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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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.

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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 (