hardy, J. S., and Randall, L. L. (1991). Science 251, 439-443.

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Holmgren, A., and Branden, C.-I. (1989). Nalure (London) 342, 248-251. Holmgren, A.. Kuehn, M. J., Branden, C.-I., and Hultgren, S. J. (1992). EMBOJ. 11, 1617-1622. Hoschuetzky, H., Lottspeich, F., and J a n n , K. (1989).Infect. Irnmun. 57, 76-81. Hull, R. A., Gill. R. E.. Hsu, P., Minshaw, B. H., and Falkow, S. (1981).In/ict. Imrnun. 33, 933-938. Hull, R. A., Hull, S. I . , and Falkow, S. (1984). Irtfect. Irnmun. 43, 1064-1067. Hultgren, S.J., and Normark, S. (1991).Cum. Opin. Grn. Dru. 1, 313-318. Hultgren, S. J., Porter, T. N., Schaeffer, A.J., and Duncan,J. L. (1985).Ivqect. Irnmun. 50, 370-377. Hultgren, S. J., Lindberg, F., Magnusson, G., Kihlberg, J., -1ennent.J. M., and Normark, S. (1989).Proc. Nall. Acad. Sci. U.S.A. 86, 4357-4361. Hultgren, S.J., Abraham, S. N., and Normark. S. (1991).Annu.Rev. Microbiol. 45,383-415. Jacob-Dubuisson, F., Heuser, J., Dodson, K., Normark, S., and Hultgren, S. J. (1993). EMBOJ. (in press). Kihlberg, J., Hultgren, S. J.. Normark, S.. and Magnusson, G. (1989).J.Am. Clirrn. Soc. 111, 6364-6368. Klein, J. 0.. and Marcy, S. M. (1976).I n “Infectious Diseases of the Fetus and Newborn Infant” (J. S. Remington and J. 0. Klein, eds.), pp. 747-802. Saunders, Philadelphia. Klemm, P., and Christiansen, G. (1990).Mol. Gen. Genrl. 220, 334-358. Kuehn, M. J., Normark, S., and Hultgren, S. J. (1991). Proc. Null. Acad. Sci. U.S.A. 88, 10586- 10590. Kuehn, M. J., Heuser. J . , Normark, S.. and Hultgren, S. 1. (1992).Nature (London) 356, 252-255. Lecker. S. H., Lill. R., Ziegelhoffer. T., Georgopoulos, C. P., Bassford, P. J.. Kumarnoto, C. A., and Wickner. W. (1989).EMBOJ. 8,2703-2709. Lecker, S. H., Driessen, A. J . M., and Wickner, W. (1990).EMBOJ. 9, 2309-2314. Lesk, A. M., and Chothia, C. (1982).,]. Mol. Biol. 160, 325-342. Levine, M. M., Kaper, J. B., Black. R. E., and Clements, M. L. (1983). Microbiol. RPU.47, 510-550. Lindberg, F. P., Lund, B., and Normark, S. (1984).EMBOJ. 3, 1167-1173. Lindberg, F. P., Lund, B.. and Normark. S. (1986). Proc. Null. Acud. Sci. U.S.A. 83, 189 1- 1895. Lindberg, F. P., Lund. B., Johansson, L., and Normark, S. (1987).Nature (London) 328, 84-87. Lindberg, F. P., Tennent, J. M., Hultgren, S. J., Lund, B., and Normark, S. (1989). J . Bacleriol. 171, 6052-6058. Lowe, M. A., Holt, S. C., and Eisenstein, B. I. (1987).J.Baclrriol. 169, 157-163. Lund, B., Lindberg, F. P., Baga, M., and Normark, S. (1985).J.Bacterial. 162, 1293-1301. Lund, B., Lindberg, F. P., Marklund. B. I . , and Normark, S. (1987).Proc. Nall. Acad. Sci. U.S.A. 84, 5898-5902. Lund, B., Marklund, B. 1.. Stromberg. N., Lindberg, F., Karlsson, K. A., and Normark, S. (1988). Mol. Microbiol. 2, 255-263. Marklund, B. I., Tenner, J. M., Garcia, E., Hamers, A., Baga, M., Lindberg, F., Gaastra, F., and Normark, S. (1992). Mol. Microbiol. 6, 2225-2242. Mooi, F. R., Classen. I.. Baaker, D., Kuipers, H., and d e Graaf, F. K. (1986). Nuclric Acids Res. 14,2443-2457. Norgren, M.. Normark. S., Lark, D., OHanley. P., Schoolnik, G., Fdlkow, S., SvanborgEden, C., Baga, M., and Uhlin, B. E. (1984). EMBOJ. 3, 1159-1 165. Norgren, M.. Baga, M., Tennent. J. M., and Normark, S. (1987).Mol. Microbiol. 1,169-178.

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Normark, S., Lark, D.. Hull, R..Norgren, M., Baga, M., O’Hanley, P., Schoolnik, G.. and Falkow, S. (1983). InIect. Irnniun. 41, 942-949. Normark, S.. Baga, M., Goransson, M., Lindberg, F. P., Lund, B., Norgren, M., and Uhlin, B. E. (1986).I n “Microbial Lectins and Agglutinins; Properties and Biological Activity” (D. Mirelman. ed.), pp. 113-143. Wilev (Interscience), New York. Plos, K.. Carter, T., Hull, S.. Hull, R., and Svanborg-Eden, C. (1990).J . Inject. Dis. 161, 518-524. Ryu. S. E.. Kwong, P. D., Trunch, A.. Porter, T. G . , Arthos, J., Kosenberg, M . , Dai, X., Xuong, N.. Axel, R.,Sweet, R. W., and Hendrickson, W. A. (1990). Nuture (London) 348, 4 19-426. Slonim, L. N., Pinkner, J. S., Branden, C.-I., and Hultgren, S. J. (1992). E M B O J . 11, 4747-4756. Uhlin. B. E., Nogren, M., Baga, M., and Normark, S. (1985). Proc. Nutl. Acud. Sci. U.S.A. 82, 1800- 1804. Wang. J., Yan, Y., Garret, T. P.. Liu, J.. Rodgers, D. W., Garlick, R.L., Tarr, G. E., Husain, Y.,Reinherz, E. L., and Harrison, S. C. (1990). Nature (London) 348, 411-418. Weis, W., Brown, j. H., Cusack, S., Paulson, j.C., Skehel, 1.1.. and Wiley, D.C. (1988). Nature (London) 333, 426-43 1. Westerlund, B., van Die, I., Kramer, C., Kuusela, P., Holthofer, H., Tarkkanen, A. M., Virkola, R., Riegman, M., Bergmans, H . , Hoekstra, W., and Korhonen, T. K. (1991). Mol. Micrabiol. 5, 2965-2975. Williams. A. F. (1982).J. T/wor. Biol. 98, 221-234. Williams, A. F.. and Barclay, A. N. (1988). Annu. Rev. lmrnuriol. 6, 381-405.

Noir ADDED I N PROOF. Additional periplasmic chaperones that belong to the same family as that described here are regularly identified and sequenced, which emphasizes the general concepts presented here. As new periplasmic chaperones have been added to the list between submission and publication of this article, we have had to revise the consensus sequence. For example, some amino acids such as Ah106 or Met172 (see Fig. 4) can no longer be regarded as conserved, and it is likely that more positions will be removed from the list of conservediinvariant amino acids. For residues such as Arg8 or Lysll2, however, the conservation throughout all twelve members of the periplasmic chaperone family strengthens the essential nature and necessary role of these residues in chaperone function. Furthermore, we cannot overemphasize that the alignment of a new sequence with the existing ones, must use as a reference the only known tertiary structure so far, PapD, to position the elements of secondary structure and avoid the insertion of gaps within these p strands.

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PROTEIN DISULFIDE-ISOMERASE: ROLE IN BIOSYNTHESIS OF SECRETORY PROTEINS By NEIL J. BULLEID Department of Biochemistry and Molecular Biology, University of Manchester, Manchester M13 QPT, England

........................

111.

IV.

V.

VI.

ies of Protein Disulfide-Isomerase A. Protein Folding in Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Enzymatic Properties and Mechanism . Cellular Properties of Protein Disulfide-Isomerase .................... A. Subcellular Localization . . . . . . . . . . . . . B. Factors Affecting Expression of Enzyme ......................... Role of Protein Disulfide-Isomerase in Intracellular Protein Folding Use of Cell-Free Systems to Study Disulfide Bond Formation at Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Multifunctionality of Protein Disulfide-Isomerase ..................... A. Sequence Information . . . . . . . . . . . . . . B. Identity with p Subunit of Prolyl4-Hydroxylase . . . . . . . . . . . . . . . . . . C. Identity with Subunit of Triglyceride Transfer Protein . . . . . . . . . . . . D. Role of Protein Disulfide-Isomerase in N-Linked Glycosylation . . . . . E. Protein Disulfide-Isomerase as Thyroid Hormone-Binding Protein and Iodothyronine 5'-Monodeiodinase . ... Conclusions ............................................ References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.

125 126 126 129 131 131 132 133 133 139 139 141 142 143 145 147 148

INTRODUCTION

Proteins that are to be secreted from the cell o r targeted to intracellular locations branching off from the secretory pathway are translocated into the endoplasmic reticulum (ER). Several co- and posttranslational modifications occur to the polypeptide chain that are necessary for subsequent exit of the protein from the ER. These include cleavage of the signal peptide, disulfide bond formation, N-linked glycosylation, folding, and assembly into oligomeric structures. Other modifications that occur to specific proteins include hydroxylation of proline and lysine residues in procollagen, the y-carboxylation of Glu residues in blood-clotting and other calcium-binding proteins, and the transfer of triglycerides to lipoproteins. Although these modifications have been identified for some time, it has proved difficult to study the enzymes involved in the catalyses due to their loss of activity on solubilization of the ER membrane. One exception to these constraints is the enzyme protein disulfideADVANCES IN PROTEIN CHEMISTRY. Vd. 44

125

Copyright 0 1993 by Academic Reu. Inc. AU righu of reproduction in any form m n l .

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NEIL J. BULLEID

isomerase (PDI). This is a soluble protein that is resident within the lumen of the EK. It has been characterized in terms of its catalytic, cellular, and molecular properties and has recently been shown to be involved not only in the formation ofdisulfide bonds in newly synthesized proteins, but also as the /3 subunit of prolyl 4-hydroxylase, and as a component of the triglyceride transfer complex. It therefore plays a key role in the co- and posttranslational modification of secretory and cellsurface proteins. T h e enzyme has also been identified as a glycosylation site binding protein, a thyroid hormone-binding protein, and an iodothyronine 5’-monodeiodinase; the functional significance of these latter activities has still to be demonstrated. ‘l‘his article will review the studies on the role of the enzyme in disulfide bond formation, and will discuss the proposed muhifunctionality of this polypeptide. 11.

(;ATALYTIC

PKOPEKTIES OF PK
A . Proteh Folding in Vilro

The native structure of a protein is formed by the linear polypeptide chain folding to form its most thermodynamically stable structure. Early studies on the refolding and renaturation of denatured proteins revealed that all the information required for folding of the polypeptide was contained within the amino acid sequence, as proteins were able to refold simply by the removal of the denaturant (Epstein et a/., 1963). This led to the “thermodynamic hypothesis” of protein folding (Anfinsen, 1973) and has contributed to the idea that, as no factors are required to fold proteins in vitro, then no factors are involved in the process in vivo. However, these early studies revealed that the time taken to refold denatured proteins (hours) was far greater than rates of folding in vivo (minutes). This observation was particularly true for proteins that contained disulfide bonds and led to the identification of an enzymatic activity present in microsomal membrane fractions that catalyzed the isonierization of disulfide bonds (Venetianer and Straub, 1963; Goldberger et al., 1963; Ramakrishna Kurup et al., 1966). The renaturation of ribonuclease was used as an assay for this enzymatic activity. This forms the basis of the most widely used assay wherein reduced, denatured ribonuclease is reoxidized in the presence of a denaturant; the resulting “incorrectly” disulfide-bonded protein, termed “scrambled” ribonuclease, is inactive but cpn be renatured in the presence of PDI. The ability to trap disulfide-bonded intermediates in the folding pathway of bovine pancreatic trypsin inhibitor (BP‘TI) enabled Creighton to

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PROTEIN DISULFIDE-ISOMERASE

ask questions about the role o f PDI in the renaturation of denatured proteins (Creighton et al., 1980). It was found that PDI catalyzed the interconversion of several intermediates, and that the catalyzed reaction followed the same pathway as the uncatalyzed reaction. Thus, PDI functions as a true catalyst in that it accelerates reactions at a molar concentration of less than 1% of the substrate, it requires disulfides or thiol reagents to break o r form disulfide bonds, and it catalyzes only the steps that are energetically favorable under the conditions used. It is also worthy to note that it did not catalyze the renaturation of proteins that do not contain disulfide bonds. These early studies concentrated on small monomeric proteins and consequently the disulfide bonds that were formed were intramolecular. However, PDI has also been shown to catalyze the formation of intermolecular disulfides (Koivu and Myllyla, 1987). This work was not carried out on denatured substrates, but rather on procollagen polypeptides that were isolated as trimeric structures. Procollagen polypeptides were generated by reduction of the trimer and the chains were allowed to refold in the presence of mixtures of oxidized and reduced glutathione and in the presence and absence of YDI. A scheme outlining the folding pathway of procollagen is shown in Fig. 1 . Acquisition of trimers was monitored by the disappearance of monomeric polypeptides with the concomitant appearance of trimeric structures as judged by sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis run under nonreprocollagen polypeptides

-

trimer formation

PDI catalyzed

formation of intermolecular disulfides

triple helix formation

procollagen

FIG. 1. Stages in the folding of procollagen.

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NEIL

1.

BULLEID

ducing conditions, and the formation of triple helical structures was determined by resistance to proteolysis by trypsin plus chymotrypsin. The results from this work are summarized in Table I. As with smaller monomeric proteins, the type I and I1 procollagens are able to form native disulfide bonds in the absence of any enzyme catalyst in the presence of an oxidizing system. However, the process is rather slow and is greatly accelerated if PDI is present during the reaction. Significantly, the rates of trimer formation and of triple helical folding in the presence of PDI correspond to those found in the cell. The ability to measure separately the rate of trimer formation and triple helical folding made it possible to demonstrate that PDI can catalyze the former, which requires disulfide bond formation, but not the latter. Thus, PDI catalyzes the refolding of procollagen in vitro at the rate observed in vivo and the step catalyzed involves the formation of native intermolecular disulfide bonds rather than folding of the triple helix. In summary, these studies on the refolding of proteins in vitro highlight the requirement for an enzyme catalyst to accelerate the rate of native disulfide bond formation to folding rates observed in vivo. Such an en-

TABLE I Effect of Protein Disulfide-Isomerase on Disulfidr Bonding and Triple Helix Formation in Types I and I1 Procollagen“ (min) of pro-y chain formation

1,

Oxidizing system”

GSH/GSSG Type 1 procollagen 10 p M , 1.0 p M 1.0 mM, 0.1 m M 20 mM, 2.0 mM 2.0 mM, 2.0 mM 0.0 mM, 5.0 mM Type I1 procollagen 10 pm, 1.0 p M 1.0 mM, 0.1 mM ~

11 (min) of

triple helix formation

Lag time (min) between pro-y and triple helix formation

Without PDI

With PDI

Without PDI

With PDI

20.6 11.2 21.1 10.7 10.9

3.7 3.7 6.1 3.7 ND

26.0 17.1 N D‘ ND ND

9.4 9.4 ND ND ND

5.4-5.7 5.7-5.9

88.2 89.1

11.6 11.6

ND ND

17.1 17.3

5.5 5.7

~

Reproduced with permission from Koivu and Myllyla (1987). Reoxidation of pro-a chains of types I and I1 procollagen into triple helical procollagen was studied using various concentrations of reduced (GSH) and oxidized (GSSG) glutathione as an oxidizing system in the reaction mixture. Pure protein disulfide-isomerase (PDI; 5ng) was used in the reaction. ND, Not determined.

PROTEIN DISULFIDE-ISOMERASE

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zyme catalyst has now been identified and has been purified to homogeneity from bovine liver (Carmicheal e l al., 1977; Lambert and Freedman, 1983).

B . Enzymatic Properties and Mechanism The availability of purified enzyme that is active in the catalysis of disulfide interchange has enabled studies to be made on the enzyme mechanism. T h e overall reaction catalyzed by PDI is an exchange between a thiol group and a protein disulfide (Freedman et al., 1984). The result of this reaction depends on the protein substrate and the redox conditions of the reaction. With a reduced protein and in the presence of a mild oxidant, PDI catalyzes the formation of native disulfide bonds. This may be a consequence of PDI accelerating the steps in the folding pathway of the protein substrate, which involve thiol/protein disulfide interchange. With an oxidized protein substrate containing nonnative bonds and in mildly reducing conditions the enzyme catalyzes rearrangement of the disulfide bonds. However, in more reducing conditions the overall effect is a net reduction of protein disulfide bonds. The activity of PDI involves an exchange between a thiol group and a protein disulfide; therefore, the mechanism of action is likely to involve an essential thiol group at the active site. This was suggested in early work on unfractionated preparations (Ramakrishna Kurup et al., 1966) and in later work on the purified enzyme (Carmicheal et al., 1979; Hillson and Freedman, 1980) that showed that the activity could be inhibited by low concentrations of Cd2+ and AsO,-. Moreover, by inactivating the essential thiol group with alkylating agents, it has been shown that there are t w o reactive groups per polypeptide, which means there are four reactive groups per dimeric enzyme (Hawkins and Freedman, 1991). The purified enzyme is not inactivated by alkylating agents unless it is preincubated with a reducing agent, suggesting that the reactive thiol group exists in the oxidized disulfide state. Thus, the active site of PDI contains an active cysteine residue, which in the purified enzyme is intramolecularly disulfide bonded to a second cysteine. The amino acid sequence of the protein, which can be inferred from the published cDNA sequence (Edman et al., 1985), reveals that each PDI polypeptide contains two domains that are closely homologous to thioredoxin. This enzyme has been characterized in detail (Holmgren, 1985) and it has been shown that the cysteine residues in the sequence WCGPCK (residues 3 1-36) act as the reactive dithiol. Chemical modification studies have shown that the enzyme, like PDI, is inactivated by alkylation at neutral pH, and that only Cys-32 is alkylated (Kallis and

130

N E I L J. BULLEID

Holmgren, 1985). The corresponding active site sequence in PDI is slightly different in that it has a histidine residue in place of the proline, i.e., WCGHCK. The reactive thiol group in both thioredoxin and PDI has a pKof 6.7 (Hawkins and Freedman, 1991),which is low for a cysteine side chain and means that at neutral pH it will exist as a nucleophilic thiolate (-S-) group. The redox potential of the two enzymes does, however, differ: -270 mV for thioredoxin (Holmgren, 1968) and - 110 mV for PDI (Hawkins et cil., 1991).Thus. PDI is a stronger oxidant and a weaker reductant than thioredoxin. Interestingly, changing the active site of thioredoxin so that it is identical to that of PDI, by mutation of the proline to a histidine, increased the redox potential of the mutant enzyme to -235 mV (Krause et al., 1991), indicating a role for this histidine in determining the redox potential of PDI. T h e redox potential calculated for PDI is such that for it to function in uiuo in the formation of native disulfide bonds, it must be present in a more oxidizing environment than that found in the cytoplasm. T h e effective redox potential present in the EK is unknown, but for secretory proteins to be able to form disulfide bonds it must be more oxidizing than the cytoplasm. How this oxidizing environment is maintained is unclear but it is interesting to note that PDI has recently been shown to reduce hydroascorbate to ascorbic acid in the presence of reduced glutathione (Wells el al., 1990). I t has yet to be shown whether this activity has any functional significance in vim. Studies carried out using cell-free systems reveal that for disulfide bonds to form in such a system, the endogenous thiol content must be altered by the addition of oxidized glutathione (Scheele and Jacoby, 1982). Thus, a system must be present within the EK for maintaining the redox conditions necessary for disulfide bond formation. It is also clear that microsonial vesicles must be present in a cell-free system for disulfide bonds to form in nascent secretory proteins (Bulleid and Freedman, 1988a), highlighting the compartmentalization of this process. Along with the active site containing the reactive dithiol, there also seems to be a binding site for peptides (Morjana and Gilbert, 1991). Whether this is at the active site or elsewhere is unclear, but due to the non-sequence-specific nature of this interaction, it seems likely that PDI recognizes and can bind to the polypeptide backbone. In this respect PDI could be acting as a molecular chaperone in a way similar to other ER resident proteins, such as immunoglobulin heavy chain binding protein (see later). The significance of this peptide binding and its relationship to the catalysis of native disulfide bonds are unclear, but it is tempting to speculate that these two properties are connected.

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131

I l l . CELLULAR PROPERTIES OF PROTEIN DISULFIDE-ISOMERASE A . Subcellulur Lorulizutioii

T h e original experiments on PDI activity were carried o u t with microsoma1 membranes, suggesting the localization ofthis protein in the endoplasmic reticulum. This has since been confirmed both by subcellular localization studies (Lambert and Freedman, 1985)and by immunochemical methods (Koch, 1987). However, these studies revealed that PDI does not behave like a typical ER membrane protein. Indeed, for some time there was a question as to whether there was more than one form of PDI due to the presence of this protein in the cytosol. This has now been discounted by studies on the latency of PDI that show that if microsomes are prepared carefully then the activity is entirely latent (Lambert and Freedman, 1985). Thus, although PDI exists as an ER protein, it is easily released from microsomes by freeze thawing, mild alkaline treatment, detergents, or sonication, suggesting that PDI is either a soluble protein or is only loosely associated with the EK membrane. An independent approach by Koch and associates (Koch, 1987; Koch el al., 1988) identified a group of proteins present within the ER lumen; the proteins shared a number of properties, including an acidic p l and the ability to bind calcium to varying degrees. These include five major proteins, which have been isolated and characterized to be of M,. 94,000, 76,000,60,000,58,000, and 55,000. T h e M,. 94,000 and 76,000 proteins are related to the family of heat-shock proteins, HSP70 and HSPSO, respectively. The M , 94,000 protein has been termed endoplasmin and is an abundant glycoprotein in the ER (Koch et al., 1986).T h e M, 76,000 protein is identical to the glucose-regulated protein GRP78, which was separately identified as immunoglobulin heavy chain binding protein (BiP) (Munro and Pelham, 1986). This protein has become the subject of intense research due to its role in the translocation of newly synthesized proteins across the ER membrane (Vogel et al., 1990) and in the folding and assembly of native multimeric proteins (Gething et al., 1986). The M, 58,000 protein is PDI and the M,. 55,000 protein is a calcium-binding protein, named calreticulin (CRP55), present in both the ER and the sarcoplasmic reticulum (Smith and Koch, 1989). Koch argues that the interior volume of the ER has, like the cytoplasm, a high concentration of resident proteins ( > l o 0 nig/nil) and should be regarded as a distinct subcellular environment, i.e., the reticuloplasni. with the proteins that reside within this environment being termed reticuloplasmins. The concentration of PDI within the ER is also likely to be high (>10 mg/nil) in

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cells active in secretion. Thus, nascent secretory proteins entering the lumen of the ER are entering an environment where they are in relatively low concentration compared to the concentration of PDI and other EK resident proteins. It is worthwhile considering this fact when evaluating results from folding studies obtained with the enzyme in vilro, where the denatured protein substrate is in vast excess of the enzyme. The cDNA sequence of PDI and other reticuloplasmins reveals that they all contain the C-terminal tetrapeptide KDEL. This sequence has been demonstrated to be necessary and sufficient for retention of proteins in the ER (Munro and Pelham, 1987). Thus, proteins that are normally secreted are retained within the ER if this tetrapeptide is fused to their C terminus. Conversely, if the KDEL sequence is deleted from PDI or other reticuloplasmins, then they are secreted. Antibodies to PDI have been used to raise antiidiotypic antibodies (Vaux et al., 1990), the idea being that antibodies recognizing anti-KDEL antibodies may recognize the receptor for KDEL. These antibodies react with a protein located in transitional vesicles between the ER and the Golgi apparatus. Any EK resident proteins that leave the ER are thought to interact with this receptor via the KDEL sequence and are recycled to the ER. Saturating this system with peptides containing the KDEL sequence causes secretion of PDI (D. Vaux, personal communication, 1991). This ability to saturate the KDEL receptor may explain the observation that PDI and other reticuloplasmins have been identified at the plasma membrane and in other organelles along the secretory pathway in rat exocrine pancreas cells (Yoshimori et al., 1990). B . Factors Affecting Expression of Enzyme

As PDI is involved in the biosynthesis of secretory proteins, it is not surprising that the levels of the enzyme are highest in tissues that are active in secretion e.g., liver and pancreas. In lymphocyte or lymphomaderived cell lines that are synthesizing a single major disulfide-bonded protein, immunoglobulin, the levels of PDI correlate with the secretion of this protein (Roth and Koshland, 1981). A similar observation can be made with fibroblast cell lines wherein the rates of synthesis of procollagen correlate with the levels of PDI (Myllyla et a/., 1983). There are also changes during development and after lipopolysaccharide-induced differentiation of lymphocytes (Paver et al., 1989a).All these observations suggest that expression of PDI is regulated and controlled by the synthesis of disulfide-bonded proteins. The enzyme is constitutively expressed in most cell types, but its expression can be induced by an increase in the synthesis of secretory proteins.

PROTEIN DISULFIDE-ISOMERASE

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A mechanism must, therefore, exist to monitor events taking place in the ER and for information to be relayed across the lipid bilayer to the nucleus. As yet no such mechanism has been identified. A genomic clone has, however, been isolated and analysis of its 5’ noncoding region reveals the presence of a number of transcription control elements, including a TATA box, six CCAAT boxes, two highly GC-rich segments, and several possible binding sites for transcription factor Spl (Tasanen et al., 1988).

IV. ROLEOF PROTEIN DISULFIDE-ISOMERASE I N INTRACELLULAR PROTEIN FOLDING Although the results described above demonstrate that PDI has the ability to catalyze the formation of disulfde bonds in denatured substrates, is present in the correct subcellular compartment for disulfide bond formation, and is more abundant in cells that are active in secretion, they d o not prove that PDI has these functions in the cell. T h e evidence is circumstantial, with more direct evidence being difficult to obtain due to the lack of specific inhibitors for this enzyme and the slow turnover of the protein (Ohba et al., 1981),which has prevented its depletion from the cell. One study that demonstrates a direct association of PDI with newly synthesized disulfide-bonded proteins in the cell has been carried out by Roth and Pierce (1987). In this study, intact lymphocytes were treated with a chemically cleavable, bifunctional cross-linking agent, lysed, and the lysates immunoprecipitated with antibodies to either PDI or immunoglobulins. PDI was found to be cross-linked to inimunoglobulins and when cells were depleted of intracellular reduced glutathione, the extent of this cross-linking increased. Thus, PDI must be in close proximity to immunoglobulins as they are being synthesized.

Use of Cell-Free Systems to Study Disuljide Bond Formation at Synthesis One way to study the role of PDI in native disulfide bond formation is to study this process using a cell-free system. T h e use ofcell-free systems to study the initial stages in the synthesis and posttranslational modification of secretory and cell-surface proteins is well established. The most popular system employs a message-dependent rabbit reticulocyte lysate that has been supplemented with dog pancreas microsomal vesicles. This system will translate exogenously added mRNA with the synthesized protein being cotranslationally translocated to the interior of the microsoma1 vesicle where processing occurs to remove the signal peptide. If the protein contains potential N-linked glycosylation sites, they may be glycosylated with high mannose, endoglycosylase H-sensitive oligosaccha-

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rides. However, folding and disulfide bond formation are not normally monitored for two reasons: (1) thiol reductants (e.g., dithiothreitol) are normally added to the reticulocyte lysate, since several components are susceptible to oxidative inactivation, and (2) the analysis of radiolabeled translation products, to determine whether signal peptide cleavage and glycosylation have occurred, is normally carried out by SDS-PAGE in the presence of reducing agents, to obtain good resolution and accurate determination of protein relative molecular weight. Hence, translation is carried out under conditions in which disulfide bonds are unlikely to form, and analysis of translation products is performed under conditions in which any disulfide bonds would be broken. Several groups have previously demonstrated that proteins synthesized in a cell-free system can fold to form molecules that have the physical properties of the native enzymes (Scheele and Jacoby, 1982; Hille et al., 1989; Sonderfeld-Fresco and Proia, 1988; Sawyer and Doyle, 1990). This is true for cytosolic proteins and secretory proteins (translated in the presence of microsomal membranes) that either do or d o not contain disulfide bonds. Proteins lacking disulfide bonds can fold under standard conditions for in vitro translation. However, to enable secretory proteins to form disulfides in a cell-free system requires alteration of the translation conditions, either by preparing lysates in the absence of exogenously added reducing agents (Bulleid and Freedman, 1988a), o r by adding an oxidant such as oxidized glutathione (GSSG) to reticulocyte lysates that contain added reducing agents (Scheele and Jacoby, 1982; Kaderbhai and Austen, 1985). The formation of disulfide bonds in proteins synthesized in vitro can be followed by measuring enzymatic activity or by an increased mobility compared to the reduced protein during SDS-PAGE. This increased mobility arises from the fact that, as disulfide-bonded proteins are intramolecularly cross-linked, they form a more compact structure and occupy a smaller hydrodynamic volume compared to the reduced protein (Goldenberg and Creighton, 1984). An illustration of this increase in mobility is shown in Fig. 2. Here the mRNA for preprolactin was translated in a cell-free system optimized for the formation of disulfide bonds, and then analyzed by SDS-PAGE. The translocated protein forms disulfide bonds under these conditions whereas the protein synthesized under the same conditions but in the absence of microsomal membranes does not form disulfide bonds. Thus the nascent protein must be translocated into microsomal vesicles for disulfide bond formation to occur. Microsomal membranes may be depleted of PDI by a number ofprocedures that exploit the solubility of the enzyme. These methods result in the permeabilzation of the membranes either by exposing the membranes to alkaline pH (Paver et al., 1989b), by treating with detergents

PROTEIN DISULFIDE-ISOMERASE

LANE...

1

2

microsornes.. . reduced... non-reduced...

+

+ +

135

+ +

+

FIG. 2. Cell-free translation of preprolactin mRNA under conditions favoring the formation of disulfide bonds. Translation products were separated by SDS-PAGE after being either reduced and carboxamidomethylated (lanes 1 and 2) or carboxarnidomethylated only (lanes 3 and 4).Lanes 1 and 3, products of translation in the absence of microsornal vesicles; lanes 2 and 4,products of translation in the presence of microsomal vesicles.

such as saponin (Bulleid and Freedman, IYYO), or by sonication. An SDS-PAGE profile of these various preparations is shown in Fig. 3. PDI is not the only protein to be depleted from these microsomes. Other proteins are extracted, including the reticuloplasmins endoplasmin and BiP. However, proteins necessary for translocation are retained within the membranes. Most methods will remove >YO56 ofthe PDI from microsoma1 preparations as judged by enzyme-linked imniunosorbent assay (ELISA). The PDI-depleted microsomes are still capable ofcotranslationally translocating nascent proteins. The ability to deplete microsomal membranes of PDI has enabled the study of the role of this enzyme in the formation of disulfide bonds at

136

NEIL J. BULLEID

LANE

1

2

3

4

5

6

97.4 68 -

PDI

+ 43 -

14.3 FIG. 3. Removal of PDI from dog pancreas microsomes. Dog pancreas microsomes after the various treatments were isolated by ultracentrifugation and separated on a 12% polyacrylamide gel in the presence of SDS. The gel was loaded as follows: lane 1. molecularweight markers; lane 2, untreated microsomes; lane 3, pH 9-washed microsomes; lane 4, saponin-washed ( I 96,w/v) microsomes; lane 5 , sonicated microsonies; lane 6, pH 9-washed microsomes reconstituted with purified PDI.

PROTEIN DISULFIDE-ISOMERASE

137

synthesis. We used the cereal storage protein, y-gliadin, as a model protein in this study due to the availability of the cloned cDNA and the difference in electrophoretic mobility between the reduced and the disulfide-bonded protein. A schematic representation of the protein is shown in Fig. 4. When this protein is synthesized in the presence of standard microsomal membranes under conditions that allow disulfide bond formation, the protein is translocated into the vesicle, where it is protected from proteolysis (lane 3, Fig. 5 ) . When the translocated product was analyzed by nonreducing SDS-PAGE, it had a greater mobility than the reduced protein, indicating the formation of intramolecular disulfide bonds (lane 4, Fig. 5 ) . To study the cotranslational formation of disulfide bonds further, time course studies were performed and translation was carried out in the presence of untreated and PDI-depleted microsomes. At various time points after translation the reaction was terminated and the translation products were treated with proteinase K to remove all products that were not translocated and segregated into the microsome vesicle interior. T h e products were then analyzed by SDS-PAGE under nonreducing conditions (Fig. 6). In the presence of native microsomes the translocated product appears as a faster migrating band compared to the reduced protein and is therefore disulfide bonded (Fig. 6a). However, in the presence of PDI-depleted microsomes there is a much lower yield of the disulfide-bonded protein, with most of the translocated protein comigrating with the reduced protein (Fig. 6b). Interestingly some bands appear with an intermediate mobility between the reduced and the fully disulfide-bonded protein. These are probably products that have formed incorrect or “nonnative” disulfide bonds. Thus the PDIdeficient microsomes are clearly defective in carrying out cotranslational disulfide bond formation. The PDI-depleted microsomes can be reconstituted with the purified enzyme by treating the microsomes with alkaline pH buffer containing high concentrations of PDI, reflecting the level of PDI in the microsomal lumen. After titrating the pH to 7.5 the microsomes are reisolated by centrifugation. When the y-gliadin mRNA was translated in the presence of PDI-reconstituted microsomes, the defect in disulfide bond formation observed with PDI-depleted microsomes was reversed (Fig. 6c). Control

138

NEIL

1

2

1. BULLELD

3

4

5

6

FIG. 5 . Cell-free translation of y-gliadin rnRNA. The y-gliadin m K N A was translated in the absence (lanes 1 and 5 ) or presence (lanes 2-4 and 6) of dog pancreas microsornes. Translation products were separated by SDS-PAGE under reducing conditions (lanes 1-3, 5 . and 6) and under nonreducing conditions (lane 4). Lanes I and 5 are products o f translation in the absence of rnicrosomal vesicles; lanes 3.4, and 6 are products oftranslation in the presence of rnicrosoinal vesicles; translation products of lanes 3 and 4 were treated with proteinase K; translation products of lane 6 were treated with I % (v/v) Triton X-100 and proteinase K.

experiments showed that activity could not be restored simply by the addition of a redox couple such as D T T and GSSG. This demonstrated that the ability of microsomes to catalyze efficiently the formation of disulfide bonds at synthesis is dependent on the presence of PDI at physiological concentrations within the microsomal lumen (Bulleid and Freedman, 198813). By using this approach we were able to show that, not only can PDI catalyze the rearrangement of disulfide bonds in denatured substrates, but PDI is also required for the formation of disulfide bonds in protein substrates as they enter the lumen of the microsomal vesicle.

139

PROTEIN DISULFIDE-ISOMEKASE

a Lane Time (mins)

1

2

3

4

5

6

5

10

20

30

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1

2

3

4

5

(mins)

5

10

20

30

40

6

C

Lane Time (mins)

1

2

3

4

5

5

10

20

30

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FIG.6. Defective cotranslational formation of disulfide bonds in PD1-deficient microsomes. Time course of disulfide bond formation in newly synthesized protein in the presence of (a) native microsomes; (b) pH 9-washed microsomes; and ( c ) pH Y-washed microsomes reconstituted with purified PDI.

V.

MULTIFUNCTIONALITY OF PROTEINDISULFIDE-ISOMERASE

A . Sequence Information PDI has been cloned and sequenced from a number of mammalian species, which has allowed comparisons to be made with other proteins and conserved regions within the protein to be identified. An amino acid

140

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sequence comparison from four mammalian species (Fig. 7) reveals the level of conservation between these species. The first level of‘ homology involves sequences within the PDI polypeptide that are similar to sequences found in other proteins. The polypeptide can be divided up into t w o pairs of internal repeats (Fig. 8).T h e a and a’ regions are homologous to thioredoxin and contain the “active site” sequence GGHC. The amino acid sequence around this site is identical in these two regions of the polypeptide and in all species studied so far. These regions are also homologous to two regions present in phosphoinositide-specific phospholipase C; the rest of the molecule shows no obvious homology with

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PDI (Bennett el al., 1988). This active site sequence has also been identified in a developmentally regulated gene from trypanosomes (Hsu et al., 1989) and in another ER protein called ERp72, which contains three copies of this sequence (Mazzarella et al., 1990). There is also homology between 43 amino acids at the carboxy-terminal side of region a and to a segment of the estrogen-binding domain of the estrogen receptor (Tsibris el al., 1989). Thus, within the sequence of PDI there are several regions of homology with other proteins. The homology of PDI with other proteins does not, however, end there. It is now clear that the PDI polypeptide has a number of functions within the cell that appear to be distinct from its role in catalyzing disulfide bond formation (Freedman, 1989). It is this diversity of function that has aroused great interest in recent years, with different functions for PDI seemingly being discovered annually. B . Identity With 0 Subunit of' Prolyl4-Hvdroxvlase

The first evidence for PDI having a different role came from Pihlajaniemi et al. (1987)during their study ofthe enzyme prolyl4-hydroxylase (P4H). This enzyme is localized in the ER and catalyzes the hydroxylation of proline residues in nascent procollagen polypeptides. T h e enzyme is a tetramer composed of two a and two subunits (Kivirikko et al., 1989). Antibodies raised against the holoenzyme were used to screen a human hgtl 1 expression library and clones were isolated that coded for the /? subunit. After sequencing it was revealed that the human /3 subunit was 94% identical with the rat PDI sequence. Southern blot analysis identified

142

NEIL J . BULLEID

only one copy of the gene in the human genome, suggesting that PDI and the j3 subunit of P4H are identical. This explains one observation that has puzzled researchers in the P4H field for some time, that is, the presence of a large excess of the P subunit. in the ER; it is now clear that this excess is PDI. This does, however, raise another question: What role does PDI have as part of the P4H tetramer? It is known that the a subunit is the catalytically active subunit and indeed the enzyme isolated from the alga Chlamydomonas reinhardii shares the same catalytic mechanism yet is monomeric and contains no protein disulfide isomerase activity (Myllyla et al., 1989). Regions within this protein could, however, be fulfilling the same role as the j3 subunit in the vertebrate enzyme. Thus the active site dithioUdisulfide couples of PDI could be involved in the various redox steps in the hydroxylation reaction. Alternatively, PDI may function to ensure that the a subunit is retained within the ER. As this subunit contains no obvious retention signal at its carboxy terminus (Bassuk et al., 1989), its retention in the ER may be dependent on its association with PDI. Another more intriguing role for PDI in the tetramer is that of a molecular support, preventing the a subunit from forming enzymatically inactive aggregates. Studies on the purified enzyme have shown that the a subunit is insoluble when dissociated from the p subunit (Tuderman et al., 1975). This insolubility prevents the renaturation of the active enzyme from the isolated subunits. Thus, in the cell PDI may bind to nascent a-subunit chains, thus preventing their aggregation and allowing them to fold to form the catalytically active subunit. C . Identity with Subunit of Triglvceride Transfer Protein

This idea that PDI may play a role in preventing the aggregation of certain polypeptides may also explain its role as a subunit in the triglyceride transfer protein (MTP). This protein catalyzes the transfer of triglyceride between membranes and may play a role in the biogenesis of plasma lipoproteins. T h e MTP complex consists of two polypeptides with apparent molecular weights of 58,000 and 88,000. T h e 58,000 molecularweight component has been identified as PDI by amino-terminal sequence analysis, peptide mapping, immunochemical characterization, and the expression of protein disulfide isomerase activity following dissociation of the two subunits (Wetterau et al., 1990, l99la). Like with P4H, PDI may be functioning to retain the 88-kDa subunit in the ER or by preventing the aggregation of this polypeptide. When the complex is dissociated, the 88-kDa subunit forms insoluble aggregates, preventing

PROTEIN DISULFIDE-ISOMERASE

143

the reassociation ofthe two subunits (Wetterau et al., 1991b). It therefore appears that for PDI to associate with the 88-kDa subunit it must do so prior to its self-association. This may mean that PDI must bind to the nascent chain during its translocation to the lumen of the ER. Thus for both the enzyme complexes MTP and P4H, the role of PDI may be to stabilize its associated subunit and prevent the formation of insoluble aggregates. D . Role of Protein Disulfide-Isomerase in N-Linked Glycosylation

The isolation and characterization of the enzyme responsible for the transfer of oligosaccharide from dolichol phosphate to the growing polypeptide chain has proved difficult due to loss of activity of the enzyme on membrane solubilization (Kaplan et al., 1987). However, insights into the location and specificity of this reaction have led to the use of photoaffinity probes modeled on the glycosylation recognition site Asn-X-Ser/ T h r (Welphy el al., 1985). These probes led to the identification and cloning of a 57-kDa ER lumenal protein, called glycosylation site binding protein (GSBP), which is labeled specifically with these probes (Kaplan et al., 1988; Geetha-Habib et al., 1988). This protein has a high degree of sequence identity with and is immunologically indistinguishable from PDI. T o determine whether PDI was involved in the transfer of oligosaccharides from dolichol phosphate to the nascent polypeptide chain, we studied the cotranslational glycosylation of interferon-y( IFN-y). When this protein is translated in a cell-free translation-translocation system containing untreated microsomal membranes, it is efficiently glycosylated, with both single- and double-glycosylated polypeptides being produced. In the presence of PDI-depleted microsomes, the synthesized protein was still glycosylated, thus demonstrating that PDI is not required directly for the transfer of oligosaccharides to the nascent polypeptide chain (Fig. 9). This has been confirmed by Lennarz and co-workers, who found that microsomes depleted of PDI were still capable of oligosaccharide transferase activity (Noiva et al., 1991). In our study, however, the glycosylation products were of a higher molecular weight than those synthesized in the presence of untreated microsomes (Bulleid and Freedman, 1990). This is probably due to either inactivation or removal of the enzyme glucosidase 11, which trims terminal glucose residues from the oligosaccharide side chain. Although PDI is not required directly for N-linked glycosylation, it may indirectly determine the extent of glycosylation of secretory proteins. In

144

NEIL J . BULLEID

1

2

3

4

5

48-

30 -

double

--+

--+ 21.5 pre--IFNr-+ core + single

-

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FIG.9. Cotranslational glycosylation in the presence of PDI depleted-microsomes. Products of translation were immunoprecipitated with a specific antibody for IFN-y and were resolved by SDS-PAGE. The gel was loaded as follows: lane 1, molecular-weight markers; lane 2, translation products in the absence of microsomes; lane 3, translation products in the presence of native microsomes; lane 4, as lane 3 but treated posttranslationally with N glycanase to remove oligosaccharide side chains; lane 5. translation products synthesized in the presence of PDI-depleted microsomes (saponin washed). The inferred products of translation are labeled as follows: x, product arising from incorrect initiation oftranslation; core, unglycosylated core polypeptide; singly and doubly glycosylated products as indicated.

PROTEIN DISULFIDE-ISOMERASE

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a recent study on tissue-type plasminogen activator (t-PA) we have shown that the number of oligosaccharide side chains added to the protein during its synthesis is dependent on the folding of the polypeptide. Thus molecules of t-PA synthesized in vitro under the appropriate conditions are enzymatically active, are responsive to natural activators and inhibitors, and are glycosylated in a pattern identical to that of the protein synthesized in vivo, with two glycoforms being produced differing in their extent of N-linked glycosylation. However, when t-PA is synthesized under conditions that prevent disulfide formation, the protein is enzymatically active and glycosylated to yield a single glycoform (Fig. 10). I t appears that the folding of a polypeptide, and therefore the Levels of enzymes that facilitate protein folding, may affect the extent of glycosylation of secretory proteins.

E . Protein Disulfide-Isomerase as Thyroid K-lormone-Binding Hormone and Iodothyronine 5’-Monodeiodinase The functions listed above all involve modification of nascent polypeptide chains. PDI has also been identified as having two further quite distinct functions. T h e first of these came to light when the gene coding for a triiodo-L-thyronine-binding protein (TSBP) was sequenced from human and bovine sources (Cheng et al., 1987; Yamauchi et al., 1987), revealing its identity with PDI. T h e binding protein was identified and purified by its labeling with a radioiodinated bromoacyl derivative of the hormone from membrane fractions derived from tissue culture cells. This protein was found to be present exclusively in the ER and nuclear envelope and is, therefore, distinct from the previously identified plasma membrane binding protein. T h e precise cellular role of TSBP has not been established but it is interesting to note that PDI does contain a sequence that is homologous to the E domain of the estrogen receptor (see above). However, until a positive link between PDI and thyroid hormone function is established, the identification of PDI as a TSBP must be treated with caution. An alternative explanation for this identification could come from the fact that PDI does have a high nonspecific binding toward alkylating reagents such as haloacetyl derivatives. The second alternative function of PDI again comes from sequence analysis. This time a clone isolated as a iodothyronine 5’-monodeiodinase (5’-MD, thyroxine deiodinase) from rat codes for a protein that is identical to PDI in all but two residues (Boado et al., 1988). T h e clone was isolated by screening a library with polyclonal antibodies raised against rat liver microsomal proteins. Clones coding for 5’-MD were selected by

146

NEIL J . BULLEID

1 97

2

3

I

68

43

29

18

FIG. 10. Cell-free synthesis of t-PA glycotorms. The mKNA coding for t-PA was translated in a rabbit reticulocyte lysate in the presence of dog pancreas iiiicrosomes. Microsomes were isolated posttranslationally and the translocated. glycosylated products were separated by SDS-PAGE. Translation was carried out under conditions that either prevented (lane 2 ) or allowed (lane 3) proper folding o f t h e t-PA molecule. yielding enzvniaticallv active protein that was sensitive to natural inhibitors and stimulators.

PROTEIN DISULFIDE-ISOMERASE

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an enzyme-binding inhibition assay. They also showed that antibodies raised against PDI were capable of inhibiting 40% of 5’-MD activity in liver microsomal proteins. Their clone obviously codes for PDI but there are several lines of evidence to question whether it codes for 5’-MD. The original screening procedure did not use antibodies to purified 5’-MD, which has previously been shown to be a basic, low-abundance protein in contrast to PDI. A study by Schoenmakers and associates (Schoenmakers el al., 1989) shows that two microsomal proteins with relative molecular weights of 28,000 and 56,000 can be labeled with radioiodinated bromoacetyl-triiodothyronine. Further characterization of these proteins demonstrated that the 28-kDa protein has 5’-MD activity wherease the 56-kDa protein does not. It would therefore seem that the biochemical evidence rules out a role for PDI as a 5’-MD. VI. CONCLUSIONS

Over the years there has been an explosion of interest in the enzyme protein disulfide-isomerase. This began with the biochemical characterization of the purified enzyme, which has provided valuable information regarding its molecular properties and catalytic mechanism. By the use of classical resolution and reconstitution experiments, a role for PDI in the formation of disulfide bonds in newly synthesized proteins has been demonstrated. Since the publication of the first sequence of PDI in 1985, several clones have been isolated from a variety of sources, including the sequence of a PDI from the yeast Saccharomyces cerevisiae (Farquhar et al., 1991; LaMantia et al., 1991). The cloning of yeast PDI will allow the genetic manipulation of this enzyme and will provide a useful tool in clearly defining the role of this protein in v i v a The identification of PDI as a multifunctional polypeptide fulfilling several roles in the cell has fueled further interest in this protein. Whether all of these activities are carried out by PDI within the cell will need to be confirmed, but it seems clear that not only is PDI involved in the formation of disulfide bonds at synthesis, but it also may play a role as a supporting subunit for polypeptides that would otherwise be insoluble. Whether this latter role is a serendipitous consequence of the ability of the protein to bind to the polypeptide backbone is unclear, but the interaction of PDI with these proteins is essential for their activity. By studying the role of PDI in facilitating the folding and maturation of nascent secretory proteins, we will be able to obtain a clearer picture of the events that take place within the ER both co- and posttranslationally prior to the exit of the protein to the Golgi appartus.

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ACKNOWLEDGMENTS This work was supported by the Royal Society, the Wellcome Trust, and the AFRC. I would like to thank Robert Freedman for his continuing support and encouragement. and David John and Rachel Middleton for their critical reading of the manuscript. Neil Bulleid is a Royal Society University Research Fellow.

REFERENCES Anfinsen, C. B. (1973). Scieticr 181, 223-230. Bassuk. J. A., Kao, W. W.-Y., Herzer, P.. Kedersha, N. L., Sever, J., DeMartino. ,I. A,, Daugherty. B. L., Mark, G. E.. 111. and Berg, R. A. (1989). Ptoc. Narl. Acrid. Sci. U.S.A. 86, 7382-7386. Bennett. C. F.. Balcarek, J. M.,Varrichio, A., and Crooke, S. 'I. (1988). Na1io.r (Lotidoti) 334,268-270. Boado, R.J.. Campbell, D. A., and Chopra, 1.1. (1988).Bioclirm. Biopliys. Krs. Coinmuti. 155, 1297-1304. Bulleid, N . J., and Freedman, R. B. ( 1988a). Biocheiri. J . 254, 805-810. Bulleid. N. J., and Freedman, R. B. (1988b). Nulute ( L o d o t i ) 335, 649-651. Bulleid, N. J., and Freedman, R. B. (1990). E M E O J . 9, 3527-3532. Carmicheal, D. F.. Morin, J . E., and Dixon, J. E. (1977).J. Biol. C h i . 252, 7163-7167. CarmicheaL D. F., Keefe. M.. Pace, M., and Dixon, J. E. (1979). ,J. B i d . C h u m . 254, 8386-8390. Cheng, S.-Y., Gong, Q.-H., Parkison, C., Robinson, E. A., Apella, E., Merlino. G . T., and Pastan, I. (1987).J. Bid. Cliem. 262, 11221-1 1227. Creighton, T. E., Hillson, D. A., and Freedman, R. B. (lYXO).J. Mol. B i d . 142, 43-62. Edman, J. C., Ellis, L., Blacher, R. W., Roth, R. A.. and Rutter, W.,]. (1985).Nalutr ( L o d o t i ) 317,267-270. Epstein. C. J., Goldberger, R. F., and Anfinsen. C. B. (1963). Cold Spvirig Hotbot Svtnp. Quanl. Eiol. 28, 439-449. Farquhar. R., Honey, N.. Murrant. S. J . , Bossier, P., Schultz, L., Montgomery, D., Ellis, R. W., Freedman, R. B., and Tuite, M. S. (1991). Gene 108, 81-89. Freedman, R. B. (1989). Cell (Cambridge, Mass.) 57, 1069-3072. Freedman, R. B.. Brockway, B. E., and Lambert, N. (1984).Bioclirm. Soc. Ttutis. 12, 929-932. Geetha-Habib, M., Noiva, R., Kaplan, K. A., and Lennarz, W. J. (1988).Crll (Cotribridge, Mas.) 54, 1053-1060. Gething. M. J., McCammon, K., and Sambrook, J. (1986). Cell (CainDtidgr, Ma.cs.) 46, 939-950. Goldberger, R. F., Epstein, C. ,I., and Anfinsen, C. B. (IY63).J. Biol. Chrtri. 238,628-635. Goldenberg, D. P.. and Creighton, T. E. (1984). Anal. Biochrm. 138, 1-18. Hawkins, H. C., and Freedman, R. B. (1991). Eiochem.J. 275, 335-339. Hawkins, H. C., de Nardi, M., and Freedman, R. B. (1991). Bioc/wm. J. 275, 341-348. Hille, A., Waheed, A., and von Figura, K. (1989).J. Eiol. Clirm. 264, 13460-13467. Hillson, D. A.. and Freedman, R. B. (1980). Biocliem. J. 191,377-388. Holmgren, A. (1968). Eur. J . Biocliem. 6,475-484. Holmgren, A. (1985). Annu. Rev. Biocliem. 54, 237-27 I . Hsu, M. P., Muhich. M. L.. and Boothroyd. J . C . (1989). Bioclwmi.sliy 28, 6440-6446. Kaderbhai, M. A., and Austen, B. M. (1985). Eur. J. Biochrm. 153, 167-178. Kallis, G. B.. and Holmgren, A. (1985).J. B i d . Chew. 255, 10261-10265. Kaplan, H. A., We1phy.J. K.,and Lennarz, W.J. (lY87).Biocliim.Biophy. Acta906, 161-173.

PROTEIN DISULFIDE-ISOMERASE

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Kaplan, H. A., Naider, F., and Lennarz, W. J. (1988).J. Bid. Chem. 263, 7814-7820. Kivirikko, K. I., Myllyla, R., and Pihlajaniemi, T . (1989). FASEBJ. 3, 1609-1617. Koch. G. L. E. (1987).J. Cell Sci. 87, 491-492. Koch, G. L. E., Smith, M., Macer. D. R. J., Webster, P., and Mortara, R. (1986).J. Cell Sci. 86,217-232. Koch, G. L. E., Macer, D. R. J., and Wooding, F. B. P. (1988).J . Cell Sci. 90, 485-491. Koivu, J.. and Myllyla. R. (1987).J. B i d . Chem. 262, 6159-6154. Krause, G., Lundstrom, J., Barea, J. L., Pueyo de la Cuesta, C., and Holmgren, A. (1991). J . B i d . Chem. 266, 9494-9500. Kreis, M., Shewry, P. R., Forde, B. G., Forde, J.. and Miflin, B. J . (1985).Oxford Suw. Planf Cell Mol. B i d . 2, 253-317. LaMantia, M.-L., Miura, T.. Tachikawa, H., Kaplan, H. A., Lennarz, W. J., and Mizunaga, T. (1991). Proc. Null. Acad. Sci. U.S.A. 88, 4453-4457. Lambert, N., and Freedman, R. B. (1983). Biochem. J . 213, 225-234. Lambert, N., and Freedman, R. B. (1985). Biochem. J . 228, 635-645. Mazzarella, R. A., Srinivasan, M., Haugejorden, S. M., and Green, M. (1990).J. B i d . Chem. 265, 1094-1 101. Morjana, N. A., and Gilbert, H. F. (1991). Biochemisfry 30, 4985-4990. Munro, S., and Pelham, H. R. B. (1986). Cell (Cambridge, Mass.) 46, 291-300. Munro, S., and Pelham, H. R. B. (1987). Cell (Cambridge, Mass.) 48, 899-907. Myllyla. R.. Koivu, J., Pihlajaniemi, T., and Kivirikko, K. 1. (1983). Eur. J . Biochem. 134, 7-11. Myllyla, R., Kaska, D. D., and Kivirikko, K. I. (1989).Biochem.J. 263, 609-61 1. Noiva, R., Kaplan, H. A., and Lennarz, W. J. (1991). Proc. Nafl. Acad. Sci. U.S.A. 88, 1986-1990. Ohba, H., Harano, T., and Omura, T. (1981). J . Biochem. (Tokyo) 89, 901-907. Parkkonen, T., Kivirikko, K. I., and Pihlajaniemi, T. (1988). Biochem. J. 256, 1005-1011. Paver, J. L., Freedman, R. B., and Parkhouse, R. M. E. (1989a). FEBS Lett. 242,357-362. Paver. J. L.. Hawkins, H. C., and Freedman, R. B. (l989b). Biochem. J . 257, 657-663. Pihlajaniemi. T., Helaakoski, T.. Tasanen, K., Myllyla, R., Huhtala, M.-L., Koivu, J., and Kivirikko, K. I. (1987). E M B O J . 6, 643-649. Ramakrishna Kurup, C. K., Raman, T. S., and Ramasarma, T. (1966).Biochim. Biophys. Acta 113,255-276. Roth. R. A., and Koshland, M. E. (1981). Biochemistry 20, 6594-6599. Roth, R. A., and Pierce, S. B. (1987). Biochemirtv 26, 4179-4182. Sawyer. J. T., and Doyle, D. (1990). Proc. Natl. Acad. Sci. U.S.A. 87, 4854-4858. Scheele, G.. and Jacoby, R. (1982).J. B i d . Chem. 257, 12277-12282. Schoenmakers, C. H. H., Pigmans, 1. G. A., Hawkins, H. C., Freedman, R. B., and Visser, T. J. (1989). Biochem. Biophy. Res. Commun. 162, 857-868. Smith, M. J., and Koch, G. L. E. (1989). E M B O J . 8, 3581-3586. Sonderfeld-Fresko, S., and Proia, R. L. (1988).J. B i d . Chem. 263, 13463-13469. Tasanen, K., Parkkonen, T., Chow, L. T., Kivirikko, K. I., and Pihlajaniemi, T. (1988). J . Biol. Chem. 263, 16218-16224. Tsibris, J. C. M., Hunt, L. T., Ballejo, G., Barker, W. C., Toney, L. J., and Spellacy, W. N. (l989).J. Biol. Chem. 264, 13967-13970. Tuderman. L., Kuuti, E.-R., and Kivirikko, K. 1. (1975). Eur. J . Biochem. 60, 399-405. Vaux, D.. Tooze, J., and Fuller, S. (1990). Nature (London) 345, 495-502. Venetianer. P., and Straub, F. B. (1963). Biochirn. Biophys. Acta 67, 166-168. Vogel, J. P., Misra, L. M., and Rose, M. D. (1990).J. CelLBiol. 110, 1885-1895. Wells, W. W., Xu, D., Yang, Y.,and Rocque, P. A. (1990).J . B i d . Chem. 265, 15361-15364.

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Welphy. J . K., Shenbagamurthi, P., Naider. F., Park, H. K., and Lennarz, W. J . (1985). J . B i d . Chrm. 260, 6459-6465. Wetterau. J. R.,Conlbs, K. A., Spinner, S. N., and Joiner. B. J . (lYYO).,/. B i d . Clietn. 265,

9800-9807. Wetterau.1. R.,Aggerbeck, L. P., Laplaud, P. M., and McLean, 1.. K. (1Y91a).Bioclmnist?~

30,4406-44 12. Wetterau, J . R..Combs, K. A., McLean, L. R.,Spinner, S. N., and Aggerbeck, L. P. (l99lb). Biochemzstiy 30, Y728-9735. Yamauchi, K.. Yaniamoto, T., Hayashi, H., Kova, S.. Takikawa, H., 'l'ovoshima, K., and Horiuchi, R. (1987).Biochrnt. Biopltvs. Ha. ~ ~ O f f I ~ t I U 146, JZ. 1485- 1492. Yoshimori, T., Semba, T., Takeinoto, H., Akagi, S., Yamanloto, A.. and Tashiro, Y. (1990). J . niol. ~ h r n t .265, 15984-15990.

SecB: A MOLECULAR CHAPERONE OF Escherichia coli PROTEIN SECRETION PATHWAY By DAVID N. COLLIER CRID, E. 1. du Pont de Nemours I Co., Wilmington, Delaware 19880

I. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . ..................................... A. Features of Presequences . . . . . . . B. Presequences Target Proteins to T r C. Components of Escherichia coli Secretion Machinery . . . . . . . . . . . . . . 111. Precursor Conformation Governing Signal Peptide Function . . . . . . A. Presequences Not Necessarily Sufficient for Translocation . . . . . . . . B. Correlation between Precursor Folding and Loss of Translocation Competence ............................ IV. SecB as Component of Secretion A. Isolation of secB Mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Phenotype of secB Mutants ............. C. SecB Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Purification of SecB ................... V. Properties of SecB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. SecB: Antifolding Activity ...................... B. SecB: Targeting Activity . . . . . . . . . . . . . . . . . . . . C. SecB: Blocking Aggregation ........................... D. Translocation Corn VI. SecB and Its Ligands Forming Isolable Complexes . . . . . . . . . . . . . . . . . . A. Interference Phenotype as Measure of SecB Binding in Vivo B. Isolation of Complexes Formed in Vivo . . . . . . . . . . . . . . . . . . . . . . . . C. Complexes Formed in Vitro . ............. VII. Nature of SecB Binding Sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Mature Moiety of Secretory Protein Sufficient to Mediate SecB Binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. SecB Binding Nonnative Proteins ........................ C. SecB Binding Positively Charged Ligands . . . . VIII. Other Chaperones and A. Genetic Selections: B. Role for GroEL/ES in Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. DnaK/DnaJ as Secretion-Related Chaperones . . . . . . . . . . . . . . . . . . . D. Trigger Factor and Role in Secretion . . . . . . . . . . . . . . . .......................... IX. Recapitulation and Speculation A. SecB as Major Secretion-Related Chaperone B. Recognition Elements as Troublemakers . . . . . . . . . . . . . . . . . . . . . . . C. Secretion of Precursors Lacking IR D. Translocation Driving Dissociation . . . . . . . . . . . . . . . . . . . . . ...................... References . . . . . . . . . . . 11.

ADVANCES IN PROTEIN CHEMISTRY, Val. 44

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152 152 153 153 154 155 155 156 157 157 158 160 162 162 162 166 168 168 169 169 170 170 171 171 174 176 180 180 182 183 184 184 184 186 187 188 189

Copyright 0 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

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I . OVERVIEW T h e molecular chaperone SecB binds a subset of Escherichia coli envelope protein precursors and promotes their efficient export from the cytoplasm by (1) maintaining them in a loosely folded or exportcompetent conformation, (2) blocking nonproductive interactions with the cytoplasmic membrane, (3) preventing aggregation, and (4)targeting them to SecA, the peripheral membrane component of the preprotein translocase. SecB is an acidic, soluble, tetramer of identical 17-kDa subunits. It comprises only 0.08% of the cytosolic proteins. The secB gene is “conditionally essential” in that secB null strains cannot grow on rich media or at subbasal levels of heat-shock protein synthesis, but can grow on minimal media. Overexpression of heat-shock proteins can partially suppress the export and growth defects in secB null strains. SecB is not a heat-shock protein, and ATP has no known effect on its function. Sequences within the mature moieties of secretory precursors are sufficient to mediate SecB binding. However, the signal peptide contributes to SecB binding by slowing the rate of precursor folding and perhaps providing an additional binding site. Although the nature of the sites to which SecB binds is not completely understood, SecB binds positively charged segments within nonnative proteins.

INTRODUCTION Compartmentalization allows cells to retain and organize the vast array of solutes and macromolecules required for life. However, life depends equally on the capacity of the cell for selective movement of molecules between distinct compartments. For example, a significant subset of proteins must be translocated from their site of synthesis in the cytoplasm, across one or more membranes, to reach their final functional destination within a topologically distinct compartment. Irrespective of the target membrane (endoplasmic reticulum, mitochondria1 outer membrane, bacterial cytoplasmic membrane, etc.), translocation generally requires that the protein destined for translocation contains a cis-acting targeting element that directs it to the proper membrane. A growing body of evidence indicates that in addition to a targeting element, membrane translocation also requires that the secretory protein assumes a loosely folded or translocation-competent state. The focus of this review is the role that SecB and other molecular chaperones play in sponsoring efficient protein secretion in E. coli. 11.

SecB: MOLECULAR CHAPERONE

153

A . Features of Presequences

Most proteins destined for translocation across membranes are synthesized as precursors that contain a cleavable, amino-terminal extension called a target peptide (for mitochondrial or chloroplast import) or signal peptide [for translocation across the endoplasmic reticulum (ER) or the bacterial cytoplasmic membrane (reviewed in Verner and Schatz, 1988; von Heijne, 1988)l. Although signal peptides of either bacterial or eukaryotic origin display little primary amino acid sequence identity, they do share three common structural features (Inouye and Halegoua, 1980; von Heijne, 1985; reviewed in Gennity et al., 1990), and are, to a degree, functionally interchangeable (Lingappa et al., 1984; Watts et al., 1983; reviewed in Briggs and Gierasch, 1986). These structural features include a hydrophilic segment at the amino terminus that contains one to three basic residues. Although not strictly required for export, a net positive charge in the hydrophilic segment is required for efficient secretion (Vlasuk et al., 1983; Puziss et al., 1989). The hydrophilic segment is followed by a sequence of 8- 15 amino acids that is characterized by a paucity of charged or hydrophilic residues. Overall hydrophobicity (Bankaitis et al., 1984; Ryan et al., 1986) and the capacity to assume an ordered secondary structure, especially a-helical (Bedouelle and Hofnung, 1981; Briggs et al., 1985; Kendall et al., 1986), are important properties of the hydrophobic core. The carboxy terminus of the signal peptide is defined by a consensus sequence that is the site of endoproteolytic cleavage by the appropriate signal peptidase (Perlman and Halvorson, 1983; von Heijne, 1984; Wu and Tokunaga, 1986). In contrast to signal peptides, target peptides contain many basic and hydroxylated residues distributed over their entire length. They are predicted to form amphiphilic p sheets or a! helices in which the charged and apolar residues reside on opposite sides of the structure (Roise et al., 1988; von Heijne, 1986). Target peptides are not a common feature of secretory proteins in bacteria. However, a single class of bacterial proteins, the integral membrane enzyme I1 components of the phosphotransferase system (sugar permeases), possesses leader sequences that resemble target peptides (Saier et al., 1988). B . Presequences Target Proteins to Translocation Apparatus

Both target peptides and signal peptides direct the precursor to the proper membrane by interaction with membrane-associated factors. In yeast, target peptides interact directly with functionally redundant receptors in the mitochondrial outer membrane (reviewed in Baker and Schatz,

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1991; Hart1 and Neupert, 1990), while targeting of most precursors to the mammalian ER is indirect via interaction of the signal peptide with the signal recognition particle (SRP), a soluble ribonucleoprotein complex (Gilmore et al., 1982; Walter and Blobel, 1982; reviewed in Walter and Lingappa, 1986). SRP also interacts with the SRP receptor, or docking protein, which is embedded in the ER membrane (Meyer et al., 1982). SRP binds to the signal peptide of a nascent precursor as it emerges from the ribosome, and depending on the experimental system employed, causes either a complete arrest in (Walter and Blobel, 1981) or a pause in translation (Meyer, 1985) that is relieved by interaction with the docking protein (Meyer et al., 1982; Walter and Blobel, 1981). The translocationarresting activity and docking protein-binding activity of SRP are separable. The latter activity is sufficient to mediate translocation (Siegel and Walter, 1985, 1986; reviewed in Siegel and Walter, 1988). Although it was proposed initially that translation and translocation across the ER membrane were obligatorily coupled, these observations, together with the demonstration of posttranslational import of polypeptides into microsoma1 vesicles (Perara et al., 1986), indicate that translation and translocation need not be mechanistically coupled. C . Components of Escherichia coli Secretion Machinery

A majority of precursors are targeted to the integral membrane components of the secretion apparatus of E. coli by the peripheral membrane protein SecA (Cabelli et al., 1988; Cunningham et al., 1989; Oliver and Beckwith, 1982; reviewed in Oliver et al., 1990).SecA has ATPase activity, which is stimulated by the simultaneous presence of an export-competent precursor and vesicles or liposomes containing both acidic phospholipids and a complex of integral membrane proteins comprising SecE, SecY, and band 1 (band 1 is a protein that copurifies with SecE and SecY, for which no gene has been identified nor any role for in wivo secretion established). In the presence of these components, the stimulated ATPase activity of SecA drives the transmembrane translocation of precursors-albeit inefficiently (Schiebel et al., 1991)-and hence has been dubbed the “translocation ATPase” (Lill et al., 1989). Since stimulation of the SecA translocation ATPase is specifically associated with bona fide secretion-related events, it can be used to determine if a given interaction is relevant to secretion (Lecker et al., 1989; Lill et al., 1989, 1990; reviewed in Wickner et al., 1991). SecA binds SecB and precursors, with both the mature region (Lill et al., 1990) and the signal peptide (Cunningham and Wickner, 1989) of the precursor participating in the interaction. SecA has a much higher

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155

affinity for SecB/precursor complexes than for either component alone (Hartl et al., 1990). Therefore SecB is involved in the targeting of certain precursors to the membrane components of the translocation machinery. Genetic (Bieker and Silhavy, 1990) and biochemical evidence (Brundage et al., 1990) indicate that SecY and SecE are components of a preprotein translocator that participates in the transfer of precursors across the membrane. Together with acidic phospholipids, SecY and SecE also mediate the high-affinity binding of SecA to membranes. When associated with these high-affinity sites, the affinity of SecA for SecBIprecursor complexes is enhanced (Hartl et al., 1990). Other integral membrane components of the secretion machinery include the protein products of the secD, secF, lep, and Isp genes (reviewed in Bieker et al., 1990; Schatz and Beckwith, 1990). Specific biochemical functions for SecD and SecF have yet to be determined. Signal peptidase I (also known as leader peptidase), product of the lep gene, and signal peptidase 11, product of the Isp gene, are responsible for the endoproteolytic removal of signal peptides from unmodified precursors and lipid-modified lipoprotein precursors, respectively (reviewed in Dev and Ray, 1990). 111. PRECURSOR CONFORMATION GOVERNING SIGNAL

PEPTIDEFUNCTION A . Presequences Not Necessarily Sufficient for Translocation Although generally necessary, target peptides or signal peptides may prove insufficient to sponsor translocation. For example, the signal peptides of various envelope proteins do not support secretion of the normally cytoplasmic protein /3-galactosidase (LacZ) in E. coli (Bassford et al., 1979; Michaelis et al., 1983; Schwartz et al., 1981). T h e inability of signal peptides to direct the secretion of LacZ is likely not due to the absence of sequences in LacZ that actively promote secretion (MacIntyre and Henning, 1990), but rather is likely due to the presence of sequences that interfere with export (Lee et al., 1989). Overproduction of GroEL or DnaK, known modulators of protein structure, enhances secretion of a LacZ-hybrid protein, suggesting that the difficulties associated with the secretion of LacZ result from the folding of this protein into an exportincompetent conformation (Phillips and Silhavy, 1990). Several additional lines of evidence indicate that presequence function is governed by conformational properties of the precursor. T h e target peptide of yeast mitochondria1 cytochrome oxidase subunit IV (COX), fused to murine dihydrofolate reductase (DHFR, a cytoplasmic enzyme),

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sponsors import of the fusion protein into isolated mitochondria, even when the DHFR moiety is in an enzymatically active state. However, if the native structure of DHFR is stabilized by the folate antagonist methotrexate, import is blocked (Eilers and Schatz, 1986). Mitochondria1 import of a target peptide-DHFR fusion is also specifically blocked in uiuo by the addition of aminopterin (Wienhues et al., 1991). When diluted from denaturant, purified precursors of the E. coli outer membrane proteins OmpA (proOmpA) and PhoE (prePhoE) are imported into E. coli inverted membrane vesicles ( IMVs), but if allowed to fold, translocation competence is lost, despite the continued presence of a functional signal peptide (Crooke et al., 1988; Kusters et al., 1989). Similar results are obtained when IMVs are added posttranslationally to precursors of the maltose-binding protein [MBP (Weiss et al., 1988)], proOmpA, o r alkaline phosphatase [prePhoA (Chen et al., 1985)l synthesized in vitro. Loss of export competence is less severe for preMBP species with altered folding properties (Weiss et al., 1989). Loss of translocation competence could result from burial of the signal peptide within the folded precursor, but at least in the case of preMBP, the signal peptide is still accessible as determined by its selective sensitivity to proteolysis and its ability to bind amphiphiles (Dierstein and Wickner, 1985). In the case of the methotrexate-stabilized COX/DHFR fusion, the COX target peptide is clearly accessible since the precursor binds to energized mitochondria (a target peptide-dependent reaction), and the target peptide is susceptible to proteolysis by partially purified matrix processing enzyme (Eilers and Schatz, 1986).

B . Correlation between Precursor Folding and Loss of Translocation Competence A correlation between the extent of preMBP folding and its export competence in vivo has also been established in E. coli (Randall and Hardy, 1986). Nonnative MBP and preMBP are sensitive to proteolysis, but both fold into proteinase K-resistant conformations (Dierstein and Wickner, 1985; Randall and Hardy, 1986). Thus resistance to proteolysis can be used as a measure of preMBP folding. When an uncoupler is used to block the translocation of precursors, preMBP trapped in the cytoplasm remains largely protease sensitive (unfolded), while preMBP14-2, a precursor carrying a defective signal peptide becomes protease resistant (folded, t l / , = 2.5 min). Whereas preMBP is rapidly and completely secreted ( t , / 2 = 30 sec), the defective signal peptide of preMBP14-2 affects both the rate and extent ofsecretion: only 40% ofpreMBP14-2 is secreted ( t , , , = 3 min). If the defective signal peptide can direct the slow secretion

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of a portion of preMBP14-2, then 100% of the preMBP14-2 should be secreted at this same slow rate unless additional parameters blocking export are manifested with time. Because the rates of preMBP 14-2 folding (protease resistance) and loss of export competence are coincident, it appears that folding is the parameter that blocks further export. If export competence is associated with a “loosely folded” precursor, then parameters that accelerate folding or stabilize folded states should impede export. This relationship has also been explored in E. coli using a DHFR fusion protein. At low levels of synthesis, a hybrid protein consisting of the signal peptide and the first 153 amino acid residues of OmpA joined to DHFR is efficiently secreted. However, addition of trimethoprim imparts a kinetic defect in the secretion rate of the hybrid protein. The effect of trimethoprim is dependent on the presence of a full length, presumably active, DHFR moiety, indicating that secretion in vzvo is inhibited by stabilization of the native DHFR structure (Freud1 et al., 1988). Conversely, factors that retard precursor folding might be expected to facilitate secretion, and, indeed, such factors have been identified. Selection for enhanced secretion of export-defective precursors has identified mutations in the mature sequence of both MBP (Cover et al., 1987; Ryan et al., 1986) and ribose-binding protein [RBP (Teschke et al., 1991)] that suppress defects in their respective signal peptides, and in the cases tested to date, the mutational alterations have been shown to slow the refolding rate of the purified proteins. Suppression is thought to result directly from the slower rate of folding (Liu et al., 1988; Teschke et al., 1991). Comparison of the refolding rates of purified precursor and mature species of MBP (Liu et al., 1989; Park et al., 1988), RBP (Teschke el al., 1991), and TEM p-lactamase [pla (Laminet and Pluckthun, 1989)] demonstrate that the signal peptide is an antifolding factor. At least in the case of MBP, the slower rate of precursor folding is significant since it allows SecB to bind at physiological temperatures (Liu et al., 1989; see Section V,A). Finally, as described below, SecB binds a subset of envelope proteins and maintains them in an export-competent state.

IV. SecB AS COMPONENT OF SECRETION MACHINERY A . Isolation of secB Mutants Hybrid proteins composed of the signal peptide and a substantial portion of the mature MBP fused to LacZ enter the export pathway but are not completely translocated (Bankaitis et al., 1985; Bassford et al.,

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1979). Incomplete translocation of the hybrid protein results in a membrane association that prevents the efficient formation of active LacZ tetramers and these cells are therefore unable to utilize lactose. Mutations that even partially block entrance of the hybrid protein into the export pathway will allow sufficient tetramerization of LacZ to sponsor growth on lactose (Oliver and Beckwith, 1981). When a large number of Lac' mutants obtained in this fashion were examined, a few were found to define the previously unidentified secB gene (Kumamoto and Beckwith, 1983; reviewed in Kumamoto, 1990). B . Phenotype of secB Mutants Loss of SecB function imparts a kinetic defect in the export of a subset of envelope proteins (see Table I), indicating that SecB is a component of the secretion machinery. T h e severity of the defect varies from precursor to precursor. For example, both the rate and extent of preMBP secretion are reduced in cells devoid of SecB, such that 40% of pulselabeled preMBP is permanently trapped in the cell [Fig. 1 (Collier et d., 1988; Collier and Bassford, 1989)l. In contrast, 100% of OmpA is secreted in the absence of SecB, but with a t1,2 of 2 min [versus 20 sec in the presence of SecB (Collier et al., 1988)l. T h e efficient secretion of other proteins, such as RBP and pla, is SecB independent (Collier et al., 1988; Kumamoto and Beckwith, 1983). While secretion of PhoA was initially thought to be SecB independent (Collier et al., 1988; Gannon et al., 1989; Kumamoto and Beckwith, 1985), the efficiency of PhoA secretion in a s e c B : : T n 5 strain shows a temperature dependence such that at 30°C secretion is slowed, while at 42°C there is no discernible SecB requirement for efficient PhoA secretion (Kusukawa et al., 1989). This result is consistent with the observations that heat-shock proteins can substitute for SecB function and that the efficient secretion of PhoA specifically requires the heat-shock proteins DnaK and DnaJ (Altman et al., 1991; Wild et al., 1992) (see Sections VIII,A and VII1,C). The observation that several uncharacterized alleles of secB have been found to weakly suppress malE signal sequence mutations (Collier and Bassford, 1989) bolsters the contention that SecB is a bona fide component of the secretion machinery. In E. colz, the signal peptides of some envelope proteins are cleaved cotranslationally, while other precursors are completely synthesized before processing can be detected. While MBP displays both modes of processing, cotranslational processing does not occur until the precursor is 80% complete (Josefsson and Randall, 1981). Since the catalytic domain of signal peptidase is on the periplasmic face of the cytoplasmic membrane (Ohno-Iwashita and Wickner, 1983), cotranslational pro-

159

SecB: MOLECULAR CHAPERONE

.rABLE 1

SecB Requireiiieiit for Serretioir

VJ

Vririora Pro/eitrs iir EsrIierirlii(i roli ~

~~

~~

Protein

Type"

SecBb

Ref.'

Alkaline phosphatase (PhoA) Maltose-binding protein (MBP) Oligopeptide-binding protein (OPPA! Penicillm-binding protein 3 (PBP 3) TEM 8-lactanlase @la) Ribose-binding protein (RBP) Lambda receptor (Lams) Outer membrane protein A (OmpA) Outer membrane protein E (PhoE) Outer membrane protein F (OmpF) Murein lipoprotein (Lpp) Phage T4 GP37 tail fiber protein Pullulanase Phage MI3 coat protein

P P P

CSD Dep Dep

3.5.6 3.4.5 ?

A P

Dep lnclep Indep Dep Dep Dep Dep Indep Dep Dep Indep

8 3.6.9

P OM 0M OM 0M OM B

c

D

3.5 1.3.5 3.6.9 2 5.6 3.10

7 9 3 ~

A, OmpA signal peptide/PBP 3 hybrid; B, OmpA signal peptide fused to 283-amino acid residues of gp37 (a nonsecreted protein); C, extracellular lipoprotein from the genus Klebsiella; D, integral membrane protein during phage infection; OM, outer membrane protein; P, periplasmic protein. SecB requirement; Dep, efficient export is dependent on SecB: Inclep. efficient export is independent of SecB: CsD. a cold-sensitive depenclence. ' Key to references: ( I ) Altman e/ (11. (19YOa); (2) de Cock r/ nl. (1992): ( 3 ) Collier e/ nl. (1988); ( 4 )Collier and Bassford (1989);( 5 ) Kunianioto and Beckwith (1985);(6) Kusukawa el 01. (1989);(7) Maclntvre el cil. (IY9l): ( 8 ) C. Piron-Fraipoint, M. Adam, and M. Nguyen-Disteche, personal communication; (9) Pugsley et al. (1991); and (10) Watanabe t t al. (1988).

'

cessing is synonymous with cotranslational translocation. This view is corroborated by the observation that processed nascent chains are externally exposed (Randall, 1983). In SecB- cells the cotranslational component of preMBP processing is completely abolished, and translocation is shifted to an entirely posttranslational mode (Collier and Bassford, 1989; Kumamoto and Cannon, 1988). However, the slow, posttranslational export demonstrated by precursors with mutationally altered signal peptides is also abolished (Altman et al., 1990b; Collier et al., 1988; Collier and Bassford, 1989; Trun et al., 1988, see fig 1). Hence SecB function is required for both temporal modes of MBP and LamB export. Nonsense mutations and T n 5 insertions in secB are tolerated in haploid cells, indicating that secB is not an essential gene. However, secB null strains are sensitive to rich media and do not form isolated colonies on LB agar plates (Kumamoto and Beckwith, 1985). Induction of the heat-

160

DAVID N. COLLIER

MBP+

MBPIG-I

0' I' 2' 4' 10'20' 0' I' 2' 4' 10' 20' -0 -m

Sec €3-:

4

P

c m

FIG. I . Export kinetics of M B P + and MBPl6-I in SecB+ and SecB- cells. Wild-type cells (top) or cells devoid of SecB (bottom) synthesizing either wild-type preMBP (left-hand column) or export-defective preMBP16-l (right-hand column) were pulse labeled with [95S]methioninefor 15 sec followed by addition of excess unlabeled methionine. Samples were removed at the indicated chase points, processed for immunoprecipitation with antiMBP antiserum, and analyzed by SDS-PAGE. The positions of precursor (p) and mature (m) MBP are indicated by arrows. From Collier and Bassford (1989).

shock response suppresses this rich media sensitivity. Since SecB itself is not a heat-shock protein (Altrnan et al., 1991), this suggests that cornponents of the heat-shock response and SecB are functionally redundant. Indeed, at subbasal levels of heat-shock protein synthesis, secB becomes an essential gene. The secretion-related activity of specific heat-shock proteins is discussed in Section VIII. C . SecB Sequence The secB gene has been cloned (Kumamoto and Beckwith, 1985) and its nucleotide sequence determined (Kumamoto and Nault, 1989). As shown in Fig. 2, the gene is predicted to encode a protein of 155 amino acid residues. T h e GenEMBL, NBRF, and Swiss Protein data bases were searched for proteins with sequences similar to the predicted SecB amino acid sequence using the TFASTA or FASTA programs of the GCG sequence analysis software package (Devereux et al., 1984). No obvious homologues of SecB were found. However, within a 108-amino acid overlap, SecB has 22.9% identity with the human fibroblast growth factor receptor 4 [residues 63 1-739, which are in the intracellular catalytic domain (Partanen et al., 1991)], and has 22% identity over 123 amino acids with the Saccharomyes cerevisiae trans-activating factor B (Rep 1). Rep1 is involved in plasmid partitioning in yeast (Hindley and Phear, 1979).

161

SecB: MOLECULAR CHAPERONE

1/1

31/11

ATG TCA GAA CAA AAC AAC ACT GAA ATG ACT TTC CAG ATC CAA CGT ATT

M e t ser g l u g l n a s n a s n t h r g l u m e t t h r phe g l n i l e g l n a r g i l e 61/21

91/31

TAT ACC AAG GAT ATC TCT TTC GAA GCG CCG AAC GCG CCG CAC GTT TTC

t y r t h r l y s a s p i l e s e r phe g l u a l a p r o a s n a l a p r o h i s

Val

phe

121/41 CAG AAA GAT TGG CAA CCA GAA GTT AAA CTT GAT CTG GAT ACG GCA TCT

g l n l y s a s p t r p g l n p r o g l u Val l y s l e u a s p l e u a s p t h r a l a s e r

151/51

181/61

TCC CAA CTG GCA GAT GAC GTA TAC GAA GTG GTA CTG CGT GTT ACC GTA

s e r g l n l e u a l a a s p a s p v a l t y r g l u Val Val l e u a r g

Val

thr val

211/71 ACG GCC TCT TTG GGC GAA GAA ACC GCG TTC CTG TGT GAA GTT CAG CAG

t h r a l a ser l e u g l y g l u g l u t h r a l a phe l e u c y s g l u

241/81

Val

gln gln

271/91

GGC GGT ATT TTC TCC ATC GCG GGT ATC GAA GGC ACC CAG ATG GCG CAT

g l y g l y i l e phe ser i l e a l a g l y i l e g l u g l y t h r g l n m e t a l a h i s

301/101

331/111

TGC CTG GGA GCA TAC TGC CCG AAC ATT CTG TTC CCG TAT GCT CGT GAG

c y s l e u g l y a l a t y r c y s p r o a s n i l e l e u phe p r o t y r a l a a r g g l u

361/121 TGC ATC ACC AGC ATG GTA TCC CGC GGT ACA TTC CCG CAA CTG AAC CTT

c y s i l e t h r ser m e t v a l ser a r g g l y t h r phe p r o g l n l e u a s n l e u

391/131

421/141

GCG CCG GTT AAC TTC GAT GCG CTG TTC ATG AAC TAT TTG CAG CAG CAG

a l a pro

Val

a s n phe a s p a l a leu phe m e t a s n t y r leu g l n g l n g l n

451/151 GCT GGC GAA GGT ACT GAA GAA CAT CAG GAT GCC TGA a l a g l y g l u g l y t h r g l u g l u h i s g l n a s p a l a OPA

FIG. 2. Predicted amino acid sequence of SecB. The nucleotide sequence of secB is shown above the predicted amino acid sequence tor SecB (Kumanioto and Nault, 1Y8Y).

SecB also has 23% identity (and 70.5%similarity) with a 61-amino acid stretch of an estrogen-regulated human 27K heat-shock protein (HSP27) (Fuqua et al., 1989; Hickey et al., 1986). Though this identity is limited to a relatively short segment, it falls in the middle of HSP27. This is the most conserved region among the small HSPs and a-crystallins (the superfamily to which HSP27 belongs), and this region has been proposed to interact with cellular components during the stress response (Hickey et al., 1986). Although SecB and the A subclass of bovine a-crystallin have 2 1 . 1 % identity within a 57-amino acid overlap, the overlap is outside

162

DAVID N. COLLIER

the conserved region. Hence the significance of these identities is unclear. SecB does not contain a predicted ATP-binding domain (Kumamoto and Nault, 1989)or any other readily identifiable structural motif. Consistent with the lack of an ATP-binding domain, the antifolding activity of SecB is not influenced by 5 mM ATP (Hardy and Randall, 1991). SecB has been detected by Western blot analysis in 17 members of the Enterobacteriaceae family. N o immunologically cross-reactive antigens were detected in 10 non-Enterobacteriaceae (de Cock and Tommassen, 1991). While these results suggest that SecB is limited to the Enterobacteriaceae family, it should be noted that the Bacillus subtilis div gene product shares 50% identity over its entire length with E. coli SecA [including regions with greater than 71% identity (Sadaie et al., 1991)], but was not detected by Western blot analysis (Yamane et al., 1991).

D. Purification of SecB SecB has been purified from haploid cells (Watanabe and Blobel, 1989b) and from cells containing a multicopy plasmid with secB under transcriptional control of the ApL promoter (Kumamoto et al., 1989) or a T 7 promoter (Weiss et al., 1988). The amino termini of the purified proteins obtained by each method (in the order given above) had the expected sequence, were missing the first two aminoacyl residues, or were blocked. All three methods yielded an oligomeric protein composed of four to six identical subunits (most likely four) that promoted translocation of precursors into IMVs. Purified SecB has a p l value of 3.95-4.1, which is in good agreement with the predicted pl of 4.1 (Weiss et al., 1988). SecB is a soluble, cytoplasmic protein (Kumamoto et al., 1989; Watanabe and Blobel, 1989b; Weiss et al., 1988) representing only about 0.08% of cytosolic protein (Watanabe and Blobel, 1989a). v . PROPERTIES

OF

SecB

A . SecB: Antifolding Activity T h e first evidence that SecB functions in the secretory pathway as an antifolding factor was obtained by pulse-chase analysis comparing the export kinetics of wild-type and mutationally altered preMBP species. In secB+ cells, the export kinetics of preMBP, preMBPAl16, preMBPA57145, and preMBP-Y283D are indistinguishable, but in cells devoid of SecB, export of the mutationally altered precursors is clearly more efficient than that of preMBP (Collier et al., 1988). PreMBPAll6 and

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preMBPA57-145 lack amino acid residues 142-150 and 57-145, respectively, of the mature MBP. These deletion derivatives have lost their inherent protease resistance, demonstrate a decreased half-life in vim, and though properly localized to the periplasm, do not support maltose transport (Collier et al., 1988; Fikes and Bassford, 1987; Weiss et al., 1989). MBP-Y283D, which contains a tyrosine-to-aspartic acid substitution at position 283 of the mature MBP, is also inherently unstable in vivo (Collier et al., 1989) and refolds after denaturation significantly more slowly than wild-type MBP (Liu et al., 1988). Hence, each of these mutations alter the ability of MBP to assume its native state. Recently it has been demonstrated that the substitution of a glycine for valine at position 8 (V8G), a cysteine for a glycine at position 19 (G19C), or a glycine for an alanine at position 276 (A276G) of the mature MBP also reduces the rate of MBP refolding after denaturation (S.-Y. Chun, S. Strobel, P. J. Bassford, Jr., and L. L. Randall, personal communication, 1992). Like preMBP-Y283D, preMBP-V8G is secreted more efficiently in secB null strains than is wild-type preMBP (S. Strobel and P. J. Bassford, Jr., personal communication, 1992). That several different mutational alterations, whose only common feature is the perturbation of MBP structure, result in a degree of SecB-independent secretion, indicates that a major function of SecB is to maintain this precursor in a loosely folded, export-competent conformation. Consistent with an antifolding role, 25% of the preMBP trapped within secB null strains by the addition of the uncoupler carbonyl cyanide mchlorophenylhydrazone (CCCP) folds into a protease-resistant conformation, while in secB+ cells, only 5% folds (Kumamoto and Gannon, 1988). Given that protease resistance is a less sensitive probe of preMBP conformation. than export competence [in vitro the translocation competence of preMBP decays more rapidly than its protease sensitivity (Weiss et al., 1989)], then this protease-resistant fraction and the fraction of preMBP that becomes export incompetent in secB null strains are quantitatively similar (25% vs. 40%, respectively). Hence the fraction of MBP that is unable to be translocated in the absence of SecB represents the folded fraction. SecB also modulates the folding of purified preMBP and preMBP synthesized in vitro. For example, 80% of the preMBP produced during 12 min of continuous synthesis in S 100 extracts devoid of SecB is protease resistant. Addition of purified SecB (Weiss et al., 1988) or synthesis of preMBP in SlOO extracts of a SecB-overproducing strain causes preMBP to remain protease sensitive during the same time period, while preMBP synthesized in extracts containing wild-type levels of SecB folds at an intermediate rate (Collier et al., 1988). T h e posttranslational addition of

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SlOO extracts rich in SecB to preMBP synthesized in the absence of SecB maintains the unfolded fraction of preMBP in a protease-sensitive conformation, but does not unfold the protease-resistant fraction (Collier et al., 1988). Protease resistance can also be used as a measure of preRBP folding (Park et al., 1988). Consistent with the SecB-independent nature of RBP secretion, purified SecB has no detectable effect on the folding of preRBP (Weiss et al., 1988). Not only does SecB modulate the conformation of preMBP synthesized in vitro, but the addition of SecB dramatically stimulates the efficiency of translocation into IMVs of a number of different precursors (Kumamoto et al., 1989; Kusters et al., 1989; Lecker et al., 1989; Watanabe and Blobel, 1989b; Weiss et al., 1988). The intrinsic fluorescence of the eight tryptophanyl residues in MBP can be used to measure its reversible unfolding/refolding reactions. When unfolded by incubation in a denaturant such as guanidinium hydrochloride, the side chains of the tryptophanyl residues are exposed to solvent and their fluorescence is quenched. On dilution of the denaturant, the subsequent refolding reaction can be monitored by the increase in fluorescence that results from burial of the side chains in the interior of the protein and their shielding from solvent (Liu et al., 1988, 1989). Compared to the folding rate of purified mMBP diluted from denaturant into buffer alone, mMBP refolding at 25°C is slower when diluted into a solution containing SecB [the molar ratio of SecB monomer to mMBP is 6 : 1 (Liu et al., 1989)l. Increasing the molar ratio to 9 : 1 results in neither a block in folding nor a further reduction in the rate of folding. However, SecB can effect a complete block in MBP refolding (operationally defined as no detectable increase in the amplitude of fluorescence during the kinetic phase) provided the inherent rate of target MBP folding is below a certain threshold. Incubation at 5°C slows the refolding of mMBP such that SecB (at the same 6 : 1 molar ratio) completely arrests refolding of a majority of mMBP (Hardy and Randall, 1991; Liu et al., 1989).The antifolding effects of the signal peptide (Park et ad., 1988) or the MBP-Y283D point mutation (Liu et al., 1988) allow SecB to block refolding of preMBP14-1 and mMBP-Y283D at 25°C (Liu et al., 1989). Conversely, for a given MBP species, increasing the temperature of the refolding reaction requires elevated SecB:MBP ratios to effect a given magnitude of blockage. For example, at 1WC, a 3.5 molar ratio (SecB monomer to MBP) results in a 50%blockage in preMBP14-1 refolding, while a molar ratio of 5 is required to mediate a 50% block at 25°C (Hardy and Randall, 1991). T h e extent to which varying amounts of SecB block the refolding of mMBP and preMBP (at 5 and 1O"C, respectively) has been determined and the relationship between the magnitude of blockage and the molar

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165

ratio of SecB to MBP was found to be slightly sigmoidal. If the MBP concentration is increased, a given magnitude of blockage is achieved at the same molar ratios, rather than at the same absolute SecB concentration. These results suggest that the sigmoidal nature of this relationship does not reflect a cooperative oligomerization of SecB, since this would be expected to depend on the absolute concentration of SecB, but rather may reflect the interaction of two or more SecB oligomers with MBP (Hardy and Randall, 1991). This interpretation is consistent with in vim mapping data suggesting that MBP (Collier et al., 1988; Cannon et al., 1989) and LamB (Altman etal., 1990a)contain two or more sites responsible for SecB binding (see Section VI1,A). Once folded, MBP is no longer a substrate for SecB binding (Randall el al., 1990), presumably because the SecB binding sites have become internalized or masked. The higher levels of SecB required to block refolding at higher temperatures and/or to block refolding of faster folding MBP species likely reflect a higher probability that the SecB binding sites will be masked when MBP encounters SecB. A temperaturedependent alteration in the affinity of SecB for its target is probably not the explanation, since the K , for SecB complexed with denatured bovine pancreatic trypsin inhibitor (BPTI) does not change over a similar temperature range. Furthermore the concentrations of MBP employed are 33- to 330-fold higher than the estimated Kd values for the MBP species (Hardy and Randall, 1991). Since substrate folding influences SecB binding, estimates of the minimum molar ratio required to block refolding will depend on the conditions employed. Dilution of the slow folding species, mMBP-Y283D, at 5°C provides the slowest refolding reaction that is experimentally measurable [(Hardy and Randall, 1991); note that preMBP14-1-Y283D, which actually refolds more slowly, cannot be used for these experiments because the sample apparently aggregates before equilibrium is reached (Liu et al., 1988)l. Refolding of mMBP-Y283D at 5°C can be completely blocked at a molar ratio of about 5 : 1 [SecB monomer to MBP (Hardy and Randall, 1991)], suggesting that a single tetramer of SecB is the minimal functional unit. Consistent with this notion is the observation that proOmpA (Lecker el al., 1989) and preMBP (Watanabe and Blobel, 1989a) cosediment with SecB tetramers as 1 : 1 complexes in linear sucrose gradients. In contrast to the refolding reaction at 25"C, the relationship between the amount of SecB and the extent of blockage of mMBP-Y283D refolding at 5°C is hyperbolic rather than sigmoidal. Additionally, a given level of blockage at 25°C requires approximately twice the molar ratio of SecB that is required at 5°C (Hardy and Randall, 1991). These observations

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suggest that if the ligand refolding rate is slow compared to the rate of SecB-ligand association, the ligand can be maintained in a loosely folded state by a single tetrarner of SecB. However, when ligand refolding is relatively rapid, a higher concentration of SecB is required to block folding. If the rate of ligand refolding is significantly greater than the association rate, as the case may be with mMBP at 25"C,then SecB cannot block refolding. B . SecB: Targeting Activity

T h e productive targeting of precursors to the transmembrane components of the secretion machinery requires the peripheral membrane protein SecA (Cabelli et al., 1991; Hartl et al., 1990; Swidersky et al., 1990). In addition to its antifolding activity SecB also enhances secretion by facilitating precursor targeting to SecA. Direct, albeit weak, interactions between purified, soluble SecA and SecB have been demonstrated by density centrifugation. T h e affinity of soluble SecA for preformed SecB-proOmpA complexes is slightly higher than for SecB alone. If associated with high-affinity sites on the membrane, then SecA mediates the specific, high-affinity binding (Kd of about 2 X lO-'M) of SecB to IMVs. Each milligram of membrane protein binds about 140 pmol SecA (SecA binds IMVs with a Kd of about 4 X 10-*M) and 117 pmol of SecB. These levels are consistent with the levels of SecY/SecE [integral membrane components of the preprotein translocase (Hartl et al., 1990)l. While proOmpA can bind IMV in the absence of SecB, the subsequent translocation reaction is significantly enhanced by the posttargeting addition of SecB. Hence SecB can promote secretion even after association of the precursor with SecA (Hartl et al., 1990). In comparison, very little preLamB binds IMVs in the absence of SecB, either posttranslationally or cotranslationally, suggesting that the targeting activity of SecB is more important for this SecB-dependent precursor than for others. SecB sponsors the posttranslational targeting to IMVs of even export-incompetent (partially folded?) preLamB (Swidersky et al., 1990), indicating that targeting and antifolding activities may be distinct. The affinity of membrane-associated SecA for proOmpA-SecB complexes (Kd = 6 X 10-8M) is about three times as high as the affinity of SecA for proOmpA alone. Consistent with the low affinity of soluble SecA for SecB, an eightfold molar excess of soluble SecA does not block the binding of proOmpA-SecB complexes to IMVs. As would be expected, preMBP-SecB complexes compete with proOmpA-SecB for SecA binding. Excess proOmpA does not, however, cause dissociation of the ternary IMV-SecA-SecB complex, suggesting that SecA contains

SecB: MOLECULAR CHAPERONE

167

discrete SecB and precursor binding sites (Hart1 et al., 1990). In whole cells, overproduction of SecB causes a noticeable defect in the export kinetics of MBP (Collier et al., 1988; Kumamoto and Beckwith, 1985), but not RBP (Collier et al., 1990). These results suggest that excess uncomplexed SecB can, by transient occupation of the putative SecB binding site on SecA, cause interference by excluding the productive interaction of SecB-precursor complexes (i.e., SecB-dependent proteins such as preMBP). Export of precursors that are not complexed with SecB (i.e., preRBP) is unperturbed since their targeting relies solely on their signal peptide or a different chaperone. Purified prePhoE, diluted from 8 M urea into buffer containing SecB, retains its competence for translocation into IMVs for several hours, while prePhoE diluted into buffer alone is competent for translocation immediately following dilution but looses this competence on further incubation (tt = 14 min). These results argue that, as is the case with preMBP, SecB slows the folding and/or blocks aggregation of prePhoE. However, significant translocation of PhoE, even immediately following dilution into buffer alone, depends on the presence of SecB in the IMV fraction (Kusters el al., 1989), suggesting that SecB is required primarily for targeting the translocation-competent precursor to the preprotein translocase. Although the previous data cannot rule out the possibility that SecB contained in the IMV fraction simply blocks further folding/aggregation of the diluted precursor on addition of the IMV fraction, a different experimental approach indicated that SecB does not maintain the translocation competence of prePhoE and therefore serves only a targeting role in PhoE secretion (de Cock and Tommassen, 1992). The key observation in these studies was that when added to an IMV fraction containing SecB, prePhoE synthesized in vitro and incubated in the absence of SecB demonstrated similar levels of translocation as prePhoE synthesized in the presence of SecB. Indeed, the translocation-competence of prePhoE synthesized in the presence or absence of SecB decayed at the same rate (4 = 14 min)-an unexpected result if SecB maintains the precursor in a competent state. That translocation of prePhoE incubated in the absence of SecB can be rescued by SecB in the IMV fraction argues that SecB plays primarily a targeting role for this precursor. As judged by coimmunoprecipitation, there was no reduction in the level of prePhoE-SecB complexes associated with the loss of translocation competence for prePhoE synthesized in vitro, suggesting that prePhoE can become export-incompetent even when complexed with SecB. Perhaps the drastic difference in half-lives of translocation competency for purified prePhoE versus in vitro-synthesized prePhoE (hours vs. minutes,

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respectively) can be explained if the residual urea (greater than 0.5 M ) present in the case of the diluted, purified prePhoE acts in concert with SecB to maintain the translocation competency of prePhoE.

C . SecB: Blocking Aggregation When diluted from denaturant, mMBP behaves as a soluble monomer. In contrast, preMBP aggregates and cannot be completely recovered following gel filtration unless it is diluted in the presence of SecB (Randall et al., 1990). Similarly, denatured proOmpA diluted into either buffer or a solution of bovine serum albumin sediments as oligomers or aggregates and is not recoverable by sucrose gradient centrifugation unless SecB is present during refolding (Lecker et al., 1989). If diluted and incubated in the absence of t.he chaperone, soluble proOmpA cannot be rescued by SecB (Lecker et al., 1990). Incubation of IMV with proOmpA under conditions in which translocation does not occur (no SecA or ATP) results in a large fraction of proOmpA that is membrane associated. However, very little of this membrane-associated precursor can be translocated on the subsequent addition of SecA and ATP. SecB eliminates this nonspecific, nonproductive binding of proOmpA (Hart1et al., 1990). Hence an important activity of SecB is to prevent aggregation and other nonproductive interactions. Consistent with its lack of ATPase activity, SecB has no detectable disaggregating activity.

D. Translocation Competency: A Terminology Disclaimer Throughout this review, terms such as unfolded, loosely folded, or exportcompetent state are bandied about (no doubt distressing some disciples of protein structure). These terms are not meant to imply a single definable conformation, or the absence of a specific secondary or tertiary structure. In fact, several lines of evidence indicate that a translocation-competent precursor can contain considerable structure. For example, efficient signal peptide function requires that the hydrophobic core assume an ahelical conformation (Jones et al., 1990), and evidence has been presented that enzymatically active DHFR (Freud1 et al., 1988) and a fully folded biotin acceptor domain (Reed and Cronan, 1991)-or conformers of these proteins that are in equilibrium with the native states-can be translocated across the cytoplasmic membrane of E. coli in vivo. Translocation into IMVs of proOmpA containing an intramolecular disulfide bond has been demonstrated (Tani et al., 1990). Exportcompetent and incompetent proOmpA have identical circular dichroic

SecB: MOLECULAR CHAPEKONE

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spectra, indicating that these two forms of proOmpA have similar levels of secondary structure. Intrinsic tryptophan fluorescence studies indicate that translocation-competent proOmpA also has significant tertiary structure and that the binding of SecB does not alter the conformation of proOmpA in the vicinity of the tryptophan residues. However, on SecB binding there is a decrease in energy coupling between tryptophans at the amino terminus of proOmpA and coumarin molecules coupled at the carboxy terminus, suggesting that SecB binding does slightly alter the conformation of the precursor. Since translocation-competent proOmpA is folded (not necessarily in its native state) and loss of competence is associated with aggregation, then the translocation-competent state in which SecB maintains proOmpA is unaggregated, rather than unfolded (Lecker et al., 1990). However, for precursors such as preMBP, assumption of the native state appears to be the predominant impediment to efficient secretion, and translocation competence is therefore primarily defined as unfolded or loosely folded. VI. SecB AND ITS LIGANDS FORMING ISOLABLE COMPLEXES A . Inte$erence Phenotype as Measure of SecB Binding in Viva

Synthesis of preLamB or preMBP species that have been rendered export-defective by mutational alterations in the signal peptide, or synthesis of MBPA323, which is missing amino acid residues 7 (of the signal peptide) through 89 (of the mature peptide), results in a kinetic defect in the export of certain other wild-type envelope proteins. This phenomenon is known as “interference” (Bankaitis and Bassford, 1984; Collier et al., 1988; reviewed in Bassford, 1990). Envelope proteins whose export kinetics are sensitive to interference also require SecB for efficient export, while interference-insensitive proteins are SecB independent, suggesting that interference is mediated at the level of SecB. Synthesis of an exportdefective interfering species does not exacerbate the secretion defects associated with the loss of SecB function, whereas overproduction of SecB suppresses interference (Collier et al., 1988). Hence interference results from the functional depletion of SecB. During the process of normal secretion, the interaction of SecB and precursors is, by necessity, transient, However, if secretion is artificially blocked, then the interaction between SecB and precursors is prolonged, as demonstrated by the isolation of SecB-precursor complexes from cells exposed to an uncoupler (Kumamoto, 1989). Since the export-defective interfering species d o not pass through the secretion pathway with nor-

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ma1 kinetics, SecB cannot be recycled at its normal rate. Therefore the free cytoplasmic pool is diminished, resulting in a kinetic defect in the secretion of other SecB-dependent species. T h e ability of an exportdefective species to interact with SecB and its ability to cause interference are correlated. For example, at physiological temperatures mMBP and SecB show little interaction, with less than 1% of mMBP coprecipitating with SecB, while about 20% of mMBP-Y283D and SecB coprecipitate (Weiss and Bassford, 1990; also see below). Synthesis of mMBP-Y283D imparts an interference phenotype, but synthesis of mMBP does not (Liu et al., 1989). Export-defective precursors of RBP, a SecB-independent protein for which SecB has very low affinity (see Section VII,B), also do not cause interference (Collier et al., 1990).

B . Isolation of Complexes Formed in Vivo A direct interaction between SecB and SecB-dependent proteins has been demonstrated by the isolation of complexes formed in vivo (Altman et al., 1990b; Kumamoto, 1989) and in vitro (Lecker et al., 1989; Randall et al., 1990; Watanabe and Blobel, 1989a; Weiss and Bassford, 1990). Complexes formed in vivo have been detected by anti-SecB affinity chromatography. Immunoprecipitation with anti-SecB antiserum of extracts from cells pulse labeled with [“5S]methionine coprecipitates preMBP, preLamB, and proOmpA, but not the cytoplasmic protein pgalactosidase (Altman et al., 1990b; Kumamoto, 1989). Immunoprecipitation of these precursor species is blocked by the addition of excess purified SecB, and does not occur in extracts of secB::Tn5 cells. T h e interaction is transient since very few radiolabeled precursors are complexed with SecB after 60 sec of chase with excess unlabeled methionine, unless export is blocked by an uncoupler. Consistent with its role in cotranslational secretion, SecB complexes containing incomplete MBP chains have been detected (Kumamoto, 1989). C. Complexes Formed in Vitro Corresponding to the ability of SecB to slow the folding of MBP species synthesized in vitro, complexes of SecB and MBP can be isolated by coimmunoprecipitation from SlOO extracts programmed to synthesize MBP. About 7% of preMBP can be recovered as a complex with SecB, and significantly higher levels of MBP variants with altered folding properties are recovered. For example, 32% of preMBP-Y283D and 42% of MBPA323 are recovered complexed with SecB (Weiss and Bassford, 1990). Sucrose gradient centrifugation following synthesis of preMBP* (a truncated version of preMBP; see Section VI1,A) in a

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171

wheat germ system supplemented with purified SecB yields 1 : 1 complexes of SecB(tetramer) and preMBP* (Watanabe and Blobel, 1989a). PrePhoE, but not prepla (a SecB-independent protein), synthesized in the presence of SecB can be specifically precipitated with anti-SecB antiserum (de Cock et al., 1992). Dilution of preMBP, proOmpA, or prePhoE from denaturant into buffer containing SecB yields stable, isolable precursor-chaperone complexes. SecB-preMBP complexes have been isolated by gel filtration chromatography (Randall et al., 1990) and SecB-proOmpA and SecB-prePhoE complexes have been demonstrated by sucrose gradient centrifugation (Lecker et al., 1989).Employing a onefold molar excess of proOmpA, essentially all of the SecB is recovered in a 1 : 1 complex [SecB(tetramer):proOmpA]. N o complexes between SecB and bovine serum albumin, ovalbumin, ribonuclease A (RNase A), lysozyme (in their native states), or SecA (either native or diluted from denaturant) can be detected by sedimentation analysis (Lecker et al., 1989). These data do not, however, rule out weak interactions between SecB and these proteins, and in fact such interactions have been demonstrated for SecA and RNase A. A shift in the sedimentation velocity of SecA occurs in response to distribution of SecB throughout the gradient and is indicative of a weak interaction between SecA and SecB (Hart1 et al., 1990). Weak interactions with SecB can also be assayed indirectly by monitoring the refolding of MBP (following the intrinsic fluorescence of tryptophans in MBP) under conditions in which SecB is expected to retard MBP refolding. Addition of a protein that competes with MBP for SecB binding will allow MBP to refold, while proteins for which SecB has low affinity d o not influence the SecB-mediated block in MBP refolding. When diluted from denaturant, several proteins, including RNase A, have been shown by this in vitro competition assay to interact with SecB. Estimated apparent dissociation constants for SecB-RNase A and SecB-MBP complexes are 8 and 1.5 nM, respectively (Hardy and Randall, 1991). VII.

NATUREOF SecB BINDING SITES

A. Mature Moiety of Secretoly Protein Sufficient to Mediate SecB Binding Since signal peptides are the most obvious discriminating feature of secretory proteins, and SecB is a component of the secretion machinery, it would seem likely that the signal peptide is the principal site for SecB interaction. Indeed, several lines of evidence support this idea. Posttranslational incubation with SecB causes a shift in the relative

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DAVID N . COLLIER

sedimentation rate of a truncated MBP species (preMBP*; the asterisk denotes a truncation), indicating that SecB binds to preMBP*. While the carboxy-terminal 49 amino acid residues have been replaced by 9 unrelated residues, preMBP* retains the entire MBP signal peptide. T h e sedimentation rate of mMBP*, the corresponding mature polypeptide lacking the signal peptide, is not influenced by incubation with SecB. SecB influences only slightly the sedimentation rate of pre*MBP*. pre*MBP* is a mix of MBP* species lacking the first 17 or 18 residues of the amino terminus of the signal peptide. These results were interpreted as indicating that SecB directly binds the signal peptide (Watanabe and Blobel, 1989a). Further support for this notion is provided by the observation that addition of SecB to a wheat germ translation system blocks the signal recognition particle-mediated arrest of preMBP* and pre*MBP* synthesis (Watanabe and Blobel, 1989a). Because SRP-mediated translational arrest is thought to result from an interaction of the SRP with the signal peptide (Kurzchalia et al., 1986; Walter et al., 1981), one interpretation of these results is that SecB binds directly to the signal peptide and blocks the interaction with SRP. However, an interaction between SecB and a domain in the mature moiety that blocks SRP binding via steric hinderance or a direct interaction between SecB and SRP could also explain these results). T h e following observations are also in keeping with the conclusion that the signal peptide is the site of SecB binding: (1) Synthesis of mMBP does not cause an interference phenotype [see Section VI,A (Liu et al., 1989)l. (2) Very little mMBP synthesized in vitro is complexed with SecB (Weiss and Bassford, 1990). (3) Deletion of 12 amino acid residues from the hydrophobic core of the LamB signal peptide dramatically reduces the amount of SecB-preLamB complexes recovered from the cell (Altman et al., 1990b). While a direct interaction between SecB and the signal peptide may occur, the signal peptide is not necessary for SecB binding. Perhaps the clearest demonstration of this is provided by the fact that both mOmpA (Lecker et af., 1989) and mMBP (Randall et al., 1990) diluted from denaturant in the presence of SecB form isolable complexes with the chaperone. Furthermore, if limiting amounts of SecB are present during the refolding of an equimolar mix of preMBP and mMBP [at ratios of 3 : 1 or 1.5 : 1 (SecB monomer to total MBP)] and the resulting complexes are isolated, mMBP is recovered at levels that suggest SecB has a similar affinity for mMBP and preMBP (Randall et al., 1990). However, as noted above (see Section V,A), SecB is able to bind to and block the refolding of mMBP only at 5°C. The SecB-mMBP complexes described here were formed and isolated at 5"C, while in vitro synthesis reactions are typically

SecB: MOLECULAR CHAPERONE

173

performed at 25 to 37°C-temperatures at which SecB cannot block mMBP refolding. Hence the inability to isolate SecB-mMBP or SecB-mMBP* complexes from in vitro synthesis reactions demonstrates that, while not strictly required for SecB binding, the antifolding activity of the signal peptide facilitates interaction with SecB at physiologically relevant temperatures. For slow-folding mature species such as mPhoE (de Cock el al., 1990) and mMBP-Y283D, complexes with SecB can be isolated at these temperatures (de Cock et al., 1992; Weiss and Bassford, 1990). In vivo mapping data corroborate the conclusion that the signal peptide is not strictly required for SecB binding. Because the binding of SecB to export-defective species within the cell results in an interference phenotype (see Section VI,A), alterations in the export-defective species that relieve interference can be used to identify elements required for SecB binding. Within a set of MBP deletion derivatives, all interfering species-including a derivative lacking the entire signal peptide-retained amino acid residues 151- 186 of the mature protein. These residues were missing from noninterfering constructs, indicating that this region of the mature, and not the signal, peptide is involved in SecB binding (Collier et al., 1988). However, deletion of residues 151-186 from an otherwise intact export-defective preMBP did not abolish interference, suggesting that mMBPcontains two or more SecB binding sites (S. Strobel, D. Collier, and P. J. Bassford, Jr., unpublished, 1989). The MBP signal peptide alone cannot be responsible for SecB binding since synthesis of a hybrid protein containing a defective MBP signal peptide fused to the RBP mature protein (a SecB-independent protein) does not cause interference, while a defective RBP signal peptide-mature MBP hybrid does (Collier et al., 1990). Hence at least in the case of MBP the mature portion is not only sufficient to mediate SecB binding, but is also necessary. The mature portion of MBP is responsible not only for SecB binding, but also determines the SecB dependence for export (Collier et al., 1990; Gannon et al., 1989), with the first 74 residues of mature MBP being sufficient to impart a SecB requirement for efficient export (Gannon et al., 1989). While secretion of the outer membrane lipoprotein (Lpp) is SecB independent, efficient secretion of a hybrid containing the signal peptide and the first 11 aminoacyl residues of the SecB-dependent mOmpF protein fused to mLpp requires SecB (Watanabe et al., 1988). Hence it is possible that the OmpF signal peptide, or the first 11 residues of the mOmpF (or both), are sufficient to impart SecB dependence on Lpp. An interfering region (IR) has been mapped to a 61-amino acid residue stretch near the carboxy terminus of the mature LamB protein [residues 320 to 380 (Altman et al., 1990a)l. Insertion of these 61 residues into

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DAVID N. COLLIER

LacZ is sufficient to convert LacZ from a noninterfering protein to a strongly interfering species. However LamB-SecB complexes cannot be isolated from cells if the LamB signal peptide contains a deletion within the hydrophobic core, despite the presence of the IR. These results suggest that LamB contains t w o or more SecB binding sites-one in the mature portion and one that overlaps, at least in part, with the signal peptide (Altman et al., 1990b). Each of several different deletions within the mature moiety, but not removal of the signal peptide, decreases the extent to which SecB and PhoE interact (de Cock et al., 1992). Together these observations suggest that several domains, located primarily or exclusively within the mature moiety of the precursor, interact simultaneously with discrete sites on a single SecB multimer (see Section VI1,C). The overall affinity of SecB for a polypeptide would therefore represent an aggregate of the individual interactions, each of which could vary widely. Hence, the affinity of SecB for residues 320-380 of LamB may be sufficiently high to impart an interference phenotype, but in the absence of an additional site around the signal peptide, the overall affinity is too low to allow isolation of a complex. Conversion of LacZ to an interfering species by insertion of the IR could be the result of the affinity of SecB for the IR alone, but it is also possible that the IR works in conjunction with low-affinity sites already in LacZ to create a hybrid target for which SecB has sufficient avidity to cause interference. B. SecB Binding Nonnative Proteins

T h e overwhelming body of evidence demonstrates that SecB binds within the mature moiety of MBP, not the signal peptide. However, two predictions of this axiom have not been met: (1) synthesis of mMBP should cause interference (it does not) and (2) mMBP synthesized in vitro should form detectable complexes with SecB (it does not). A resolution of this apparent paradox lies in the ability of the signal peptide to influence the rate of precursor folding and therefore indirectly affect SecB binding. Employing an in vitro competition assay (see Section VI,C), denatured BPTI, RNase A, the a subunit of tryptophan synthase, and an unstable mRBP variant were each found to compete with mMBP for SecB binding (Hardy and Randall, 1991). Half-maximal competition occurred at molar ratios (competitor:MBP) of about 2.5,4,6, and 25, respectively. A dissociation constant of approximately 5 nM for the SecB-BPTI complex was calculated from the binding curve obtained from stepwise addition of carboxamidomethylated BPTI (R-BPTI) to a solution of SecB (the reaction is monitored by the increase in fluorescence that results when SecB

SecB: MOLECULAR CHAPERONE

175

interacts with R-BPTI). From these values apparent relative dissociation constants of 1.5, 8, 20, and 50 nM were calculated for SecB complexed with mMBP, RNase A, the (Y subunit of tryptophan synthase, and mRBP, respectively. When assayed in their native states, these proteins did not compete with MBP for SecB binding (stabilization of the folded form of the mRBP variant by addition of ribose was required for loss of competitiveness). These results indicate that SecB specifically recognizes nonnative structures and are corroborated by the observation that once folded, neither preMBP nor mMBP is a substrate for SecB (Randall et al., 1990; Weiss and Bassford, 1990). Since SecB specifically recognizes nonnative states, then the ability of the signal peptide-even an export-defective signal peptide-to slow the folding of the precursor relative to the mature protein (Laminet and Pluckthun, 1989; Liu et al., 1989; Park et al., 1988; Teschke et al., 1991) facilitates the interaction with SecB. If the primary role of the signal peptide in SecB binding is to prolong the lifetime of' nonnative states, then mutational alterations in the mature moiety that favor nonnative states should also enhance SecB binding. For MBP this is indeed the case, since mMBP-Y283D, which folds more slowly than mMBP (Liu et al., 1988), is an interfering species (Liu et al., 1989) and complexes with SecB in vitro, while mMBP does not (Weiss and Bassford, 1990). Although it is possible that the tyrosine-to-aspartic acid substitution in MBP-Y283D creates a new SecB binding site, or directly enhances an existing weak site, this is unlikely since mutationally altered species as diverse as the MBPA323 deletion derivative [missing amino acids 7 (of the signal peptide) through 89 (of the mature peptide)] also cause interference and bind SecB in vitro (Weiss and Bassford, 1990). Because SecB apparently recognizes features displayed by polypeptides in their nonnative state, and precursors such as preMBP are estimated to fold from 20 to 660 times more slowly than mMBP or cytosolic proteins, it has been proposed that the relatively slow rate of presecretory protein folding is a discriminating feature of proteins destined for export (Hardy and Randall, 1991; Randall and Hardy, 1989). Slow folding would favor interaction with secretion-related chaperones such as SecB, which in turn would facilitate targeting to the secretion machinery (Liu et al., 1989). If an important property of the signal peptide is simply to slow folding, then a diverse array of peptide sequences might be expected to fulfill this role. Indeed, when substituted for a wild-type signal peptide, over 20% of essentially random peptide sequences support, to varying degrees, secretion of invertase in yeast (Kaiser et al., 1987), and an unexpectedly high percentage of quasi-random DNA fragments of bacterial

176

DAVID N. COLLIER

origin encode polypeptides that sponsor secretion in B . subtilis and E. coli (Smith et al., 1987,1988),or translocation into yeast mitochondria (Baker and Schatz, 1987). Under certain circumstances even the vestiges of a signal peptide are not required for export, as demonstrated by the low level of mMBP (synthesized without a signal peptide) secreted by E. coli harboring the prlA666 allele of secY. However, this secretion is completely SecB dependent (Puziss et al., 1992), reinforcing the ideas that (1) SecB binds within the mature portion of the protein and (2) modulators of folding can sponsor a modicum of secretion even in the absence of a normal signal peptide. The rate of precursor folding clearly influences the ability of SecB to bind. However, despite a t,,, of refolding of about 12 min for prepla (Laminet and Pluckthun, 1989), SecB neither retards the rate of prepla folding (Laminet et al., 1991) nor complexes with prepla (de Cock et al., 1992). Similarly, SecB has no detectable influence on the rate of preRBP folding (Weiss et al., 1988) and does not bind preRBP in vivo (Collier et al., 1990) despite similar relaxation times for preRBP and preMBP (Liu et al., 1988; Teschke et al., 1991; Park et al., 1988). Hence the ligand selectivity of the SecB cannot be based solely on the rate of folding of a target protein. Rather, a SecB ligand must comprise a sufficiently slowfolding species that also contains sequence or structural elements not common to all polypeptides. C . SecB Binding Positively Charged Ligands

The propensity of SecB to bind a broad range of denatured proteins-including nonsecretory proteins [phage T 4 tail fiber (MacIntyre et al., 1991) and the CY subunit of tryptophan synthase (Hardy and Randall, 1991)] and proteins not native to E. coli [BPTI and RNase A (Hardy and Randall, 199l)]-suggests that SecB recognizes rather degenerate features commonly displayed by many polypeptides in their nonnative states. Exposed hydrophobic domains are frequently posited as the sites of chaperone binding (Hemmingsen et al., 1988; Pelham, 1986), but in the case of SecB, hydrophobic interactions are likely not the primary source of binding selectivity. First, regions within mMBP and mLamB implicated in SecB binding include the most hydrophilic stretches of the protein. Second, the hydrophobic core of the signal peptide is clearly the most hydrophobic domain of the protein, yet the signal peptide plays primarily an indirect role in SecB binding. Third, binding studies (described below) employing oligopeptides indicate that a local net positive charge is required for SecB binding (Randall, 1992). Unliganded SecB is in a “loose” conformation in which the carboxy

SecB: MOLECULAR CHAPERONE

177

terminus is susceptible to cleavage by proteinase K. When complexed with ligands or in high-ionic-strength buffer, SecB adapts a more compact structure as evidenced by changes in its circular dichroic spectra and a decreased protease sensitivity. Employing protection from proteolysis as an assay, Randall (1992) has surveyed a set of polypeptides ranging in size from 7 to 260 aminoacyl residues for their ability to bind SecB (see Tables I1 and 111). A feature common to protective peptides is a net positive charge. Negatively charged [for example, poly(~-Glu)] or neutral [poly(Pro-Gly-Pro)] oligopeptides showed no protection. Bradykinin and [Met,Lys]bradykinin, containing 9 and 1 1 residues and net positive charges of 2 and 3, respectively, show no protection, while somatostatin and mastoparan, containing 14 residues and net positive charges of 2 and 4, respectively, protect SecB from proteolysis. This result suggests that in addition to a positive charge, a SecB ligand must contain a minimum of 12 to 14 residues. However, POIY(L-LYS) containing as few as 8 residues, but not 7 residues, affords protection. Hence increased charge density appears to compensate for length and a minimum length is therefore probably 8 aminoacyl residues for peptides with a high charge density. To protect 1 mol of SecB tetramer requires 4 mol of denatured BPTI (R-BPTI), indicating that the functional SecB unit has multiple substrate-binding sites (the SecB concentration in the assay was about 120 times the estimated dissociation constant for R-BPTI, i.e., 600 vs. 5 nm). It has been shown that 4 o r 5 mol of SecB tetramer are protected y ~ ) 260 residues), demby 1 mol of high-molecular-weight p ~ l y ( ~ - L(about onstrating that a single polypeptide ligand can bind several SecB multimers. Approximately 14 residues of p ~ l y ( ~ - Lligand y ~ ) are apportioned for each binding site of SecB. OmpA, MBP, and LamB are each SecB ligands, yet have acidic isoelectric points and are therefore expected to carry net negative charges ranging from -3 to -17 at normal cytoplasmic pH values [7.5-7.8 (Maloney, 1987)l. However, the mature domains of these proteins contain three or more segments of 14 or more aminoacyl residues carrying a net positive charge of 2 or more (these segments are generally devoid of acidic residues). Although the regions within MBP and LamB implicated in SecB binding (see Section V1,A) have a net negative charge, a segment with well-isolated positive charges is found within the interfering region of mLamB, and one is found very close to the region of mMBP implicated in SecB binding. In contrast, RBP and Pla, which are not SecB substrates, carry only two such segments, and within these segments the charge separation is less pronounced. Therefore it is likely that these charged patches play a role in the selective binding of natural substrates by SecB. T h e interaction of SecB and ligands has also been examined with

Concentration for 50% protection Peptide pa SI

SIh +

-I 92

s4 Melittin Zinc finger (no zinc) Defensin HNP-I Defensin NP-I Defensin NP-5 Somatostatin Mastoparan

(PM)

ti <3 <3

Net charge

Scq11cntc

+2

K\'FYNAKA(;IKQTF VIEVVQGAYKAIKHIPRKIR DRC'IEVVQGAYRAIKHI PRRIRQG h."N'I'RKSIRIQKGPGKAF~~l~I(;KI(; < ; I ( ; . ~ V I . K \ ' I . ~ I T ( ~ L P A L l SKRKRQQ-NH2 ~l RSFV<:EV<~TKAFAKQEHI.I(RHYRSH-INFK A<:Y(:KII'A<:IA<;IRR~~;-T<:I\'Q<;RI.WAF(;C VV<:.4(;KKAI.<:I.PREKRA(;F(:KIR(;RIHPL(;~;RR VF(:~I'<:K(;Fl.(;<;S<;ERAS~;S~~~I~l NGVRHTLCCRR AG<:liN FFM'KI'FI'SC INI.KAI.AAI.AKKlI.-SH.,

i-4

4

+4 +6 +6 +4 +3 +Y +4 +2

12

+4

1

3.5 <4

2 <3 <3

' Incubation mixtures (0.4 nil 10 m M HEPES, pH 7.6) containing 4 p g purified SecB with test peptides \wrc incuhatcd on ice for 20 min with or without proteinase K (0.3 pg/ml). Proteolvsis was detected hv electrophoresis on SDS-I5% poI!.;icrylamitlc gel. l'@ is Kroin BPI'I; SI. Slh, and S4 are from H I V gp41, and the zinc finger is from ADRI. For calculating net charge. histidine residues were siniply ignored. Adapted from Randall (1992). Copyright 1992 hv the AAAS.

Concentration tested (piM)

Peptide

Net charge +2

Bradvkinin [Met,Lys]Bradykinin SO26-B

9 2-8 70

Pa

1-100

Glucagon GCN4-pI FOS-p 1

1-7

-1

3-50

0 -5

~ ~ ~ _ _ _ _ _~ _ ______

0 0

0.4-40 ~~

KPPGFSPFK MKKPPGFSPFK Ac-SLNAAKSEl.I)KAI(;-N Hz N N FKSAEDC:hl KI'A(;<;A HSEG~I'~rSI)~SK~l.DSKKAQl~~\'~~Lh~N.r Ac- KM KQLEI) KV EELLS K NY H LEN EVA KLK KLVGER Ac-CGCLTDTLQAETDQLEDKKSALQTEI ANLLKEKEKLEFILAAY

+s

~____

~

~~

~

~~

~

~ _ _ _

~

~

~~

Protection was determined as described in Table 11. S026-B is lroin '1.4 lysozvnie, P, is lroni B P H .GNC4-pI and Fos-pl are leucine zippers; Ac, acetyl. Histidines were ignored in determining net charge. Adapted Irom Randall (1992). Copyright 19W by the AAAS. "

180

DAVID N. COLLIER

the fluorescent probe l-anilinonaphthalene 8-sulfonate (ANS) (Randall, 1992). Because the intensity of ANS fluorescence increases when it associates with hydrophobic domains in polypeptides, ANS can be used to probe the surface of proteins for hydrophobic patches. Interestingly, ANS fluorescence increases in the presence of SecB complexed with ligands, but only when the binding sites on SecB are saturated. This suggests that full occupation of SecB induces a conformational change that exposes a hydrophobic domain. This domain may provide an additional type of ligand binding site that accommodates hydrophobic elements in the substrate, or it may facilitate interaction with other components of the secretion machinery. Hydrogen bonding between the backbone of the polypeptide chain exposed in extended structures and hypothetical 0 strands in SecB has also been proposed to sponsor SecB binding (Hardy and Randall, 1991). In a comparison of the three-dimensional structures of MBP and RBP (Spurlino et al., 1991; Teschke et al., 1991), de Cock et al. (1992) have pointed out that the putative interfering region of MBP is contained in an antiparallel 0 sheet that is not present in RBP and propose that this structure might be the salient feature of MBP that is recognized by SecB.

VIII. OTHERCHAPERONES A N D PROTEIN SECRETION A. Genetic Selections: Heat-Shock Proteins Partially Replacing SecB What is the likelihood that SecB is the sole secretion-related chaperone? If one assumes that the efficient export of each envelope protein requires a chaperone, then the low abundance of SecB and the SecB-independent export of several envelope proteins argue that there must be additional secretion-related chaperones. However, there is no a priori reason to expect that the efficient export of each secretory protein requires a chaperone, since the folding pathways or other properties of different precursors are likely to be variable. In fact, the observation that minor changes in the primary sequence of preMBP can virtually eliminate the SecB dependence of this protein (Collier and Bassford, 1989; Section IX,A) challenges the assumption that secretion of each envelope protein must be chaperoned by SecB or a SecB analog. To determine if additional chaperones are involved in secretion, two different genetic selections have been employed. One selection exploited the inability of a malE16-1, secB::Tn5 mutant to utilize maltose as sole carbon source (Mal- phenotype). Since growth on maltose requires the proper localization of MBP to the periplasm, the complete block in

SecB: MOLECULAR CHAPERONE

181

preMBP16-1 export that results from loss of SecB function (see Fig. 1) imparts a Mal- phenotype. It was postulated that selection for growth on maltose (Mal’, due to restoration of MBP export) would yield mutants in which the function of an alternative chaperone was recruited, either by its overproduction and/or a broadening of its specificity, to include preMBP16-1 as a substrate. Although Mal’ revertants were easily obtained, all of the revertants that were analyzed resulted from either mutational alterations in the signal peptide coding region of malE, or a “precise excision” of the T n 5 from within the secB::Tn5 allele (Collier and Bassford, 1989). Hence this selection did not identify additional chaperone candidates. It is probable that the selection was too stringent, and that a selection based on secretion of preMBP species whose signal peptides are less dysfunctional than that of preMBP16-1 might prove fruitful. Mal+ revertants were obtained in which t w o (T16, T17) or three (T16, T17, M18) aminoacyl residues of the MBP signal peptide had been deleted. In sharp contrast to wild-type preMBP, the preMBP derivatives with truncated hydrophobic cores displayed a high degree of cotranslational processing in SecB- cells, with a significant increase in both the efficiency and extent of MBP secretion. T h e mechanism by which the truncated signal peptides reestablish cotranslational secretion in the absence of SecB is not understood, but it is likely that cotranslational secretion suppresses the requirement for SecB simply by allowing translocation to occur before the precursor is able to assume an exportincompetent conformation (Collier and Bassford, 1989). In this context it is interesting to note that secretion of the SecB-independent species prepla, preRBP, and preMBP4mRBP occurs entirely posttranslationally (Collier et al., 1990; Randall, 1983). Therefore, cotranslational secretion is not a general mechanism employed by SecB-independent species to circumvent SecB function. The Y283D substitution in mMBP, in cis to the signal peptide with the three-residue deletion, further stimulated secretion. Presumably the antifolding effects of Y283D extend the posttranslational competence of the fraction of molecules that are not cotranslationally secreted. A second selection exploited the inability of cells harboring the secB::Tn5 allele to form isolated colonies on rich media. Again, it was postulated that the overproduction and/or a broadening of ligand specificity of other chaperones could partially supplant the lost SecB function and restore the ability to grow on rich media. A majority of the revertants isolated employing this selection contained mutations that map to rpoH, the gene encoding the RNA polymerase sigma factor u3*. Since d2is responsible for the high-level transcription of the heat-shock genes, this

182

DAVID N. COLLIER

result indicates that heat-shock proteins can, at least partially, substitute for SecB. Indeed, induction of the heat-shock response by overproduction of u3*not only alleviates the rich media sensitivity, but partially suppresses the defects in MBP, LamB, and OmpA export caused by the loss of SecB (Altman et al., 1991). Interestingly, disruption of secB induces constitutive synthesis of heat-shock proteins at five to six times normal levels (Wild et al., 1993, cited in Wild et al., 1992), but apparently even this elevated level of HSP expression is not high enough to allow growth on rich media. These results suggest that, to a certain extent, HSP and SecB functions overlap. If this were so, then low levels of HSPs, which could be tolerated by cells producing haploid levels of SecB, might prove insufficient in the absence of SecB. Indeed, basal levels of heat-shock proteins are required for viability of a secB::Tn5 mutant under all conditions, including growth on minimal media (Altman et al., 1991; Wild et al., 1992). While a majority of the mutations allowing growth on rich media mapped to rpoH, additional mutants were obtained in which the compensating mutations mapped elsewhere. These mutations fell into two distinct groups, neither of which mapped to known heat-shock o r secretionfactor loci. Hence it is tempting to speculate that these mutations have identified genes encoding novel secretion-related chaperones (Altman et al., 1991). B . Role for GroELIES in Secretion GroEL facilitates the secretion of LamB-LacZ hybrids in vivo (Phillips and Silhavy, 1990) and has “chaperoning activity” that stimulates the translocation of precursors into IMV in vitro (Bochkavera et al., 1988; Lecker et al., 1989), thus it would not be unreasonable to expect that GroEL/ES is responsible for the suppressing effect associated with the heat-shock response. However, overproduction of GroEL andlor GroES [under lacUV5 promoter control on a multicopy plasmid (Kusukawa et al., 1989)or under groE promoter control on a multicopy plasmid (Altman et al., 1991)] suppressed neither the secretion defect nor the rich media sensitivity associated with the loss of SecB function. Consistent with the inability of GroELIES to suppress the secB null defect, functional depletion of GroEL o r GroES does not cause pleiotropic secretion defects. Among the proteins examined to date only the export of j3la is adversely affected by mutational alterations in GroEL or GroES [note that GroEL or GroEL/ES can block the refolding of denatured j3la or effect a net unfolding of native @la (Laminet et al., 1990)], while the secretion of OmpA, MBP, OmpF, LPP, PhoA (Kusukawa et al., 1989),and RBP (Wild et al., 1992) is unaffected.

SecB: MOLECULAR CHAPERONE

183

Although overproduction of GroEL/ES suppresses both the growth and secretion defects associated with the heat-sensitive secA5I and secY24 alleles, overexpression of GroELlES does not sponsor growth at nonpermissive temperatures of a strain in which the synthesis of SecA is heat sensitive (Van Dyk et al., 1989). This implies that, at least in the case of SecA, the chaperones do not directly supplant the Sec protein function. Hence a direct role for the GroELlES chaperones in normal in uivo secretion is likely limited to a few (maybe just one?) secretory proteins. C . DnaKIDnaJ as Secretion-Related Chaperones

Stress-70 or HSP70 homologs have been shown to be broadly involved in protein trafficking in a variety of organisms (reviewed in Ang et al., 1991; Gething and Sambrook, 1992). In yeast, for example, the antifolding activities of the cytosolic stress-70 homologues Ssal p and Ssa2p are required for translocation of precursors across the endoplasmic reticulum and mitochondria1 membranes (Chirico et al., 1988; Deshaies et al., 1988), while stress-70 proteins residing in the matrix of mitochondria and the lumen of the endoplasmic reticulum also modulate the folding, assembly, and trafficking of proteins (Gething and Sambrook, 1992). Since DnaK is the HSP70 homologue of E. coli, DnaK is a likely candidate among the HSPs to supplant SecB function. Several lines of evidence suggest that DnaK is indeed involved in protein secretion in E. coli. Overproduction of DnaK has been shown to relieve the lethal “jamming phenotype” associated with the abortive entrance into the export pathway of certain LamB-LacZ hybrid proteins. Presumably DnaK facilitates export of the hybrid protein by maintaining it in a secretion-competent conformation (Phillips and Silhavy, 1990). Overproduction of DnaJ [a HSP that stimulates DnaK activity (Liberek et al., 1991)] and DnaK together (Wild et al., 1992), but not DnaK alone (Altman et al., 1991; Wild et al., 1992), sponsors growth of some, but not all, secB null strains on rich media and partially suppresses the defects in MBP and LamB, but not OmpA export (Wild et al., 1992). Consistent with the inability of DnaK to suppress the defect in OmpA secretion in vivo, DnaK does not stimulate the translocation of proOmpA into IMV (Lecker et al., 1989). However, the defect in OmpA secretion associated with loss of SecB function can be partially suppressed by full induction of the heat-shock regulon (Altman et al., 1991). While dominant missense alleles of dnaK and dnaJ have been found to impart a kinetic defect in the processing of PhoA, depletion of DnaK and DnaJ in a secB+ background has little influence on the processing of LamB, MBP, o r OmpA. However, the processing of both MBP and LamB in cells devoid of SecB is severely affected by reduction in DnaK/DnaJ levels. Interestingly, as

184

DAVID N. COLLIER

is the case in secB null strains, loss of DnaK function does not affect the rate of RBP secretion (Wild et al., 1992).

D. Trigger Factor and Role in Secretion Trigger factor is an abundant ribosomally associated protein (Lill et al., 1988) that was identified and purified based on a chaperoning activity that stimulates the translocation of proOmpA into IMV (Crooke et al., 1988; Crooke and Wickner, 1987). However, unlike the case of cells lacking SecB, cells depleted of trigger factor do not exhibit pleiotropic secretion defects. T h e in vivo secretion of OmpA is unaffected by the depletion of trigger factor (Guthrie and Wickner, 1990) despite the fact that, in vitro, trigger factor stabilizes a translocation-competent conformation of proOmpA (Crooke et al., 1988; Crook and Wickner, 1987; Lecker et al., 1989). Even in cells devoid of SecB, trigger factor depletion caused no additional defect in the secretion of OmpA (Guthrie and Wickner, 1990). Hence trigger factor does not appear to play a role in the secretory pathway in vivo, either as a primary or a secondary secretion-related chaperone.

IX. RECAPITULATION AND SPECULATION A . SecB as Major Secretion-Related Chaperone Efficient secretion of most E. coli envelope proteins depends on molecular chaperones. T h e heat-shock proteins GroEL/GroES and DnaK/ DnaJ serve as the primary secretion-related chaperones for pla and PhoA, respectively. DnaK/DnaJ also play a secondary role in the secretion of other envelope proteins, a role that is partially redundant with SecB function. The binding of a chaperone such as SecB to a precursor may sponsor efficient export by (1) slowing the rate of precursor folding and/or (2) blocking aggregation and/or (3) preventing nonproductive interactions with the membrane. Despite its low abundance (0.08% of the cytosolic protein), SecB is the primary secretion-related chaperone for a majority of E. coli envelope proteins because of its ability to target precursors to the preprotein translocase. Targeting is via specific, highaffinity interactions between SecB-precursor complexes and membranebound SecA. While a chaperone such as GroEL is able to maintain precursors in a translocation-competent state, GroEL has no significant targeting activity as evidenced by the inability of GroEL/proOmpA and SecA to form ternary complexes (Hart1 et al., 1990). T h e readiness with which proOmpA is exchanged from preformed

SecU: MOLECULAR CHAPERONE

185

GroEL complexes into complexes with SecB demonstrates that SecB binds proOmpA with higher affinity than does GroEl (Lecker et al., 1989). Though a comparison of the relative affinities of SecB and other chaperones for additional precursors has not been reported, it is possible that SecB binds many secretory precursors with a higher affinity than do other chaperones. If this is so, then selective binding of precursors by SecB would ensure that the relatively small pool of SecB can compete with the more abundant chaperones (which lack targeting activity). A lack of targeting activity could explain why the overproduction of DnaK and DnaJ only partially suppresses the export defects caused by the loss of SecB. As is the case with the slow-folding mMBP-Y283D mutant, suppression of the secB::Tn5 defect by overproduction of DnaK/ DnaJ is primarily mediated by enhanced posttranslation chase (Wild et al., 1992), which results from maintaining an export-competent conformation. In contrast, certain mutational alterations in the signal peptide of MBP result in a high degree of SecB-independent export of this protein by dramatically increasing the level of cotranslational processing. Export of an MBP species that carries such a signal sequence mutation and the Y283D mutation approaches the efficiency with which wild-type MBP is secreted in SecB' cells. While it is possible that these alterations in the primary sequence have converted this M BP species to a substrate for another chaperone(s), it is most likely that the intrinsic properties of the precursor have been altered such that its secretion has a less stringent requirement for a chaperone. Secretion of RBP has been found to be completely independent of SecB, GroEL/GroES, or DnaK. An intriguing question is whether there is an additional chaperone(s) that facilitates secretion of RBP, or whether preRBP is simply a model precursor whose efficient secretion does not require a chaperone function. Because alterations within the preMBP signal peptide can impart a degree of SecB-independent MBP export, then it is plausible that the RBP signal peptide is responsible for the SecB-independent export of this protein. However, the SecBindependent export of RBP cannot be ascribed exclusively to its signal peptide because secretion of an MBPaRBP chimera is SecB independent, whereas efficient secretion of an RBPaMBP chimera is SecB dependent. Tetramers of SecB contain multiple binding sites that are selective for polypeptides with exposed extended stretches carrying a local net positive charge and a minimal length of 8-14 aminoacyl residues (depending on the charge density). Precursors for which SecB has no significant affinity (preRBP, prepla) contain fewer such sequences within their mature domains than precursors that are bound by SecB (preMBP, preLamB). By virtue of its basic amino terminus, the signal peptide could comprise such

186

DAVID N. COLLIER

a site. However, at least in the cases of MBP and KBP, the signal peptide is neither sufficient nor strictly necessary for SecB binding. T h e antifolding effect of the signal peptide does, however, favor interaction of SecB and the precursor, since SecB specifically binds nonnative proteins. While it seems oxymoronic that an antifolding factor requires a slow-folding ligand, it is likely that the comparatively slow rate of precursor folding helps to distinguish them from cytoplasmic proteins and contributes to the overall specificity of the interaction of a precursor with the secretion machinery. In this light the degeneracy of signal peptides is more easily understood. On full occupation of the peptide ligand binding sites, the SecB tetramer undergoes a conformational shift that further exposes a hydrophobic patch. Perhaps this patch represents a second type of ligand binding site that interacts with hydrophobic domains within ligands, or it may participate in interactions with components of the secretion machinery such as SecA. When SecB is complexed with mMBP, the patch is no longer exposed, arguing that it is probably occupied by the ligand and therefore unavailable for direct interaction with SecA. It is tempting to speculate that an interaction between the hydrophobic core and the patch keeps the signal peptide accessible and therefore plays a role in targeting. Several different domains within a single precursor molecule are likely to interact simultaneously with the multiple binding sites on a single tetramer of the chaperone. The overall affinity will therefore be the result of several interactions of varying strength, some of which may be quite weak individually. This type of binding could be described as a “zipper model” in the sense that the strength of a zipper is the result of many weak interactions. Mutational alterations in mMBP, whose only common feature is the perturbation of MBP structure, partially supplant SecB function during MBP secretion. However, a similar alteration in mLamB does not suppress the defect in LamB secretion associated with the loss of SecB. Unlike preMBP, proteins such as OmpA, LamB, or PhoE display little tendency to assume their native state outside the context of the outer membrane. Therefore antiaggregation and/or targeting activities of SecB, rather than antifolding properties, may represent the predominant activities that sponsor secretion of these outer membrane proteins, while its antifolding activity is also essential for efficient MBP secretion.

B . Recognition Elements as Troublemakers The basis of the selectivity of SecB in binding is likely the result of a complex interplay between several factors, including relatively degenerate structural elements, their spacing and sequence context, and the

SecB: MOLECULAR CHAPERONE

187

inherent rate of substrate folding. It is interesting to note that the preference of SecB for ligands carrying positive charge is in sharp contrast to that of BiP (a HSP70 of the ER lumen), which preferentially binds ligands enriched in hydrophobic aliphatic residues and specifically excludes from its binding site ligands containing basic residues (Flynn et al., 1991). In fact, copolymers of Lys and Val or Lys and Leu were only half as effective as poly(Lys) in protecting SecB from proteolysis (Randall, 1992). Since exposed hydrophobic domains are thought to favor aggregation, and BiP selectively binds hydrophobic ligands, it is tempting to speculate that the features of a substrate that are recognized by a given chaperone actually define, in part, the features that cause the substrate to “misbehave” when unchaperoned. This hypothesis would imply that patches of unmasked positive charge, which may provide the basis for selective binding by SecB, inhibit secretion. Although there is no direct evidence of this, salt bridging with the acidic phospholipids of the membrane could provide the mechanism by which translocation is inhibited. In fact, there is a strong electrostatic component in the interaction of a synthetic PhoE signal peptide (with its positive amino terminus) and phospholipid monolayers containing cardiolipin (Batenburg et al., 1988), demonstrating that such interactions do occur. consistent with this hypothesis, proOmpA binds to vesicles in a state that is translocation incompetent in the absence of SecB (Hart1 et al., 1990). However, the nature of this binding is not known.

C . Secretion of Precursors Lacking IR If a precursor were relieved of its SecB-binding sites, would its secretion in SecB+cells resemble secretion of the wild-type precursor in SecBcells? For MBP, the sites contributing to SecB binding have not been clearly identified or cleanly removed (perhaps an impossible task). Furthermore, SecB binding and SecB dependence are partially dissociable, since mutations that alter folding both suppress the SecB requirement for export and actually enhance SecB binding. Based on the loss of the interference phenotype, a discrete region (albeit 6 1 aminoacyl residues) within mLamB has been implicated in SecB binding. Surprisingly, in SecB’ cells, secretion of LamB lacking this interfering region is indistinguishable from that of LamB’ (Altman et al., 1990b). However, if the rate of LamB secretion is slightly lowered by virtue of a weakly defective LamB signal peptide, then loss of the IR imparts a significant defect in the export kinetics in SecB’ cells. Furthermore, preLamB carrying the IR, but not preLamB lacking the IR, trapped in the cytoplasm by the uncoupler CCCP is competent for posttranslational export when the translocation block is relieved by the addition of 2-mercaptoethanol (Altman et al., 1990b). Evidence that the LamB signal peptide directly

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participates in SecB binding suggests that SecB interacts with LamB signal peptide mutants lacking the IR, but with insufficient affinity to impose an interference phenotype.

D. Translocation Driving Dissociation For many combinations of chaperones and substrates, discharge of the ligand requires the binding and/or hydrolysis of ATP (reviewed in Gething and Sambrook, 1992). However, since ATP seems to play no role in SecB function, ATP hydrolysis cannot directly drive the discharge of ligands from SecB. Each bound domain of the ligand exists in equilibrium with an unbound state, as demonstrated by (1) the ability of excess unliganded SecB to strip ligand from preformed complexes (Randall, 1992) and (2) the ability of a competing species to displace MBP from SecB-MBP complexes (Hardy and Randall, 1991). SecBligand complexes appear to result from several interactions, some of which could be weak. If transiently unbound domains were to complex with other proteins such as SecA or SecY, they would be unavailable for rebinding SecB and an “unzippering” would lead to discharge from SecB. Translocation of domains associated with SecA-SecY would render them inaccessible to SecB and therefore drive the “unzippering” to completion. Consistent with this hypothesis is the observation that the prolonged interaction between export-defective preMBP species and SecB, which is manifested as an interference phenotype, is relieved by extragenic suppression of the defective signal peptide by prlA mutations [alleles of secY (Bankaitis and Bassford, 1984)]. Relief of interference results from freeing the small pool of SecB (Collier et al., 1988) and therefore can be considered as a measure of the rate of discharge or unzippering, while suppression is a measure of the rate of domain translocation. Furthermore, the extent to which the interfering phenotype is relieved is strictly correlated with the strength of suppression (Bankaitis and Bassford, 1984). These genetic experiments suggest that SecA alone is insufficient to trigger discharge (rather a functional interaction with SecY is required) and are corroborated by the observation that preformed precursor-SecB complexes form ternary complexes with soluble SecA in vitro, rather than dissociating in the presence of SecA. ACKNOWLEDGMENTS This review is dedicated to the memory of’PhillipJ.Basstord,Jr. who died on December 22. 1991. at the age o f 44. His death is a tremendous loss to the field and his many friends and colleagues.

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I thank colleagues who shared unpublished results, acknowledge the many stimulating discussions that I have had with Phil Bassford. Sharon Strobel, Elliot Altman, and Lin Randall, and I especially thank Lin Randall for her critical reading of this manuscript.

REFERENCES Altman, Altman. Altman. Ang, D..

E.. Bankaitis. V. A.. and E m . S. D.(lY90a).J. B i d . Clrutri. 265, 18148-18153. E.,Emr. S.D., and Kuinamoto. C. A. (lYYOb).j. B i d . Cl/utr/. 265, 38154-18160. E.. Kumanioto, C. A., and Emr, S. D. (1991).E M B O J . 10, 239-245. Liberek. K.. Skowyra. D.. Zylicz, M.. and Georgopodos, C. P. (199I).,]. B i d . Clietn.

266, 24233-24236. Baker. A.. and Schatz, G. (1987).Pror. Nrr/l. Arad. Sri. U.S.A. 84, 31 17-3121. Baker. K. P..and Schatz, G . (1991).Nalrrru (Lotidoti) 349, 205-208. Bankaitis. V. A.. and Bassford, P.,I.. Jr. (1Y84)./.Bid. Clierri. 259, 12193-12200. Bankaitis. V. A.. Rasniussen. €3. A.. and Bassford, P. ,I.. J r . (1984).Cull (Cartrbridgu, Mass.)

37, 243-252. Bankaitis. V. A., Ryan, J. P.. Rasniussen. B. A., and Bassford, P.J., Jr. (1985). Curr. 7 u p . Membr. Tratup. 24, 105-150. Bassford, P. 1.. Jr. (lY90).J.Biorrierg. Biomurrd~t.22, 401-439. Bassford. P.1.. Jr., Silhavy. T. J., and Beckwith.]. R. (1979).J.Bar/rriol. 139, 19-31. Batenburg, A. M., Demel, R. A., Verkleij, A. J., and de Kruijff. B. (1988).Biorlirrtiisl?p 27,

5678-5685. Bedouelle, M.. and Hofnung, M. (1981).f n “Membrane Transport and Neuroreceptors” (D. Oxender, ed.). pp. 309-343. Liss, New York. Bieker, K. L., and Silhavy. T. J . (1990).Cell (Carrhidgu, Moss.)61, 833-842. Bieker. K. L., Phillips, C . J., and Silhavv, T..J.(lYYO).J. Biourwrg. Biorr/rtr/bt. 22, 291-310. Bochkareva, E.S., Lissin, N. M.. and Girshovich, A. S. (1988).Nolriru (Loridor/)336,254-257. Briggs, M. S., and Gierasch, L. M. (1986).Adif. Proleiri C l i m . 38, 109-380. Briggs. M. S.,Gierasch, L. M.. Zlotnick. A.. Lear,,]. D.. and Degrddo. W. F. (1985).Sciericr

228, 1096-1099. Brundage. L., Hendrick, J. P.. Schiebel, E., Driessen, A. J . M., and Wickner, W. (1990). Cell (Cambridge, Mass.) 62, 649-657. Cabelli. R.J.. Chen. L., Tai, P. C., and Oliver, D. B. (1988). Cull (Carrrbridgu,Mass.) 55,

683-692. Cabelli. R. J., Dolan. K. M., Qian, L., and Oliver, D. 13. (1991).J . B i d . C/tert/. 266,

24420-24427. Chen. L., Rhoads, D., and Tai, P. C. (1985).J. Bmlrriol. 161, 973-980. Chirico, W. J., Waters. M. G.. and Blobel, G . (1988).Naliiru (Lortdotr) 322, 805-810. Collier. D. N., and Bassford, P.J., Jr. (1989).J . Barfrriol. 171, 4640-4647. Collier. D. N., Bankaitis, V. A., Weiss,J. B., and Bassford, P . J . , J r .(1988).Cell (Cambridge, MUSS.)53, 273-283. Collier. D. N., Strobel. S. M.. and Bassford, P. J., Jr. (lYYO).J. Burleriol. 172,6875-6884. Cover. W. H.,Ryan, J. P., Bassford, P. ,I., Jr., Walsh. K. A.. Bollinger, J., and Randall, L. L. ( 1987).J . Bacleriol. 169, 1794- 1800. Crooke. E., and Wickner, W. (1987).Pror. Nall. Acad. Sri. U.S.A. 84, 5216-5220. Crooke, E.,Brundage, L., Rice, M., and Wickner, W. (1988).E M B O J . 7, 1831-1835. Cunningham, K.. and Wickner, W. (1989).P w r . Natl. Arad. Sri. U.S.A. 86, 8630-8634. Cunningham, K., Lill, R., Crooke, E., Rice, M., Moore, K., Wickner, W., and Oliver, D. (1989). EMBV J . 8, 955-959.

190

DAVID N. COLLIER

d e Cock. H., and Tommassen, J. (1991).FEMS Micrubiol. 80, 195-200. d e Cock, H., and Tommassen, J. (1992).Mol. Mirrvbiol. 6, 599-604. d e Cock, H., Hekstra, D., and Tommassen, J. (1990). Bioclrinrie 72, 177-182. de Cock, H., Overeem. W., and Tommassen, J. (1992).J.Mu/. B i d . 224, 369-379. Deshaies, R. J., Koch, B. D., Werner-Washburne. M.,Craig, E. A., and Schekman. R. (1988). Nature (Lvtrdoti) 332, 800-805. Dev, 1. K., and Ray, P. H. (1990).J. Biuetierg. Bionie~rib.22, 271-290. Devereux, S., Haeberli, P., and Smithies, 0. (1984). Nucleic Acids Res. 12, 387-395. Dierstein. R.. and Wickner, W. (1985).J. B i d . C/retn. 260, 15919-15924. Eilers, M.. and Schatz. G. (1986). Nature (Lvtidotr) 322, 228-232. Fikes. J. D., and Bassford, P. J., Jr. (1987). J . Buclrriul. 169, 2352-2359. Flynn. G. C., Pohl. J.. Flocco, M. T., and Rothman, J. E. (1991). Nutiire (Lotidvti) 353, 726-730. Freudl, R., Schwarz. H.. Kramps. S.. Hindennach, I., a n d Henning, U.(l988).J. B i d . Chetn. 263, 17084- 17091. Fuqua. S. A. W.. Blurn-Salingaros. M.. and McGuire. W. L. (1989). Cuticer Res. 49, 4 126-4 129. Gannon. P. M.. Li. P.. and Kumamoto. C. A. (I989).J. Baclrriol. 171, 813-818. Bioetierg. Biottteabt. 22, 233. Gennity, J., Goldstein. J.. and Inouye, M. (1990).J. Cething. M.-J.. and Sanibrook, J. (1YY2). Nutiire (Lotidon) 355, 33-45. C h o r e , R., Walter. P., and Blobel. G. (1982).J.Cell B i d . 95, 470-477. Guthrie, B., and Wickner, W. (1990).J. Bucteriol. 172, 5555-5562. Hardy, S. J. S., and Randall, L. L. (1991). Scietice 251,439-443. Hartl, F. U.,and Neupert, W. (1990).Scietice 247, 930-938. Hartl, F. U.,Lecker, S.. Schiebel, E., Hendrick, J. P., and Wickner, W. (1990).Cell ( C a w bridge, Muss.) 63, 269-279. Hemmingsen, S. M.,Woolford, C., van der Vies, S. M.,Tilly, K., Dennis, D. T., Georgopoulos, C. P., Hendrix, R., a n d Ellis, R. J. (1988). Nature (London) 333, 330-334. Hickey, E.. Brandon, S. E.,Potter, R.. Stein. G.. Stein, J., and Weber. L. A. (1986).Niirleic Aridr RPS.14,4 127-4 145. Hindlev. J., and Phear, G . A. (1979). Niicletc A d s Res. 7, 361-375. Inouye. M.. and Halegoua. S. (1980). CRC CtY/. Rev. Bidrein. 7 , 339-37 1. Jones. J. D., McKnight. C.J.. and Gierasch, L. M. (1990).J. Biuetierg. Bionirnibr. 22,2 13-232. Josefsson. L. G., a n d Randall, L. L. (1981).Cell (Cuttibridge, Moss.) 25, 151-157. Kaiser. C. A., Preuss, D.. Grisaf, P.. and Botstein. D. (1987). Srietrre 235, 3 12-3 17. Kendall. D. A.. Bock. S. C., and Kaiser, E. T. (1986).Nutiire (Lotrifvtr) 321, 706-708. Kumarnoto, C. A. (1989). Prof. Null. Acnd. Sci. U.S.A. 86, 5320-5324. Kumamoto. C. A. ( 1990).J. Bioetirrg. Biontrttrbt. 22, 337. Kumamoto. C. A., and Beckwith. J. (l983).J. Bucteriol. 154, 253-260. Kumamoto, C. A.. and Beckwith, J. (1985).J. Burleriul. 163, 267-274. Kumamoto. C. A.. a n d Gannon, P. M. (1988).J. B i d . Chetn. 263, 11554-1 1558. Kumamoto, C. A., a n d Nault, A. K. (1989). &tie 75, 167-175. Kumamoto. C. A., Cheni, L., Fandl,.]., and Tai, P. C. ( 1989).,]. B i d . Chetn. 264,2242-2249. Kurzchalia, T. V., Wiedmann, J., Cirshovich. H. S., Bochkareva, E. S., Bielka. H., and R a p p o r t , T. A. (1986).Nature (Lotido~i)320, 634-636. Kusters, R.. de Vrije. T.. Breukink, E.. and d e Kruijff, 8. (1989). J . B i d . Chenr. 264, 20827-20830. Kusukawa, N., Yura. T., Ueguchi. C., Akiyania. Y.. and Ito, K. (1989). EMBO J . 8, 35 17-352 1. Laminet, A. A., and Pluckthun, A. (1989). E M B O J . 8, 1469-1477.

SecB: MOLECULAR CHAPERONE

191

Laminet, A. A., Ziegelhofter. T., Georgopoulos. C. P., and Pluckthun, A. (lY90). EMBOJ. 9,2315-2319. Laminet, A. A., Kumamoto, C. A., and Pluckthun, A. (lYY1). Mol. itlirrobiol. 5, 117-122. Lecker, S. H., Lill, R., Ziegelhoffer, T., Ceorgopoulos. C. P., Bassford. P . , l . . J r . .Kumamoto, C. A., and Wickner, W. (lY89). EMBO J. 8,2703-270Y. Lecker, S. H., Driessen, A. J. M., and Wickner, W. (lY90). EMBOJ. 9,2309-2314. Lee, C., Li, P., Inouye, H.. Brickman, E. R., and Beckwith, J. (lY8Y). J . Baclniol. 171, 4609-46 16. Liberek. K., Marsza1ek.J.. Ang, D., Georgopoulos, C. P.. and Zylicz, M. (l9Yl). Proc. Null. Acud. Sci. U.S.A. 88, 2874-2878. Lill, R., Crooke, E., Guthrie, B., and Wickner, W. (1988). Cell (Cambridge, Mass.) 54, 1013- 1018. Lilt, R., Cunningham. K., Brundage, L. A.. Ito, K., Oliver, D., and Wickner. W. (1989). E M B O J . 8,961-966. Lill, R., Dowhan. W., and Wickner. W. (I9Y0). Cell (Cambridgt, Mass.) 60, 271-280. Lingappa, V. R.. Chaidez, I., Yost, C. S., and Hedgpeth. J . (1984). Proc. Nutl. Acud. Sci. U.S.A. 81,456-460. Liu, G . , Topping, T. B.. Cover, W. H.. and Randall, L. L. (lY88). J . B i d . Chem. 263, 14790- 14793. Liu, G.. Topping, T. B., and Randall, L. L. (1989). Proc. Null. Acad. Scz. U.S.A. 86, 92 13-92 17. Maclntyre, S., and Henning, U. (1990). Biocliimie 72, 157-167. Maclntyre, S., Mutschler, B.. and Henning, U. (1991). Mol. Gun. Gene!. 227, 224-228. Maloney, P. C. (1987). In “Escliericliiu coli and Saliiionellu tvpvpliinzuriuvi’’(F. C. Neidhardt, ed.in-chief), pp. 222-243. Am. SOC.Microbiol., Washington, D.C. Meyer, D. 1. (1985). EMBOJ. 4,2031-2033. Meyer, D. I., Krause, E., and Dobberstein, B. (1982). Nature (London) 297, 647-650. Michaelis, S. H.. Inouye, H.. Oliver, D., and Beckwith, J. (1Y83).J. Bacleriol. 154,366-374. Ohno-lwashita, Y., and Wickner, W. (1983).J.B i d . Chetn. 258, 1895-IYOO. Oliver, D. B., and Beckwith, J. (1981). Cell (Cambridge, Mass.) 25, 765-772. Oliver, D. B., and Beckwith, J. (1982).J. Bacterial. 150,686-69 1. Oliver, D. B., Cabelli, R. J., and Jarosik, G. P. (1990). J . Bioenerg. Bzomembr. 22, 31137. Park, S., Liu, G., Topping. T. B., Cover, W. H., and Randall, L. L. (1988). Science 239, 1033- 1035. Partanen, J. M., Maekelae, T . P., Eerola, E., Korhonen, J., Hirvonen, H., Claesson-Welsh, L., and Alitalo, K. (1991). EMBOJ. 10, 1347-1354. Pelham, H. R. (1986). Cell (Cambridge, Mass.) 46, Y59-961. Perara, E., Rothman, R. E., and Lingappa, V. R. (lY86). Science 232, 348-352. Perlman, D.. and Halvorson, H. 0. (1983).J. Mol. B i d . 167, 3Y 1-40Y. Phillips, G . J., and Silhavy. T. J. (lY90). Nature (London) 344, 882-884. Pugsley, A. P., Kornacker. M. G., and Poquet, I. (1991). Mol. Mirrolhl. 5, 343-352. Puziss, J. W., Fikes, J. D., and Bassford. P. J., Jr. (lY8Y). J . Bucirriol. 171, 2303-231 I. Puziss, J. W.. Strobel, S. M.. and Bassford, P. J.,Jr. (1YY2).J. Bacletrol. 174, Y2-101. Randall, L. L. (1983). Cell (Cornbridge, Mass.) 33, 231-240. Randall, L. L. (1992). Science 257, 241-245. Randall, L. L., and Hardy, S. J. S. ( I 986). CP//(Cainbridgt, Mass) 46, Y! 1-928. Randall, L. L.. and Hardy, S. J. S. (198Y). Science 243, 1156-1 15Y. Randall, L. L.. Topping. T. B., and Hardy, S. J. S. (IYYO). Science 248, 860-863. Reed, K. E.,and Cronan, J. E., Jr. (IY91).J. B i d . Clietii. 266, 11425-1 1428.

192

DAVID N. COLLIER

Roise, D.. Theiler, F., Horvath. S. ,I.. Toniich. J. M., Kichards, ,I. H.. Allison. I). S.. and Schatz, G. (1988). EMBOJ.7, 649-653. Ryan, J. P., Duncan, M. C . , Bankaitis. V. A.. and Bassford. P. ,I., Jr. ( l 9 8 6 ) . / . BifJl. Cltein.

261,3389-3395. Sadaie. Y.. Takaniatsu, H.. Nakdniura, K.. and Yaniane, K. (1991). Gcttc 98, 103-105. Saier, M. H., Jr., Yamada. M.. Suda, K.. E r i k B., Kak, B., Lengeler. 1.. Stewart, G. C . , Waygood, E. B., and Rapoport. G . (1988).Biorliiifiir 70, 1743-1748. Schatz. P. J., and Beckwith. J. (1990). Attttii. th. Grttr/. 24, 215-248. Schiebel. E.. Driessen, A,,]. M.. Hartl. F. U.,and Wickner, W. (I99 I ) . Crll(CNfN6ridgu.Mriss.) 64,927-939. Schwartz. M.,Roa. M.. and I)ebdrbouille, M. (19x1). Pror. N d . Accid. Sri. U.S.A. 78, 2937-294 1. Siegel, V., and Walter, P. (1985).J. Crll B i d . 100, 1913- ly:! I . Siegel, V., and Walter. P. (1986). Na/iou (Lotidoti) 320, 81-84. Siegel. V.. and Walter, P. (1988). Tretids Bior/irttt. Sci. 13, 314-316. Smith, H., Bron. S., Van EE,,J.. and Venenia, G. (1987).J. Borteriol. 169, 3321-3328. Smith, H., d e Jong, A., Bron. S., and Venenia. G. (3988). Griw 70, 35 1-361. Spurlino, J. C., Lu, G.-Y., and Quiocho, F. A. (l99l).,/. B i d . C/trin. 266, 5202-5219. Swidersky. U. E.,Hoffschulte. H. K.. and Muller, M. (1990). E M B O J . 9, 1777-1785. Tani, K.. Tokuda, H., and Mizushinia, S. (1990).J . B i d . C h i . 265, 1734 I- 17347. Teschke, C.. M..Kim. J., Song. T.. Park. S., Park, C., and Kandall, L. L. (1991). J. B i d . Chem. 266, 11789- 1 1796. T r u n , N.J., Stader..J., Lupas. A.. Kumanioto, C., and Silhavy,T.,I. (l988).J. B ~ r t e t i ~170, ~l. 5928-5930. Van Dyk. T. K.. Gatenbv. A. A., and LdRossa. R. A. (1989). N d u r r ( L u n c h ) 342,451-453. Verner. K.. and Schatz, G . (1988).Srirtice 241, 1307-1313. Vlasuk, G. P., Inouve, S.. Ito. H.. ltakara, K., and Inouve, M. (l983).,/.Biol. Cliutn. 258, 7 14 1-7 148. von Heijne. G. (1984). J . Mol. B i d . 173, 243-251. von Heijne. G. (1985).J. Mol. B i d . 184, 99-105. \'on Heijne, G. (1986). E M B O J . 5, 1335-1342. von Heijne, G . (1988). Biorltini. B i o p h y . Actn 947, 307-333. Walter, P.. and Blobel, G. (1981).J. Crll B i d . 91, 557-561. Walter, P., and Blobel, G. (1982). Nature (London) 299, 196-198. Crll Bi01. 2, 499-516. Walter, P.. and Lingappa. V. R. (1986). Aritiii. fh. . 545-590. Walter, P., Ibrahirni, I., and Blobel, G . ( I Y X I ) . J . Crll B i ~ l 91, Watanabe, M., and Blobel. G. ( 1989,). Crll (Cattibridge, Mass.)58, 699-705. Watanabe. M., and Blobel, G . (1989b). Pror. Natl. A d . Sri. U . S . A . 86, 2728-2732. Watanabe, T., Hayashi. S., and Wu. H . C . (l988).J. Bartrrid. 170, 4001-4007. Watts, C.. Wickner. W., and Zimnierman. K. (1983). h J C . N o / / . Arnd. Sri, U.S.A. 80, 2809-28 13. Weiss. J. B., and Bassford, P. J., Jr. (1990).J . Bnr/urio/. 172, 30'234029. Weiss. J. B.. Ray, P. H., and Bassford. P. J.. Jr. (1988). Ptor. N d . Arcicl. Sri. U.S.A. 85, 8978-8982. Weiss,J. 8..MacGregor. C . H.. Collier. L). N.. Fikes. .I. I)., Kay. P. H., and Basslord, 1'. ,I., Jr. (1989).J. B i d . Clirttt. 264, 302 1-3027. Wickner. W., Driessen. A.J. M.. and Hartl. F. U. (1991). A f t t i t i . H c i ~ Biocliriti. . 60, 101-124. Wienhues. U.. Becker, K.. Schlever. M.. Guiard. B.. Tropschug. M.. Horwich. A. L., Pfanner. N.. and Neupert. W. (lY91).J. Cell BkJl. 115, 1601-l(iO9.

SecB: MOLECULAR CHAPERONE

193

Wild, 1.. Altrnan, E., Yura. T., and Gross, C. A. (lYY2). Guws Duii. 6, 1165-1 172. Wild, J., Altrnan, E., Water, W., and Gross, C. (1993). Submitted. Wu, H. C., and Tokunaga, M. (1986). Cum. Top. Microbiol. lmmunol. 125, 127-157. Yarnane, K., Nakarnura, K., Nakarnura, A., Takarnatsu, H., Furna, S., Irnai, T., and Sadaie, Y. (1991). Abstr. Int. Conf. Bacilli, 6th, July 29, 1991. p.9.

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AUTHOR INDEX Numbers in italics refer to the pages on which the complete references are listed.

Abrahan, S. N., 100, 101, 102, I22 Acton, S. L., 86, 93 Adler, M., 29, 62 Aggerbeck, L. P., 142, 150 Akagi, S., 132, 150 Akiyama, Y., 158, 159, 182, 190 Albers, M. W., 14, 17, 18, 21, 22, 23 Alexndrescu, A. T., 28, 62 Alfano, C., 69, 89, 96 Alger, T. D., 3, 23 Alitalo, K., 160, 191 Allen, B. L., 120, I21 Allison, D. S., 153, 192 Altman, E., 158, 159, 160, 170, 173, 174, 182, 183, 184, 185, 187, 188,189, 193 Amir-Shapira, D., 68, 93, 96 Amit, A. G., 112, 115,121 Anderson, S., 31, 63 Andrews, P. R., 27, 63 Anfinsen, C. B., 126, 148 Ang, D., 80, 89, 90, 95, 96, 98, 183, 189, 191 Apella, E., 145, I48 Arndt, K. T., 90, 96 Arthos, J., 114, 115, 122 Astbury, L., 68, 94 Atencio, D. P., 90, 93 Austen, B. M., 134, 148 Axel, R., 114, 115, 122

6 Baaker, D., 120, 122 Babul, J., 30, 62 Bachinger, H.-P., 35, 55, 56, 62, 63 Badwin, R. L., 29, 30, 59, 65 Baga, M., 101, 102, 103, 118, 121, I22

Baker, A., 176, 189 Baker, C. H., 34, 65 Baker, E. K., 34, 62 Baker, K. P., 153, 189 Balcarek, J. M., 141, 148 Balch, W. E., 80, 94 Baldwin, R. L., 26, 27, 28, 29, 30, 40, 59, 61, 63, 64, 65 Ballejo, G., 141, I49 Bang, H., 1, 8, 9, 11, 12, 21, 22,23, 26, 31, 32, 33, 34, 42, 63 Bankaitis, V. A,, 153, 157, 158, 159, 162, 163, 164, 165, 169, 173, 188, 189, 192 Barclay, A. N., 111, 112, 114, 116, 122 Bardwell, J . C., 68, 89, 93 Barea, J. L., 130, 149 Barker, W. C., 141, 149 Barnett, B. J., 37, 43, 44, 64 Bassford, P. J., 117, 122 Bassford, P. J., Jr., 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 167, 168, 169, 170, 171, 172, 173, 175, 176, 180, 181, 182, 183, 184, 185, 188,189, 190, 191, 192 Bassuk, J. A., 142, 148 Basten, A., 68, 94 Batenburg, A. M., 187, 189 Bates, D. J., 75, 94 Bazan, J. F., 111, 121 Becker, K., 156, 192 Beckmann, M. P., 111, 115, 121 Beckmann, R. P., 70, 9? Becktel, W. J., 84, 96 Beckwith, J. R., 155, 157, 189 Beckwith, J., 154, 155, 158, 159, 160, 167, 190, 191, 192 Bedouelle, M., 153, 189 Beintem, J. J., 30, 65 Bennett, C. F., 141, 148 195

196

AUTHOR INDEX

Berarek, V., 7, 22 Brodsky, F. M., 86, 93 Berg, R. A., 142, 148 Bron, S., 176, 192 Berger, A., 5, 20, 22,23, 24 Brot, N., 68, 92, 93, 96 Berger, E., 8, 9, 1 1 , 21, 23 Brown, J. H., 117, 122 Berget, P. B., 30, 65 Brown, R. D., 111, 51, 62 Bergmans, H., 103, 122 Bruckner, P., 55, 56, 62, 65 Bieker, K. I.., 155, 189 Brundage, L. A , , 154, 191 Bielka, H., 172, 290 Brundage, L., 155, 156, 184, 289 Birkelund, S., 68, 93 Brunham, R. C., 68, 94 Bjorknian, P. J., 115, 121 Bryant, C., 28, 64 Blacher, R. W., 129, 148 Buchner, J., 35, 62 Black, R. E., 101, 122 Bulleid, N. J., 51, 62, 130, 134, 135, 138, Blackburn, E. C., 51, 63 143, 148 Blaschek, H., 28, 40, 61, 65 Bycroft, M., 30, 64 Bleicher, F.. 91, 97 Blobel, G., 70, 94, 154, 162, 164, 165, 170, C 171, 172, 183, 189, 190, 192 Cabelli, R.J., 154, 154, 166, 189, 191 Blum-Saingaros, M., 161, 190 Blumberg, H., YO, 93 Cdldwell, C. G., 8, 11, 21, 23 Campbell, D. A., 145, I 4 8 Boado, R.J., 145, 148 Campbell, L., 68, 95 Bochkareva, E. S., 172, 182, 189, 190 Canel, C., 78, 94 Bock, S. C., 153, 190 Caplan, A. J., 90, 93 Bole, D. G., 80, 95 Carlsson, L., 91, 93 Bollinger, J., 157, 189 Carmicheal, D. F., 129, I 4 8 Boorstein, W., 69, 94 Booth, R. J., 68, 94 Caroni, P., 34, 51, 62 Carter, T., 101, 122 Boothroyd, J. C . , 141, 148 Chaffotte, A,, 38, 64 Bossier, P., 147, 148 Boswell, B. A , , 56, 63 Chaidez, I., 153, 191 Boswell, D. R., 114, 122 Challis, B. C., 5, 23 Botstein, D., 175, 190 Challis, J. A., 5, 23 Botterman, J.. 74, 94 Chamoux, M . , 34, 65 Bovey, F. A , , 3, 23, 27, 62 Chang, C., 68, 96 Braell, W. A., 70, 86, 87, 97 Chappell, T. G., 70, 74, 79, 81, 93, 94 Bram, R.J., 62, 63 Chazin, W. J., 27, 31, 51, 62, 64 Branden, G I . , 104,106, 107,110,111,112, Chem, L., 162, 164,190 115, 120, 121,122 Chen, C. M., 21,23 Brandon, S. E., 161, 190 Chen, L., 154, 156, 189 Brandts, J . F., S,23, 26, 27, 29, 30, 34, 48, Cheng, H. N., 3,23, 27, 62 49, 51, 53, 59, 62, 6f Cheng, S.-Y., 145, 248 Braum, W., 18,24 Chiang, A,, 90, 97 Brennan, M., 3, 23, 27, 29, 30, 59, 62 Chirico, W. J., 70, 94, 183, 189 Breukink, E., 156, 164, 167, 190 Chopra, I. J., 145, 148 Brewer, C. F., 51, 62 Chothia, C., 114, 121, 122 Brickman, E. R., 155, 191 Chow, L. T., 133, 149 Briggs, M . S., 153, 189 Christeller, J. T., 35, 63 Brinton, C. C., Jr., 101, I22 Christiansen, G., 68, 93, 120, 122 Britton, W. J., 68, 94 Chuman, L., 34, 65 Brockway, B. F,., 129, 148 Claesson-Welsh, L., 160, 191 Brodin, P., 27, 62 Clardy, J., 19, 20, 22, 24

AUTHOR INDEX

Classen, I., 120, I 2 2 Clegg, S., 101, 120, 121 Cleland, W. W., 14, 15, 2 3 Clements, M. L.,101, 122 Colley, N . J., 34, 62 Collier, D. N., 158, 159, 160, 162, 163, 164, 165, 167, 169, 170, 173, 176,180, 181, 188,189, 192 Colon-Bonilla, E., 16, 21, 22,23, 32, 54, 64 Combs, K. A,, 142,149, 142,150 Cook, K. H., 28, 30, 40, 61, 63 Copeland, C. S., 80, 95 Cortay, J . C., 91, 97 Corthesy, B., 62, 63 Cosman, D., 111, 115, 121 Cover, W. H., 157, 163, 164, 165, 175, 176, 189, 191

Cozzone, A. .J., 91, 97 Crabtree, G. R., 62, 63 Craig, E. A , , 68, 69, 70, 9-3, 94, 95, 97, 98, 183, 190 Craig, E., 89, 93 Craik, D. J . , 27, 63 Creighton, T. E., 43, 64, 127, 134, 148 Cronan, J. E., Jr., 168, I 9 1 Crooke. E., 154, 156, 184, 189, 191 Crooke, S. T., 141, 148 Crowther, R. A,, 86, 94 Cunningham, K., 154, 189, 191 Cusack, S., 117, 122

Dahlquist, H.-I., 3, 5, 12, 20, 22, 23 Dai, X., 114, 115, 122 Dalie, B., 68, 93, 96 Danilition, S. LA.,68, 94 Daugherty, B. L., 142, 1 4 8 Davis, J . M., 56, 63 Davis, M. M., 115, 121 d e Cock, H., 159, 162, 167, 171, 173, 174, 176, 180,189, 190 De Graaf, F. K., 101, 120, 121, 122 de Jong, A., 176, 192 d e Kruijff, B., 156, 164, 167, 187, 189, 190 d e Silva, A. M., 80, 94 De Vos, A,, 11 1, 115, 121 d e Vrije, T., 156, 164, 167, I90 Debarbouille, M., 155, 192 Degrado, W. F., 153, 1 8 9

197

Delmas, O., 34, 65 DeLuca-Flaherty, C., 75, 81, 82, 87, 94 DeMartino, J. A., 142, 148 Demel, R. A., 187, 189 Demolder, J., 74, 94 Dempster, G., 101, 121 Denecke, J., 74, 94 Dennis, D. T., 176, 190 DeRocher, A. E., 68, 96 Deshaies, R. J., 183, 190 Dcshaies, T. I., 70, 94 Dev, I. K., 155, 190 Devereux, S., 160, 190 Dierstein, R., 156, 190 Dixon, J. E., 129, 148 Dobberstein, B., 154, 191 Dobson, C. M., 27, 28, 63 Dobson. K . , 102, 103, 104, 118, 120, 121, 122 D o h , K. M . , 166, 189 Donelson, J. E., 68, 94 Douglas, M. G., 90, 93 Dowhan, W., 154,191 Doyle, D., 134, 149 Drakenberg, T., 3, 5, 12, 20, 22,23, 27, 31, 51, 62, 64 Driessen, A . J . M., 117, 122, 154, 155, 168, 169, 189, 191, 192 Drugge, R. J., 32, 34, 63 Duguid, J . P., 101, 121 Duncan, J. L., 101, 122 Duncan, M. C., 153, 157, 192

Edman, J . C., 129, 148 Edmunds, P. N., 101, 121 Eerola, E., 160, 191 Eikenberry, E. F., 55, 56, 6 2 Eilers, M., 156, 190 Eisenberg, E., 86, 87, 95 Eisenstein, B. 1.. 117, 122 Eisenstein, A. J., 101, 121 Ellar, D.J., 68, 78, 95 Ellis, L., 129, 148 Ellis, R. J., 176, 190 Ellis, R. W., 147, 148 Emr, S. D., 158, 159, 160, 170, 173, 174, 182, 183, 187, 188, 189 Engeborghs, Y., 38, 64

198

AUTHOR INDEX

Engel, J. N., 90, 94 Engel, J., 55, 62, 63 Engman, D. M., 68, 94 Epstein, C. J., 126, 148 Erni, B., 153, 192 Evans, C. A,, 3 , 2 3 Evans, P. A., 27, 28, 63 Exnor, O., 4, 17,23

F Falkow, S., 101, 102, 103, 121, 122 Fandl, J., 162, 164, 190 Farquhar, R., 147, 148 Feiss, M., 69, 89, 95, 98 Fersht, A. R., 29, 30, 59, 63, 64 Fersht, A , , 12,23 Fikes, J. D., 153, 156, 163, 191, 190, 192 Fink, A. L., 79, 80, 83, 85, 97 Fischer, G., 1, 8, 9, 11, 12, 21, 22, 23, 26, 31, 32, 33, 34, 35, 41, 42, 46, 48, 49, 50, 55, 59, 60, 63, 64, 65 Fitzgibbon, M. J., 19, 20, 24 Flaherty, K. M., 75, 78, 82, 94 Flajnik, M. F., 78, 94 Flanagan, W. M., 62, 63 Fletterick, R.J., 75, 94 M. Fr., 84, 94, 187,190 FIOCCO, Flynn, G. C., 81, 84, 94, 187, 190 Forde, B. G., 137,149 Forde, J., 137, 149 Forsen, S., 3, 5, 12, 20, 22, 23, 27, 31, 51, 62, 64 Fox, R. O., 27, 28, 63 Framer, J. D., Jr., 62, 64 Freedman, R. B., 46, 51, 62, 63, 127, 129, 130, 131, 132, 134, 135, 138, 141, 143, 147, 148,149 Freudl, R., 157, 168, 190 Friedman, J., 62, 64 Fuchs, M., 35, 62 Fujihara, H., 8, 23 Fujii, W., 34, 63 Fuller, S., 132, 149 Fuma, S., 162, I 9 3 Fuqua, S. A. W., 161,190

G Gaastra, F., 102, 122 Gaastra, W., 101, 121

Gaat, A., 1 , 2 3 Gaitanaris, G. A., 71, 94 Galardy, R. E., 3, 23 Galat, A., 18, 24, 32, 63 Ganem, D., 90,94 Gannon,P.M., 158, 159, 163, 165,173,190 Gao, J., 27, 63 Garcia, E., 102, 122 Garel, J. R.,26, 29, 63 Garel, J.-R., 30, 35, 59, 64, 65 Garlick, R. L., 114, I22 Carrels, J. I., 68, 96 Garret, T. P., 114, 122 Garsia, R. J., 68, 94 Gatenby, A. A,, 35, 63, 183, 192 Geetha-Habib, M., 143, I48 Gennity, J., 153, 190 Georgopoulos, C. P., 69, 71, 80, 83, 85, 89, 90, 95, 96, 97, 98, 117, 122, 154, 164, 165, 168, 170, 171, 176, 182, 183, 184, 185,189, 190, 191 Georgopoulos, C., 89, 93 Gerig, J . T., 5, 6, 20, 22, 23 Gerlach, G.-F., 120, 121 Gething, M. J., 68, 69, 73, 95, 97, 131, 148 Gething, M.-J., 183, 188, 190 Gierasch, L. M., 84, 95, 153, 168, 189, 190 Gilbert, H. F., 130, 149 Gill, R. E., 102, 122 Gilmore, R., 154, 190 Girshovich, A. S., 182, 189 Girshovich, H. S., 172, 190 Glass, J. R., 34, 51, 63 Gober, J. W., 68, 95 Godbout, M., 34, 51, 63 Goldberger, R. F., 126, 148 Goldenberg, D. P., 134, 148 Goldman, M. H., 74, 94 Goldstein, J., 153, 190 Goloubinoff, P., 35, 63 Gomes, B., 69, 89, 96 Gomez, S. L., 68, 95 Gong, M., 103,121 Gong, Q.-H., 145, 148 Goodwin, R. G., 111, 115, 121 Goransson, M., 101, 102, 103, 122 Goto, Y., 28, 29, 30, 63 Gottesman, M. E., 71, 94 Grafl, R., 30, 63 Grastoph, G., 3, 23 Grat, R., 30, 65

AUTHOR INDEX

Grathwahl, C., 27, 51, 63 Grcia, P. D., 80, 95 Green, M., 141, 149 Green, N. M., 78, 97 Greene, L. E., 86, 87, 95 Grimsley, G. R., 37, 43, 44, 64 Grisafi, P., 175, 190 Gross, C. A., 158, 182, 183, 184, 185, 193 Gross, C., 182, 193 Grundstrom, T., 27, 62 Grunert, H.-P., 30, 37, 39, 40, 42, 64 Grunwald, E., 17,23 Guiard, B., 156, 192 Guthrie, B., 184, 190, 191

Haas, I. G., 68, 69, 95 Haeberli, P., 160, 190 Haendler, B., 34, 63, 65 Hagerman, P. J., 28, 63 Hahn, U., 28,29, 30, 36,37,38,39,40, 42, 52, 64 Halavorson, H. O., 153, 191 Halegoua, S., 153, 190 Hall, J. G., 27, 63 Hamaguchi, K., 28, 29, 30, 63 Hamers, A., 102,122 Handschumacher, R. E., 32, 34, 63 Harano, T., 133, 149 Harding, M. W., 1,23, 32, 34, 63 Hardy, J. S., 117, 121 Hardy, J., 102, 121 Hardy, S. J. S., 156, 162, 164, 165, 168, 170, 171, 172, 174, 175, 176, 180,190, 191 Harrington, W. F., 5, 20, 22, 24 Harris, S. L., 34, 65 Harrison, R. K., 1, 3, 5, 8, 11, 13, 14, 15, 16, 21, 22, 23, 32, 54, 63 Harrison, S. C., 114, 122 Hartl, F. U., 154, 155, 166, 167, 168, 171, 184, 187, 190, 192 Hasel, K. W., 34, 51, 63 Hasumi, H., 26, 34, 48, 49, 51, 53, 64 Hatfull, G., 27, 28, 63 Haugejorden, S. M., 141,149 Havorsonk, H. R.,27, 30, 59, 62 Hawkins, H. C., 51, 63, 129, 130, 135, 147, 148,149 Hayano, T., 1, 24, 32, 34, 63, 65 Hayashi, H., 145, 150

199

Hayashi, S., 159, 173, 192 Hearne, C. M., 68, 78, 95 Hedgpeth, J., 153, 191 Heinernann, U., 36, 37, 63, 64 Hekstra, D., 173, 190 Helaakoski, T., 141, 149 Helenius, A., 80, 94, 95 Hellqvist, L., 68, 94 Hemrningsen, S. M., 176, 190 Hendershot, L. M., 80, 92, 95 Hendrick, J. P., 155, 166, 167, 168, 171, 184, 187,189, 190 Hendrickson, W. A , , 114, 115, 122 Hendrix, R., 176, 190 Henning, U., 155, 157, 159, 168, 176, 190, 191 Herning, T., 29, 63 Herzer, P., 142, 148 Heuser, J., 88, 95, 102, 104, 118, 120, 122 Hickey, E., 161, 190 Hiestand, P. C., 34, 65 Higgins, K. A,, 27, 63 Hill, B. L., 81, 87, 94 Hille, A., 134, 148 Hillson, D. A., 127, 129, 148 Hinck, A. P., 28, 62 Hindennach, I., 157, 168, I 9 0 Hindley, J., 160, 190 Hinz, H.-J., 37, 64 Hirvonen, H., 160, 191 Hiskey, R. G., 51, 64 Hoekstra, W., 103, 122 Hofer, E., 34, 63 Hofer-Warbinek, R.,34, 63 Hoffschulte, H. K., 166, 192 Hofnung, M., 153,189 Holmes, K. C., 75, 78, 82, 94, 95 Holrnes, S., 68, 94 Holrngren, A,, 104, 106, 107, 120,121,122, 129, 130,148,149 Holt, S. C., 117, 122 Holthofer, H., 103, 122 Homer, R. B., 5 , 2 3 Honey, N., 147,148 Hoover-Litty, H., 80, 95 Horiuchi, R., 145, 150 Horowitz, P. M., 35, 64 Horvath, C., 3, 23 Horvath, S. J., 153, I92 Horwich, A. L., 156, 192 Hoschuetzky, H., 116, 122

200

AUTHOK INDEX

Hoskins, J., 70, 89, 98 Hsu, M. P., 141, 148 Hsu, P., 102, 122 Huhtala, M.-L., 141, 149 Hull, R. A,, 101, 102, I22 Hull, R., 101, 102, 122 Hull, S. I., 101, 122 Hull, S., 101, 122 Hultgren, S. J., 100, 101, 102, 103, 104, 106, 107, 110, 111, 116, 117, 118, 120,121, I22 Hung, S. H. Y., 1, 24, 32, 65 Hunt, L. T., 141, 149 Hurle, M. R., 31, 63 Hurtey, S. M., 80, 95 Husain, Y., 114, 122 Hwang, S. T., 70, 97

I Ibrahimi, I., 172, 192 Idzerda, R. L., 111, 115, I21 Ihara, S., 30, 63 Imai, T., 162, 193 Inouye, H., 155, 191 Inouye, M., 153, 190, 192 Inouye, S., 153, 192 Ishihara, T., 30, 64 Itakar, K., 153, I92 Ito, H., 153, 192 Ito, K., 154, 158, 159, 182, 190. I91

J Jackson, S., 29, 30, 59, 63 Jacob-Dubuisson, F., 102, 104, 118, 120, 121, I22 Jacobson, J., 3, 23 Jacoby, R., 130, 134, 149 Jaenicke, R., 30, 35, 62, 63, 64 Jann, K., 116, 122 Jarjour, W. N., 69, 98 Jarosik, G. P., 154, 191 Jencks, W. P., 12,23 Johansson, S., 102, 122 Johnson, C., 83, 85, 96 Johnson, R. B., 5 , 2 3 Joiner, B. J., 142, 149 Jones, J. D., 168, I 9 0 Jordan, R., 84, 95

Jorgensen, W. L., 18, 23, 27, 63 Josefsson, L. G., 158, 190

Kabiling, A. N., 92, 96 Kabsch, W., 75, 78, 82, 94, 95 Kaderbhai, M. A , , 134, 148 Kaiser, C. A., 175, I90 Kaiser, E. T., 153, 190 Kallen, J., 33, 63 Kallis, G. B., 129, 148 Kamamoto, C. A., 154, 164, 165, 168, 170, 171, 176, 182, 183, 184, 185, 191 Kang, P. J., 69, 70, 94, 95, 97 Kao, W. W.-Y., 142, 148 Kaper, J. B., 101, I22 Kaplan, H. A., 143, 147, 148, I 4 9 Karlsson, K. A , , 101, 122 Karplus, M., 19,23 Karplus, P. A,, 19, 20, 22, 24 Kasahara, M., 78, 94 Kaska, D. D., 142, 149 Kassenbrock, C. K., 79, 80, 95 Katchalski, E., 5 , 20, 22, 24 Kato, N., 34, 63 Kato, S., 29, 63 Kaufman, R. J., 73, 98 Kautz, R. A., 27, 28, 63 Kawamukai, M., 34, 63 Kedersha, N. L., 142,148 Keefe, M., 129, 148 Keegstra, K., 68, 96 Keler, R., 34, 65 Kelleher, K., 73, 98 Kelley, R. F., 28, 30, 35, 63, 64 Kellis, J. T., 30, 64 Kelly, R. B., 79, 80, 95 Kendall, D. A , , 153, 190 Kiefhaber, T., 1, 9, 21, 23, 28, 29, 30, 32, 35, 37, 38, 39, 40, 42, 52, 62, 63, 64 Kihlberg, J., 102, 103, 111, 116, 117, 122 Kikuchi, M., 29, 63 Kim, J., 157, 175, 176, 180,192 Kim, P. S., 26, 27, 64 King, J . , 89, 93, 96 Kirchhoff, L. V., 68, 94 Kishore, V., 16, 21, 22,23, 32, 54, 64 Kivirikko, K. I., 132, 133, 140, 141, 142, 148,149

20 1

AUTHOR INDEX

Klein, J. O., 101, 122 Klemm, P., 101, 120, 121, 122 Koch, B. D., 70, 94, 183, 190 Koch, G. L. E., 131, 148, 149 Kocher, H. P., 34, 65 Kochler, K. A., 51, 64 Koenig, S. H., 51, 62 Koepke, J., 36, 64 Kofron, J. L., 16, 21, 22, 23, 32, 54, 64 Kohler, H.-H., 28, 64 Kohno, K., 73,97 Koivu, J., 127, 128, 132, 141, 149 Komano, T., 34, 63 Konforti, B. B., 74, 79, 93 Kong, N., 57, 58, 64 Kordel, J., 27, 31, 51, 62, 64 Korhonen, J., 160,191 Korhonen, T. K., 103, 122 Kornacker, M. G., 159, 191 Kornak, J., 68, 95 Koshland, M. E., 132, 149 Kosic-Smithers, J., 68, 94 Kossiakoff, A., 111, 115, 121 Kovach, I. M., 12, 21,23 Koya, S., 145, 150 Kozutsumi, Y., 73,97 Kramer, C., 103, 122 Kramer, J., 68, 78, 94 Kramps, S., 157, 168, 190 Krause, E., 154, 191 Krause, G., 130, 149 Krebs, H., 30, 64 Kreis, M., 137, 149 Krieg, U. C., 70, 97 Kriz, R.,73, 98 Kuehn, M. J., 106, 107, 120,122 Kuehn, P., 102, 103, 117, 122 Kuipers, H., 120, 122 Kumarnoto, C. A,, 117, 122, 158, 159, 160, 161, 162, 163, 164, 165, 167, 169, 170, 173, 174, 182, 183, 187, 188, 189, 190 Kumarnoto, C., 159, 192 Kuntz, I. D., 31, 63 Kuo, C., 68, 95 Kurihara, T., 90, 97 Kurzchailia, T. V., 172, 190 Kusters, R.,156, 164, 167, 190 Kusukawa, N., 158, 159, 182, 190 Kuusela, P., 103, 122 Kuuti, E.-R., 142, 149

Kuwajima, K., 30, 64 Kuzmic, P., 16, 21, 22, 23, 32, 54, 64 Kwong, P. D., 114, 115,122

L Laarrivee, D. C., 34, 65 Labhardt, A. M., 29, 65 Lam, B., 69, 95 LaMantia, M.-L., 147, 149 Larnbert, N., 129, 131, 148, 149 Laminet,A.A., 157, 175, 176, 182,190,191 Landry, S. J., 84, 95 Lane, W. S., 62, 64 Lang, K., 1, 9, 21,23, 26,30, 32, 34, 35,46. 48, 49, 50, 60, 63, 64 Laplaud, P. M., 142, 150 Lark, D., 102, 103, 121, 122 LaRossa, R. A., 183, 192 Laurents, D. V., 37, 64 Law, D. T., 71, 96 Lazarides, E., 91, 93 Le Bras, G., 35, 65 Lear, J. D., 153, 189 Lecker, S. H., 117,122, 154, 164, 165, 168, 169, 170, 171, 182, 183, 184, 185,191 Lecker, S., 155, 166, 167, 168, 171, 184, 187,190 Lee, A. S., 73, 92, 95, 98 Lee, C., 155, I91 Leffler, J. E., 17, 23 Lengeler, J., 153, 192 Lennarz, W. J., 143, 147, 148, 149 Lesk, A. M., 114, 121, 122 Leustek, T., 68, 92, 93, 96 Levine, M. M., 101, 122 Levitt, M., 30, 64 Lewis, M. J., 74, 96 Li, P., 155, 158, 165, 173, 190, 191 Liakopoulou-Kyriakides, M., 3, 23 Liberek, K., 80, 83, 85, 89, 90, 95, 96, 98, 183, 189, 191 Lill, R., 117, 122, 154, 164, 165, 168, 170, 171, 182, 183, 184, 185, 189, 191 Lin, C. S., 32, 65 Lin, C., 1 , 24 Lin, L. N., 3 , 2 3 Lin, L.-N., 3, 23, 26, 29, 34, 48, 49, 51, 53, 62, 64 Lindberg, F. P., 101, 102, 103, 122

202

AUTHOR INDEX

Lindberg, F., 102, 103, 111, 116, 122 Matouschek, A., 30, 64 Lingappa, V. R., 153, 154, 191, 192 Matsuda, H., 34, 63 Lipinska, B., 89, 96 Matsumura, M., 84, 96 Lissin, N. M., 182, 189 Matthews, B. W., 84, 96 Liu, G., 157, 163, 164, 165, 170, 172, 175, Mayer, S., 32, 41, 49, 55, 59, 65 176, 191 Mazzarella, R. A,, 141, 149 Liu, J., 21,23, 34, 62, 64 McCall, J. M., 15, 23 Lodish, H. F., 57, 58, 64 McCammon, K., 131,148 Loomis, W., 91, 96 McCarty, J. S., 79, 80, 83, 91, 96, 97 Lorimer, G. H., 35, 63, 64 McGlynn, E., 34, 51, 62 Lottspeich, F., 116, 122 McGuire, W. L., 161, 190 Love, A. L., 3 , 2 3 McIntyre, J., 69, 98 Lowe, M. A., 117,122 McKay, D. B., 75, 78, 81, 82, 87, 94 Lowenstein, A,, 5, 20, 22, 23 McKenney, K., 70, 89, 98 Lu, G.-Y., 180, 192 McKeon, F. D., 34, 65 Luke, M. M., 90, 96 McKnight, C. J., 168, 190 Lumry, R., 17, 22, 23 McLean, L. R., 142,150 Lund, B., 101, 102, 103, 122 McMacken, R., 69, 84, 89, 95, 96 Lundemose, A. G., 68, 93 Mech, C., 1, 9, 12, 22, 23, 26, 31, 32, 33, Lundquist-Heil, A,, 69, 95 42,6? Lundstrom, J., 130, 149 Meiboom, S., 5, 20, 22, 23 Lupas, A., 159, 192 Melander, W., 3, 23 Lyman, S. D., 111, 115,121 Melillo, D., 8, 11, 21, 23 Memmert, K., 33, 65 M Mendoza, J. A., 35, 64 Mensa-Wilmot, K., 69, 89, 96 MacArthur, M. W., 27, 64 Merlino, G. T., 145, 148 Macer, D. R. J., 131, 149 Meyer, D. I., 154, I91 MacGregor, C. H., 156, 163, I92 Michaelis, S. H., 155, 191 MacIntyre, S., 155, 159, 176, 191 Michnick, S. W., 19, 23 Maclean, I. W., 68, 94 Miflin, B. J., 137, 149 Maekelae, T. P., 160, 191 Milarski, K. L., 75, 96 Maes, P., 34, 65 Mingjie, J . , 34, 65 Magnusson,G., 102, 103, 111, 116,117,122 Minshaw, B. H., 102, 122 Maki, N., 34, 63 Misra, L. M., 71, 74, 97, 98, 131, 149 Makowsky, J., 103, 121 Mitchell, H. K., 67, 98 Maloney, P. C., 177, 191 Miura, T., 147, I49 Mannherz, H. G., 75, 95 Mizunaga, T., 147, 149 March, C. J., 111, 115, 121 Mizushima, S., 168, 192 Marcy, S. M., 101, 122 Mizzen, L. A., 68, 70, 92, 93, 96 Mariller, C., 34, 65 Montgomery, D., 147, 148 Mark, G. E., 111, 142, 148 Montreuil, J., 34, 65 Markley, J. L., 28, 62 Mooi, F. R., 101, 120, 121, 122 Marklund, B. I., 101, 102, 122 Moore, J. M., 19, 20, 24 Marriuzza, R. A,, 112, 115, 121 Moore, K., 154, 189 Marsh, H. C., 51, 64 Morimoto, R. I., 75, 96 Marshall, J. S., 68, 96 Morin, J. E., 129, 148 Marszalek, J., 80, 89, 96, 183, 191 Morjana, N. A., 130, 149 Martin, R. B., 5, 23 Mortara, R., 131, 149 Maslowska, M., 36, 64 Movva, R., 34, 65

203

AUTHOR INDEX

Miicke, M., 43, 44, 46, 64 Muhich, M. L., 141, 1 4 8 Muller, M., 166, 192 Munro, S., 68, 73, 96, 131, 132, 149 Murrant, S.J., 147, 148 Muthukrishnan, K., 30, 65 Mutschler, B., 159, 176, 191 Myllyla, R., 127, 128, 132, 141, 142, 148, 149

Naider, F., 143, 148, 149 Nakagawa, A., 30, 62, 162, 19? Nakamura, K., 162, 192, 193 Nakatsuka, M., 18, 24 Nall, B. T., 28, 29, 30, 59, 64, 65 Nardi, M . , 130, I48 Nault, A. K., 160, 161, 162, 190 Nemethy, G., 17, 24 Neupert, W., 70,95,97, 154, 156, 166,190, 192 Nguyen, T. H., 71, 96 Nicoet, C. M., 68, 94 Nishimura, S., 89, 97 Nogren, M., 102, 122 Noiva, R., 143, 148, 149 Norgren, M., 101, 102, 103, 118, 122 Normark, F., 102, 122 Normark, S., 100, 101, 102, 103, 104, 111, 116, 117, 118, 120, 121, 122 Normington, K., 73, 97

O’Hanley, P., 102, 103, 121, 122 Ohba, H., 133,149 Ohki, M., 89, 97 Ohno-Iwashita, Y., 158, 191 Okamur, M., 29, 6? Okayama, N., 30, 64 Old, D. C., 101, 121 Oliver, D. B., 154, 158, 166, 189, 191 Oliver, D., 154, 155, 189, 191 Olsen, R. K., 3, 23 Olsson, O., 102, 121 Omura, T., 133, 149 Ono, T., 34, 65 Oobtake, M., 37, 43, 64 Ooi, T., 30, 37, 43, 63, 64

Ostermann, J., 70, 95, 97 Overeem, W., 159, 171, 173, 174, 176, 180, 190 Ozaki, M., 34, 65

P Pace, C. N., 36, 37, 43, 44, 64 Pace, M., 129, 148 Page, M. I., 12, 23 Pai, E. F., 75, 95 Pak, W. L., 34, 65 Palleros, D. R., 79, 80, 83, 85, 97 Palter, K. B., 70, 93 Papavassiliou, A. G., 71, 94 Parham, P., 81, 87, 94 Park, C., 157, 175, 176, 180, 192 Park, H. R., 143,149 Park, L. S., 111, 115, 121 Park, S., 157, 164, 175, 176, 180, 191, 192 Parkhouse, R. M. E., 132, 149 Parkison, C., 145, I48 Parkkonen, T., 133, 140, I49 Partanen, J. M., 160, 191 Pastan, I. H., 69, 97 Pastan, I., 145, 148 Paulson, J. C., 117, 122 Paver, J. L., 132, 135, 149 Pease, B. M. F., 86, 94 Peattie, D. A,, 19, 20, 24 Peeling, R., 68, 94 Pelham, H. R. B., 68, 74, 96, 97, 131, 132, 149 Pelham, H. R., 68, 73, 96, 176, 191 Perara, E., 90, 94, 154, 191 Perersen, L., 12, 24 Perlman, D., 153, 191 Pfanner, N., 70, 95, 97, 156, 192 Phear, G. A., 160, 190 Phillips, G. J., 155, 182, 183, 189, 191 Phillips, S. E., 112, 115, 121 Pierce, S. B., 133, 149 Pigmans, I. G. A,, 147, 149 Pihlajaniemi, T., 132, 133, 140, 141, 148, I49 Pinkner, J. S., 107, 110, 11 1, 122 Plos, K., 101, 122 Pluckthun, A., 157, 175, 176, 182, 190, 191 Poe, M., 1, 24, 32, 65 Pohl, J., 84, 94, 187, 190

AUTHOR INDEX

Poljak, R. J., 112, 115, 121 Pollack, J., 90, 94 Poquet, I., 159, 191 Porter, T. G., 114, 115, 122 Porter, T. N., 101, 122 Potter, R., 161, 190 Preuss, D., 175, 190 Price, E. R., 34, 65 Prockop, D. J., 55, 62, 63 Proia, R. L., 134, I 4 9 Pueyo de a Cuesta, C., 130, 149 Pugsley, A. P., 159, 191 Puziss, J. W., 153, 176, I 9 1

Q Qian, L., 166, 189 Quaas, R., 28, 29, 30, 37, 38,39,40, 52,64 Quinn, D. M., 5, 12, 16, 21,23, 24 Quiocho, F. A,, 180, 192

R Rabenstein, D. L., 3, 23 Radford, A. J., 68, 94 Radzicka, A., 12, 24 Rajender, S., 17, 23 Rak, B., 153, 192 Ramakrishna Kurup, C. K., 126, 129,149 Raman, T. S., 126, 129,149 Ramasarma, T., 126, 129, I49 Ramdas, L., 30, 65 Randall, L. L., 117, 121, 156, 157, 158, 159, 162, 163, 164, 165, 168, 170, 171, 172, 174, 175, 176, 177, 178, 179, 180, 181, 187, 188,189, 190, 191, 192 Rapoport, G., 153, 192 Rapoport, T. A., 172, 190 Rasmussen, B. A., 153, 157, 188, 189 Ray, P. H., 155, 156, 162, 163, 164, 190, I92 Reed, K. E., 168, 191 Reid, K. L., 79, 83, 97 Reinherz, E. L., 114, 122 Renner, M., 37, 64 Rhoads, D., 156,189 Rice, J., 32, 34, 63 Rice, M., 154, 156, 184, 189 Rich, D. H., 16, 21, 22, 23 Rich, D., 32, 54, 64

Richards, F. M., 35, 63 Richards, J . H., 153, 192 Ridge, J. A., 29, 65 Riegman, M., 103, I22 Rieul, C., 91, 97 Rippmann, F., 78, 97 Ritchie, C. D., 7, 24 Roa, M., 155, 192 Robinson, E. A., 145, I 4 8 Rocque, P. A., 130, 149 Rodgers, D. W., 114, 122 Roise, D., 153, 192 Rose, M. D., 71, 74, 97, 98, 131, 149 Rosegay, A , , 8, 11, 21, 23 Rosen, M. K., 18, 19, 23, 24 Rosenberg, M., 114, 115, 122 Roth, R. A., 129, 132, 133, 148, 149 Rothbard, J. B., 78, 97 Rothblatt, J., 90, 97 Rothenluh, A., 34, 51, 62 Rothman, J. E., 70, 74, 79, 81, 84, 86, 87, 93, 94, 97, 187, 190 Rothman, R. E., 154, 191 Rubock, P., 71, 94 Rudolph, C. F., 69, 95 Rudolph, R., 35, 62 Rutter, W. J., 129, 148 Ryan, J. P., 153, 157, 189, 192 Ryu, S. E., 114, 115, 122

Sadaie, Y., 162, 192, I93 Sadler, I., 90, 97 Saenger, W., 36, 37, 63, 64 Sager, W. F., 7, 24 Saier, M. H., Jr., 153, 192 Saito, H., 89, 97, 98 Sambrook, J., 68, 69, 73, 95, 97, 131, 148, 183, 188,190 Sarkar, A,, 27, 50, 65 Sawyer, J. T., 134, 149 Schaeffer, A. J., 101, 122 Schatz, G., 70, 97, 153, 156, 176, 189, 190, 192 Schatz, P. J., 155, I92 Scheele, G., 130, 134, 149 Scheknian, R., 70, 94, 183, 190 Scheraga, H. A., 29, 62 Scherer, P. E., 70, 97

AUTHOR INDEX

Schesinger, M. J., 70, 93 Schiebel, E., 154, 155, 166, 167, 168, 171, 184, 187,189, 190, 192 Schleyer, M., 156, 192 Schlossman, D. M., 70, 86, 93, 97 Schmid, F. X., 1, 9, 21, 23, 26, 27, 28, 29, 30, 32, 34, 35, 37, 38, 39, 40, 41, 42, 43, 44, 46, 48, 49, 50, 51, 52, 53, 55, 59, 60, 61, 62, 63, 64, 65 Schmid, S. L., 70, 74, 79, 86, 93, 97 Schmid, S., 86, 87, 97 Schmidt, J., 91, 96 Schmidt, M., 35, 62 Schneider, C., 34, 51, 62 Schoenmakers, C. H. H., 147, 149 Schonbrunner, E. R., 32,41,49, 51,52,53, 55, 59, 65 Schoolnik, G., 102, 103, 121, 122 Schowen, R. I.., 8, 17, 23, 24 Schreiber, S . L., 1, 14, 17, 18, 19, 20, 21, 22, 23, 24, 32, 63 Schreiber, S. S., 62, 64, 65 Schultz, L., 147, 148 Schumann, W., 90, 98 Schwartz, M., 155, 192 Schwarz, H., 157, 168, 190 Seaby, R., 69, 89, 96 Seavello, S., 34, 65 Sela, M., 5, 20, 22. 24 Sell, S. M.. 69, 98 Semba, 'r., 132, 150 Serrano, L., 30, 64 Setlow, P., 68, 78, 98 Seurinck, J . , 74, 94 Seyer, J., 142, 148 Shapiro, I.., 68, 95 Shenbagamurthi, P., 143, 149 Sherman, F., 30, 65 Shewry, P. R.,137, 149 Shieh, R.-H., 34, 65 Shilling, J., 68, 70, 94, 95 Shimamoto, N., 29, 63 Shiu, R. P. C., 69, 97 Shneuwly, S., 34, 65 Shortridge, R. D., 34, 65 Shossman, D. M., 86, 87, 97 Siddall, T. H., 2 , 24 Siegel, V . , 154, 192 Siekierka, J. J , , 32, 65 Sierkierka, J . J., 1, 24

205

Sigal, N. H., 1, 24, 32, 65 Signon, L., 35, 65 Silhavy, T. J., 155, 157, 159, 182, 183, 189, 191,192 Silver, P. A,, 90, 93 Silver, P., 90, 97 Silverstein, S. J., 71, 94 Skehel, J. J., 117, 122 Skowyra, D., 69, 71, 83,85, 96, 97, 98, 183, 189 Slonim, L. N., 107, 110, 11 1, 122 Smith, H., 176, 192 Smith, I. W., 101, 121 Smith, M. J., 131, 149 Smith, M., 131, 149 Smithies, O., 160, 190 Sonderfeld-Fresko, S., 134, 149 Song, T., 157, 175, 176, 180,192 Speicher, D. W., 32, 34, 63 Spelacy, W. N., 141, I49 Spik, G., 34, 65 Spinner, S. N., 142, 149, 150 Spitzfaden, C., 33, 63, 65 Spurlino, J. C., 180, 192 Srinivasan, M., 141, 149 Stader, J., 159, 192 Stamnes, M. A,, 34, 62, 65 Standaert, A. G., 18, 24 Standaert, R. F., 19, 20, 22, 24 Stedman, K., 34, 65 Steer, C. J., 88, 95 Stein, G., 161, 190 Stein, J., 161, 190 Stein, R. L., 1, 3, 5, 8, 11, 13, 14, 15, 16, 20, 21, 22, 23, 24, 32, 54, 63 Steinberg, I. Z., 5, 20, 22, 24 Steinmann, B., 56, 65 Steitz, T. A,, 75, 94 Stellwagen, E., 28, 30, 62, 63, 64 Stewart, D. E., 27, 50, 65 Stewart, G. C., 153, 192 Stewart, W. E., 2, 24 Stone, D. E., 68, 98 Straub, F. B., 126, 149 Strobel, S. M., 167, 170, 173, 176, 181, 189, 191 Stromberg, N., 101, 122 Suck, D., 75, 95 Suda, K., 153, 192 Sugai, S., 30, 64

206

AUTHOR INDEX

Sunshine, M., 89, 98 Supertifurga, A,, 56, 65 Sussman, M. D., 68, 7 8 , 9 8 Sutcliffe, J. G., 34, 51, 63 Sutton, A., 90, 96 Sutton, L. D., 5, 16, 24 Suzuki, M., 1, 24, 32, 34, 63, 65 Svanborg-Eden, C., 101, 102, 103, 122 Sweet, R. W., 114, 115, 122 Swidersky, U. E., 166, 192

1 Tachikawa, H., 147, 149 Tai, P. C., 154, 156, 162, 164, 189, 190 Takahashi, N., 1,24, 32, 34,41,49, 55, 59, 63, 65 Takahashi, S., 37, 43, 64 Takarnatsu, H., 162, 192, 193 Takemoto, H., 132, 150 Takikawa, H., 145, 150 Tamura, F., 89, 97 Tani, K., 168, 192 Taniyama, Y., 29, 63 Tarkkanen, A. M., 103, 122 Tarr, G. E., 114, 122 Tartar, A., 34, 65 Tasanen, K., 133, 141, 149 Tashiro, Y., 132, 150 Taylor, W. R., 78, 97 Tennent, J. M., 102, 1 0 3 , l l I , 116,118,122 Teschke, C. M., 157, 175, 176, 180, 192 Theiler, F., 153, 192 Thompson, J. A., 19, 20, 24 Thomson, J. A,, 37, 43, 44,64 Thornton, J. M., 27, 64 Thulin, E., 27, 51, 62, 64 Tilly, K., 89, 93, 176, 190 Timpl, R., 55, 62 Ting, J., 73, 92, 95, 98 Tissikres, A., 67, 98 Toedo, H., 92, 96 Tokuda, H., 168,192 Tokunaga, M., 153, 193 Tomich, J. M., 153, I92 Tommassen, J., 159, 162, 167, 171, 173, 174, 176, 180,189, 190 Toney, L. J., 141, 149 Tooze, J., 112, 115, 121, 132, 149

Topping, T. B., 157, 163, 164, 165, 168, 170, 171, 172, 175, 176, I91 Toyoshima, K., 145, 150 Traber, R., 18,24 Tracy, U.M., 67, 98 Trent, R. J., 68, 94 Tropschug, M., 32, 41, 49, 55, 59, 65, 156, I92 Trun, N. J., 159,192 Trunch, A., 114, 115,122 Tsibris, J. C. M., 141, 149 Tsong, T. Y., 30, 65 Tuderrnan, L., 142,149 Tuite, M. S., 147, 148

U Uchida, H., 89, 97, 98 Ueguchi, C., 158, 159, 182,190 Uehling, D. E., 1 , 2 3 Ueling, D. E., 32, 63 Uhlin, B. E., 101, 102, 103, 122 Ulrich, E. L., 28, 62 Ultsch, M., 111, 115, 121 Utiyama, H., 29, 63 Utsumi, R., 34, 63

V Vaisnys, G., 3, 23 van der Vies, S. M., 176, 190 van Die, I., 103, 122 Van Duyne, G. D., 19, 20, 22,24 Van Dyk, T. K., 183,192 Van EE, J., 176, 192 Varrichio, A., 141, 148 Vaucheret, H., 35, 65 Vaux, D., 132,149 Venema, G., 176,192 Venetianer, P., 126, I49 Verkej, A. J., 187, 189 Verner, K., 153, 192 Vestweber, D., 70, 97 Vierling, E., 68, 96 Virkola, R., 103, 122 Visser, T. J., 147, 149 Vlasuk, G. P., 153, 192 Vogel, J. P., 71, 74, 97, 98, 131, 149 von Figura, K., 134, I48

207

AUTHOR INDEX

von Freyberg, B., 18,24 von Heijne, G., 153,192 Voos, W., 70, 97

Wabl, M., 68, 95 Wachter, E., 32, 49, 65 Waheed, A,, 134,148 Wal, J., 103, 121 Walker, G. C., 79, 80, 83, 91, 96, 97 Walkinshaw, M. D., 33, 63 Walsh, C. T., 14, 17, 18, 21,22, 23, 34, 64, 65 Walter, P., 80, 95, 154, 172, 190, 192 Wampler, J . E., 27, 50, 65 Wandless, T. J., 19, 23 Wang, J., 114, 122 Wash, K. A., 157, 189 Watanabe, M., 162, 164, 165, 170, 171, 172, I92 Watanabe, T., 159, 173, 192 Waters, M. G., 70, 94, 183, 189 Watts, C., 153, 192 Way, J., 90, 97 Waygood, E. B., 153, 192 Weber, C., 18, 24 Weber, L. A,, 161, I90 Webster, P., 131, 149 Weis, W., 117, 122 Weiss, J. B., 156, 158, 162, 163, 164, 165, 169, 170, 172, 173, 175,189, 192 Weissbach, H., 68, 92, 93, 96 Weissrnan, I., 62, 64 Welch, W. J., 68, 70, 79, 80, 83, 85, 92, 93, 96, 97 Wells, W. W., 130, 149 Welphy, J. K., 143, 148, 149 Werner-Washburne, M., 68,70,94,98, 183, 190 Westerlund, B., 103, I22 Wetterau, J. R., 142, 149, 150 Wetzstein, M., 90, 98 Wheeler, S., 91, 96 White, T. B., 30, 65 Wickner, S. H., 69, 70, 89, 98 Wickner, W., 117, 122, 153, 154, 155, 156, 158, 164, 165, 166, 167, 168, 169, 170,

171, 182, 183, 184, 185, 187, 189, 190, 191, 192 Wider, G., 18, 24, 33, 65 Widmer, H., 18, 24, 33, 63, 65 Wiedrnann, J., 172, 190 Wienhues, U., 156, 192 Wild, J., 158, 182, 183, 184, 185, I93 Wiley, D. C., 117, 122 Williams, A. F., 111, 112, 114, 116, 122 Williams, D. B., 71, 96 Wilson, J., 28, 64 Wilson, M. I., 101, 121 Winfield, J . B., 69, 98 Winston, S., 68, 94 Wittrnann-Leibold, B., 1, 9, 21, 23, 32, 63 Wold, M. C., 69, 89, 96 Wolfenden, R., 12, 24 Wood, L. C., 30, 65 Wooden, S. K., 73, 98 Wooding, F. B. P., 131, I49 Woolford, C., 176, 190 Wrba, A., 30, 63, 65 Wu, H. C., 153, 159, 173, 192, 193 Wurthrich, K., 3 , 2 3 Wiithrich, K., 18,24, 27, 33, 51, 63, 65

Xu, D., 130, 149 Xuong, N., 114, 115, 122

Y Yaffe, M. P., 90, 93 Yamada, M., 153,192 Yamarnoto, A,, 132, I50 Yamamoto, K., 30, 64 Yamamoto, T., 69, 89, 98, 145, 150 Yamane, K., 162,192, 193 Yamauchi, K., 145, 150 Yan, Y., 114,122 Yang, Y., 130,149 Yochern, J., 69, 89, 95, 98 Yoshirnori, T., 132, 150 Yost, C. S., 153, 191 Yura, T., 158, 159, 182, 183, 184, 185,190, 193 Yutani, K., 29, 63

208

AU'I'HOK INDEX

z Ziegelhoffer, T., 117, 122, 154, 164, 165, 168, 170, 171, 182, 183, 184, 185, 191 Zimmerman, R., 153, 192

Zlotnick, A,, 153, 189 Zuker, C. S., 34, 62, 65 Zydowsky, L. D., 34, 65 Zylicz, M., 69, 71, 80, 83, 85, 89, 90, 93, 95, Y6, 97, 98, 183, 189, 191

SUBJECT INDEX

A Acid catalysis, of nonenzymatic prolyl cis-trans isomerization, 5-6 Actin, consensus sequence with HSC70, 78 Adaptor structures, in P pili, 104 Adhesins chaperone-bound, conformation, 116-1 I7 PapC, 103, 116 pilus, 101 Adhesive properties, of P pili, 102 Affinity chromatography, PapD pilus protein recognition site, 116 Aggregate formation, PDI role, 142-147 Amide rotation in N,N-dimethylacetamide linear free energy correlation, 7 organic solvent effects, 5 para substituent effects, 7 solvent deuterium effects, 4-5 temperature effects, 2-3 Amino acid composition, PapD-PapC complex, 116 Amino acid sequences PapD chaperone, 108 PDI homologies, 139-141 protein disulfide-isomerase, 129- 1 30 SecB gene, 161 ATPase, rates of stress-70 proteins, 80-83 Autophosphorylation, stress-70 proteins, 91-92

B Bacterial proteins, target peptides, 153 Bacteriophages, PI replication, Escherichia coli dnaK role, 69 209

Base catalysis, of nonenzyrnatic prolyl cis-trans isomerization, 5-6 Beta sheets, immunoglobulin, 1 1 1-1 16 Beta strands, of pilus chaperones, 106-107 Binding protein ATPase activity, 8 1 autophosphorylation in uivo and in uitro, 92 labeling by [3H]adenosine in uiuo, 91-92 role in protein translocation into endoplasmic reticulum, 7 1 temperature-dependent structural transitions, 79-80 BiP, see Binding protein

C Calreticulum, 131 Capping proteins, PapD as, 118 Cell-free systems, PDI role in disulfide bond formation, 133-139 Cellular folding CsA effects on collagen folding, 55-57 on transferrin maturation, 57-58 prolyl isomerase role, 54-58 prolyl isomerization in, 60-61 Chaperones molecular DnaK/DnaJ, 183-184 heat-shock proteins, 180- 182 PDI, 130 SecB, see SecB trigger factor, 184 pilus /3 strands, 106-107 consensus sequences, 106- 107

210

SUBJECT INDEX

conserved structures in superfamily, 104-111 PapD, 3D structure, 104-105 papD gene encoding of, 103 salt bridges, 107 surface-exposed invariant residues, 107-1 10 Clathrin cages, in vitro disassembly by HSC70, 70, 86-88 Clathrin light chain peptides, stimulation of HSC70 ATPase activity, 81-83 Clathrin uncoating reaction, 86-88 Collagen folding, CsA effects, 55-57 Concanavalin A, conformational transitions, PPI effects, 5 1 Consensus sequences pilus chaperones, 106-108 in stress-70 proteins, 78 Cotranslational glycosylation, of IFN-a, 143 a-Crystallin, amino acid identities with SecB, 161 CsA, see Cyclosporin A Cyclophilins catalysis of prolyl cis-trans isomerization enthalpy-entropy compensation, 17-18 Eyring plots for, 16-17 cis-to-trans isomerization of Suc-AlaAla-cis-Pro-Phe-pNA, 16 cysteine role in catalysis, 2 1 efficiency of PPI catalysis, 48-50 family of, 33-34 inhibition by cyclosporin A, 18 by immunosuppressive agents, 61-62 PPI activity, 1, 12- 13 sequence specificity, 32 3D structure (human cytoplasm), 33 Cyclosporin A effects on cellular protein folding, 55 collagen, 55-57 transferrin, 57-58 inhibition by CyP, 18 structure, 19 Cytochrome c folding catalyzed by PPI, 35 iso-1 and iso-2 folding, 30

Cytochrome oxidase subunit IV-dihydrofolate reductase fusion protein, 155- 156

D Desolvation, distortion mechanism in PPI catalysis, 22 Deuterium isotope effects secondary, on prolyl cis-trans isomerization, 7-9 solvent, on prolyl cis-trans isomerization enzymatic isomerization, 7-9 nonenzymatic isomerization, 4-5 N,N-Dimethylacetamide amide rotation, linear free energy correlation, 7 amide rotation rate effects of para substituents, 7 organic solvent effects, 5 C-N rotation, secondary deuterium isotope effects, 7-9 Distortion, catalysis of prolyl cis-trans isomerization by, 9- 10, 2 1-22 Disulfide-bonded proteins, synthesis effects on PDI, 132-133 Disulfide bonds catalysis by PDI, 51-54 folding catalysis without, 42-44 in hCHbp, 114 intermolecular, PDI catalysis, 127 intramolecular, PDI catalysis, 127 PDI effects, 129-130 RNase T1,36-37 and folding, 51-53 dnaJ protein effects on dnaK protein, 89 and initiation of A replication, 89 modulation of dnaK interaction with substrates, 89-90 as secretion-related chaperone, 183-184 dnaK protein ATPase mechanism, 80-81 autophosphorylation in vim and in uitro, 91 conformational change, two-state model, 85-86 dnaJ and grpE effects, 89-90

211

SUBJECT INDEX

in A phage replication, 69 in PI bacteriophage replication, 69 primary structure, 74 renaturation activities, 7 1-72 as secretion-related chaperone, 183- 184 temperature-dependent structural transitions, 79-80 Drosophila rnelanogaster, NinaA protein, 32

E Electron microscopy, P pili structure, 102-104 Electrostatics, distortion mechanism in PPI catalysis, 22 Endoplasmic reticulum, PDI localization, 131- 132 Endoplasmin, 131 Enthalpy-entropy compensation, for PPI catalysis, 17- 18 Escherichia coli dnaJ protein effects on dnaK protein, 89 and initiation of A replication, 89 modulation of dnaK interaction with substrates, 89-90 as secretion-related chaperone, 183-184 dnaK protein ATPase mechanism, 80-8 1 autophosphorylation in vivo and in vitro, 91 conformational change, two-state model, 85-86 dnaJ and grpE effects, 89-90 and in A phage replication, 69 and P1 bacteriophage replication, 69 primary structure, 74 renaturation activities, 7 1-72 as secretion-related chaperone, 183-184 temperature-dependent structural transitions, 79-80 grpE protein effects on dnaK protein, 89 and initiation of A replication, 89 modulation of dnaK interaction with substrates, 89-90 pup gene cluster, 101-102

secretion machinery, 154- 155 SecB component, 157-162 Eukaryotic cells, stress-70 proteins in, 68

F Fingerprinting, consensus, stress-70 proteins, 78 FK506 inhibition of FKBP, 18 structure, 19 FK506-binding proteins (FKBPs) cis-to-trans isomerization of Suc-AlaLeu-cis-Pro-Phe-pNA, 16 efficiency of PPI catalysis, 48-50 inhibition by FK506, 18 by immunosuppressive agents, 61-62 sequence specificity, 32 solution structure, 19-20 FKBP:FK506 complex, structure, 20 Freeze-etched electron microscopy, P pili structure, 102-104

G 0-Galactosidase, secretion, 155 y-Gliadin, cotranslational formation of disulfide bonds, 137-138 Glucokinase, consensus sequence with HSC70, 78 Glycerol kinase, consensus sequence with HSC70, 78 Glycosylation, N-linked, PDI role, 143-145 GroEIlES, role in secretion, 182-183 GRP78, see Binding protein grpE protein effects on dnaK protein, 89 and initiation of A replication, 89 modulation of dnaK interaction with substrates, 89-90

Heat-shock proteins as molecular chaperones, 180- 182 70-kDa, see Stress-70 protein family

212

SUBJECT INDEX

HepG2 cells, CsA effects on transferrin secretion, 57-58 Hexokinase, consensus sequence with HSC70, 78 hCHbp, see Human growth hormonebinding protein Histocompatibility antigen proteins, peptide-binding domain structure, and HSC70 proteins, 78-79 HSC proteins, see Stress-70C proteins, cognate HSC70s, see Stress-70 proteins, cognate HSP27, amino acid identities with SecB, 161 HSP70s, see Stress-70 proteins, inducible Human fibroblast growth factor receptor 4, amino acid identities with SecB, 160 Human growth hormone-binding protein disulfide bonds, 114 molecular recognition via immunoglobulin fold, 112- 1 13 Hydrophobicity, signal peptides, 153

I Immunoglobulin fold /3-barrel motif, 1 1 1 - 1 16 hCHbp use of, 112-1 13 structural variations, 114 Immunoglobulin light chain C, fragment folding, 30 folding catalyzed by PPI, 35 Interference phenotype, for SecB binding in vivo, 169-170 Interference region near carboxy terminus of LamB protein, 173-174 secretion of precursors lacking, 187-188 Interferon-y, cotranslational glycosylation, 143 Inverted membrane vesicles, importation of OmpA and PhoE, 156 Iodothyronine S'monodeiodinase, PDI as, 145-146

L LamB, SecB ligand, 177 Ac1857 protein, renaturation, dnaK role, 71-72 A phage replication dnaJ and grpE roles, 89 dnaK role, 69 Leader peptidase, 155 lep gene signal leader peptidase, 155 Ligands, positively charged, SecB binding, 176- 180 Linear free energy, correlation for amide rotation in DMA, 7 Lymphoma-derived cells, PDI expression, 132- 133

Macromolecular complexes, disassembly, role on stress-70 proteins, 70 Maltose-binding protein folded, and SecB binding, 165 preMBP export kinetics, 158-160, 162-163 preMBP folding and export competence, 156-157 reversible unfolding/refolding reactions, 164 SecB ligand, 177 Mechanical distortion, role in PPI catalysis, 22 Microsomes, PDI-deficient, cotranslational sulfide bond formation, 137-139 Molecular chaperones DnaK/DnaJ, 183-184 heat-shock proteins, 180-182 PDI, 130 SecB, see SecB trigger factor, 184 Molecular ushers, 103, 118-120 Mutagenesis, cysteine role in CyP catalysis, 2 1 Mutations, site-directed, PapD chaperones, 109-1 11

SUBJECT INDEX

NinA protein (Drosophilu), 32 N-linked glycosylation, PDI role, 143-145 Nucleophilic catalysis, of prolyl cis-trans isomerization, 1, 9- 10 Nucleotide hydrolysis, coupling with peptide-binding in stress-70 proteins, 85-86 Nucleotide sequences, SecB gene, 161 Nulceophilic catalysis, of enzymatic prolyl cis-trans isomerization, 9- 10

0 Oligomeric complexes, disassembly, role of stress-70 proteins, 69-70 OmpA importation in IMVs, 156 SecB ligand, 177 Organic solvents, effects on amide rotation rate constant in DMA, 5 prolyl cis-trans isomerization, 4-5

P Pancreatic RNase, folding, 30 PapA, 102- 103 PapC as molecular usher, 103, 118-120 production of adhesive P pili, 118 recognition by PdpD-PapA complexes, 118 papC gene, and assembly ofpilus subunits into pili, 103 PapD immunoglobulin-like domain, 113-114 modulation of pilus assembly, 103 roles of, 1 14-1 15 PapD chaperone aniino acid sequence, 108 p strands, 106-107 binding paradigm for, 115-1 16 conserved structures in superfamily, 104-1 11

213

C-terminal domain, 115 difference from classic immunoglobulin fold, 115 effector functions, 115-1 16 and folding of pilus subunit proteins, 110- 1 11 interactive surfaces, 107 prevention of misassembly by, 117-118 as reversible capping protein, 118 salt bridges, 107 site-directed mutations, 109- 111 space-filling model of, 109 3D structure, immunoglobulin-like, 104-105 uncapping, 120 papD gene, encoding of periplasmic chaperone protein, 103 PapD-PapA complex, 118 PdpD-PapG complex, 116-1 18 PapE characterization, 102 function, 103 PdpF, characterization, 102 PapC adhesin, 103, 116 PapD-PapG complex, 116 specificity of binding, 1 16- 117 pup gene cluster, 101-102 PupPC gene, and Gala( 1-4)Gal binding of P pili, 102 PapK protein, characterization, 102 PDI, see Protein disulfide-isomerase Pepsinogen, folding catalyzed by PPI, 35 Peptide bonds, prolyl, 27-28 Peptidylprolyl cis-trans isomerases and aggregation, 35 catalysis of KNase T 1 folding, 40-41 of slow-folding steps, 34-35 in unfolding proteins, 44-46 catalytic efficiencies, 48-50 cyclophilin-type, 32 enzymatic, enthalpy-entropy compensation, 17- 18 FK506-binding proteins, 32 function, evolutionary conservation, 54-55

214

SUBJECT INDEX

inhibition by immunosuppressive agents, 61-62 lack of catalysis, 59-60 and oxidative folding of reduced proteins, 51-54 reaction with Suc-Ala-Xaa-Pro-PhepNA-type substrates, 13 sequencing, 32 simultaneous actions with PDI, 51-54 and slow conformational transitions of ConA and prothrombin, 51 structural studies, 18-2 1 substrate concentration dependence, 46-48 substrate specificity, 12- 13 Periplasmic pilus chaperone protein amino acid sequence, 108 p strands, 106-107 binding paradigm for, 115-1 16 consensus sequences, 106-107 conserved structures in superfamily, 104-111 C-terminal domain, 115 difference from classic immunoglobulin fold, 115 effector functions, 115-1 16 and folding of pilus subunit proteins, 110-1 11 interactive surfaces, 107 papD encoding of, 103 PapD prototype, 104- 105 prevention of misassembly by, 117-118 as reversible capping protein, 118 salt bridges, 107 site-directed mutations, 109-1 11 space-filling model of, 109 surface-exposed invariant residues, 107-1 10 3D structure, immunoglobulin-like, 104-105 uncapping, 120 pH effects on enzymatic prolyl cis-trans isomerization, 14-15 on nonenzymatic prolyl cis-trans isomerization, 5-6 PhoE importation in IMVs, 156 secretion, SecB role, 167

Pili assembly modulation by PapD, 103 papC and papD roles, 103 classification, 101 folding domain-domain interactions, 104 of subunit domains, 110- 111 PapD binding, 116 Polyacrylamide gel electrophoresis, PapD-PapG complex, 116 Polypeptides folding, role of stress-70 proteins, 70-7 1 recognition by stress-70 proteins, 83-84 Posttranslational modifications, of stress70 proteins, 91-92 PPI, see Peptidylprolyl cis-trans isomerase P pili adaptor structures, 104 adhesive properties, 102 biosynthesis and expression, genes involved with, 101- 102 composite structure, 102- 104 evolution, 103-104 and Gala( 1-4)Gal binding, papG gene role, 102 PapD chaperones p strands, 106-107 consensus sequences, 106- 107 conserved structures in superfamily, 104-1 11 papD gene encoding of, 103 salt bridges, 107 surface-exposed invariant residues, 107-110 3D structure, 104-105 pap gene cluster, 101-102 Precursors folding, 157 lacking IR, secretion, 187-188 transmembrane translocation, 154- 155 Presequences functions, and precursor conformation, 155-156 signal peptides, 153

SUBJECT INDEX

targeting of proteins to translocation apparatus, 153-154 target peptides, 153 Primary structure, stress-70 proteins, 73-75 Procollagen, folding pathway, 127- 128 Proline residues classification, 30-3 1 types I, 11, and 111, 30-31 Xaa-Pro sequences as targets for PPI in refolding, 42 Prolyl cis-trans isomerization activation parameters, 3 catalysis in unfolding proteins, 44-46 in cellular folding, 60-61 enzymatic catalysis by distortion, 9-10, 21-22 desolvation, 22 electrostatics, 22 mechanical distortion, 22 nucleophilic catalysis, 9-10, 150 pH dependence, 14-15 secondary deuterium isotope effects, 11-12 substrate specificity, 12-14 thermodynamics, 16017 FKBP:FK506 complex structure and, 20 nonenzymatic acid and base catalysis, 5-6 one-step mechanism for, 9 rate acceleration in acidic solution, 9 in organic solutions, 9 secondary deuterium isotope effects, 7-9 substituent effects, 7 thermodynamics, 2-4 prolyl peptide bonds, 26-28 properties, 59 protein folding reactions, 28-30 fast, 27-28 slow, 27-28 RNase T 1 folding catalysis, 40-41 kinetics, 37-40 solvent effects, 4-5 transition state structure for, 8 Prolyl 4-hydroxylase, p subunit, PDI amino acid identities with, 141-142

215

Protein disulfide-isomerase active site, 129-130 amino acid sequence homologies, 139- 141 binding to polypeptide backbone, 130 catalysis, of intra- and intermolecular disulfide bonds, 127 C-terminal tetrapeptide KDEL, 132 disulfide bond formation in cell-free systems, 133- 139 effects on disulfide bonding, 126-129 triple helix formation, 128- 129 enzymatic properties and mechanism, 129-130 expression, 132-133 in formation of disulfide bonds at synthesis, 135-139 identification with prolyl4-hydroxylase p subunit, 141-142 with triglyceride transfer protein subunit, 142-143 as iodothyronine 5'-monodeiodinase, 145-146 localization, 131-132 as molecular chaperone, 130 multifunctionality of, 139- 147 in N-linked glycosylation, 143-145 oxidizing environment of, 130 prevention of aggregates, 142- 147 and protein folding in vitro, 126-129 redox potential, 130 regulation by disulfide-bonded protein synthesis, 132-133 as reticuloplasmin, 131- 132 simultaneous actions with PPI, 51-54 solubility, 134- 135 as triiodo-L-thyronine-binding protein, 145 Protein folding catalysis without disulfide bonds, 42-44 CsA effects on collagen, 55-57 on transferrin, 57-58 fast, 26-27 intracellular, PDI role, 133- 139 in vitro, protein disulfide-isomerase role, 126- 129

2 16

SUBJECI' lNDEX

large proteins, 35 pili domain-domain interactions for folding, 104 subunit domains for pilus assembly, 110-111 PPI catalysis in unfolded proteins, 44-46 precursor folding and export competence, 156- 157 procollagen, 127-128 prolyl isomerization in, 28-30 reduced proteins, PPI effects, 51-54 reversibility, 35 RNase TI kinetics, 37-40 PPI-catalyzed, 40-4 1 simultaneous actions of PPI and PDI, 5 1-54 slow RNase A, 26-27 RNase A by PPI, 34-35 and stress-70 proteins, 7 1 thermodynamic hypothesis, 126 Xaa-Pro targets of PPI, 42 Prothrombin, conformational transitions, PPI effects, 5 1

Redox potential, protein disulfideisomerase, 130 Renaturation, role of stress-70 proteins, 71-72 Reticuloplasniins, 131 RNase, scrambled, 126 KNase A , folding catalyzed by PPI, 34-35 RNase T1 concentrations, and PP1 catalysis, 46-48 disulfide-reduced and carboxymeth ylated PPI-catalyzed refolding, 42-44 unfolding catalysis, 46 disulfides, and PPI catalysis, 48 folding catalyzed by PPI, 35, 42-44 kinetics, 37-40

linkage to disulfide bond formation, 51-53 stability, 36-37 structure, 36-37

S Succhuromyces cerevisiae trans-activating factor B, amino acid identities with SecB, 160 Salt bridges in pilus chaperones, 107 translocation inhibition mechanism, 187 SecA SecY-SecA complexes, and translocation, 188 as translocation ATPase, 154 SecB aggregation prevention by, 168 antifolding activity, 162-166 binding interference regions, 173- 174 i ? ~ vzvo, interference phenotype for, 169- 170 nonnative proteins, 174- 176 positively charged ligands, 176-180 signal peptide as site for, 171-173 complexes formed in uitro, 170- 17 1 formed in uivo, 170 functions as chaperone, 184- 186 hydrophobic patch, 186 mutants isolation, 157- 158 phenotype, 158- 160 proteolysis, protecting peptides, 178 purification, 162 targeting activity, 155, 166-168 SecB gene, sequence identities with other proteins, 160-1 162 SecD, functions, 155 SecE, functions, 155 SecF, functions, 155 Secondary deuterium isotope effects on enzymatic prolyl cis-trans isomerization, 1 I - 12 on nonenzymatic prolyl cis-trans isomerization, 7-9

217

SUBJECT INDEX

Secretion chaperones, see Chaperones, molecular trigger factor role, 184 SecY, functions, 155 70-kDa heat-shock proteins, see Stress-70 protein family Signal peptidases 1 and 11, 155 Signal peptides, 153 as antifolding factor, 154, 186 SecB binding, 17 1- 173 and translocation competence, 156 Signal recognition particles, 154 docking protein-binding activity, 154 translocation-arresting activity, 154 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, disulfide bonds synthesized in vztro, 134- 135 Solubility, protein disulfide-isomerase, 134- 135 Solvents deuterium isotope effects on enzymatic prolyl cis-trans isomerization, 15- 16 nonenzyniatic prolyl cis-trans isomerization, 4-5 organic, effects on amide rotation rate in DMA, 5 prolyl cis-trans isomerization, 4-5 Stress-70 protein family accessory proteins, 89-90 ATPase function of, 70, 75 ATPase mechanism, 80-83 clathrin uncoating reactions, 86-88 common mechanism for activities, 72 conformational change, two-state model, 84 consensus fingerprinting, 78 disassembly of macromolecular complexes, 69-70 high- vs. low-peptide affinity states, 85 interactions with polypeptides, 70-7 1 peptide-binding activity, 83-84 peptide-binding domain structure, 78-79 posttranslational modifications, 9 1-92 primary structure, 73-75 renaturation activities, 7 1-72 role in protein folding, 71 temperature-dependent structural transitions, 79-80

Stress-70 proteins cognate (HSC70s) ATPase activity, 8 1 ATPase fragment, 75-79 classification, 68 clathrin uncoating reaction, 86-88 disassembly of clathrin cages, 70 interactions with polypeptides, 71 primary structure, 73-75 tertiary structure, 75-79 inducible (HSP70s) classification, 68 primary structure, 73-75 Substrate specificity BiP peptide-binding sites, 84 of' prolyl cis-trans isomerization, 12-14 Suc-Ala-Gly-cis-Pro-Phe-p-nitroanilide, cis-to-trans prolyl isomerization, secondary deuterium isotope effects, 8-9 Suc-Ala-Xaa-Pro-Phe-p-nitroanilide, substrates, PPI reactions, 13-14, 16-17 Sulfide bonds, cotranslation formation in PDI-deficient microsomes, 137-139

T Target peptides, 153 Temperature effects on prolyl cis-trans isomerization activation, 2-3 on structural transitions of stress-70 proteins, 79-80 Tertiary structure, stress-70 proteins, 75-79 Thermodynamic hypothesis, of protein folding, 126 Thermodynamics enzymatic prolyl cis-trans isomerization, 16-18 nonenzymatic prolyl cis-trans isomerization, 2-4 Thioredoxin active site, 130 folding, 30 PPI lack of effect, 35

218

SUBJECT INDEX

Three-dimensional structures human cytoplasmic cyclophilin, 33 PapD chaperone, 104-105 Tissue-type plasminogen activator, glycoforms, cell-free synthesis, 145-146 Transferrin folding, CsA effects, 57-58 Transition state structure in enzymatic prolyl cis-trans isomerization, 11- 12 for nonezymatic prolyl cis-trans isomerization, 8 Translocation inhibition by salt bridging, 187 ligand discharge and, 188 of precursors, 154-155 presequence targeting of proteins, 153-154 signal peptides, 153 signal recognition particle role, 154 target peptides, 153 Translocation competence, 156 and precursor folding, 156- 157

presequence function and, 155- 156 terminology for, 168-169 Trigger factor, role in secretion, 184 Triglyceride transfer protein, subunit, PDI amino acid identities with, 142-143 Triiodo-L-thyronine-binding protein, PDI as, 145 Triple helical folding, protein disulfideisomerase effects, 128- 129 Triskelion, of clathrin cages, 70, 86-87

U Unzippering reaction, 188

X Xaa-Pro bonds cis-trans isomerization, 27-28, 59 targets of PPI in refolding, 42

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