COMMUNICATION:
Conditions of Forming Protein Complexes with GroEL Can Influence the Mechanism of Chaperonin-assisted Refolding*

(Received for publication, June 4, 1994, and in revised form, October 25, 1996)

Boris M. Gorovits and Paul M. Horowitz Dagger

From the Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78284

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The interaction of GroEL with urea-unfolded dihydrofolate reductase (DHFR) has been studied in the presence of DHFR substrates by investigating the ability of GroES to release enzyme under conditions where a stable GroES-GroEL-DHFR ternary complex can be formed. In these circumstances, GroES could only partially discharge the DHFR if ADP was present in the solution and approximately half of the DHFR remained bound on the chaperonin. This bound DHFR could be rescued by addition of ATP and KCl into the refolding mixture. The stable ternary complex did not show any significant protection of bound DHFR against proteolysis by Proteinase K. These results are in contrast to those observed with the GroEL-DHFR complex formed by thermal inactivation of DHFR at 45 °C in which GroES addition leads to partial protection of bound DHFR. Thus, the method of presentation influences the properties of the bound intermediates. It is suggested that the ability of GroES to bind on the same side of the GroEL double toroid as the target protein and displace it into the central cavity depends on the way the protein-substrate is presented to the GroEL molecule. Therefore, the compact folding intermediate formed by thermal unfolding can be protected against proteolysis after GroES binds to form a ternary complex. In addition, structural changes within GroEL induced by the experimental conditions may contribute to differences in the properties of the complexes. The more open urea-unfolded DHFR binds on the surface of chaperonin and can be displaced into solution by the tighter binding GroES molecule. It is suggested that the state of the unfolded protein when it is presented to GroEL determines the detailed mechanism of its assisted refolding. It follows that individual proteins, having characteristic folding intermediates, can have different detailed mechanisms of chaperonin-assisted folding.


INTRODUCTION

The chaperonin GroEL,1 together with the co-chaperonin GroES, has been shown to facilitate protein folding (1). The initial step of the chaperonin-assisted folding cycle is interaction of the unfolded polypeptide with GroEL (2). The mechanism of the subsequent release of the bound protein from the complex has been studied intensively (3, 4, 5, 6). GroEL is an oligomer in which its 14 identical subunits are arranged as two, stacked, 7-membered rings to form a cylindrical structure with a central cavity. It has been shown that during the chaperonin cycle two distinct types (cis and trans) of the GroEL-GroES-protein substrate complex can be formed (4). In case of the trans complex, unfolded polypeptide is bound to the GroEL ring opposite to the ring occupied by GroES. Release and rebinding of GroES during the chaperonin cycle results in formation of a cis complex in which the GroES is bound on the same GroEL ring occupied by the protein-substrate (4). When this cis complex is formed, it has been suggested that the unfolded polypeptide is displaced into the central cavity which is capped by the bound GroES, thus placing the previously bound protein in a protected space. It has been suggested that final release of the protein-substrate occurs from the cis complex only (4, 7). These studies have used unfolded proteins that have high affinity for GroEL and require for successful folding the complete chaperonin system including co-chaperonin GroES, ATP, and KCl. There are several proteins that are able to at least partially dissociate from their complexes with GroEL without ATP hydrolysis (8, 9, 10). These proteins generally display low affinity for GroEL. It has been suggested that both GroES and the target proteins use linked binding sites on the surface of GroEL (11). Assuming that the strength of GroEL-GroES interaction does not depend on the nature of the protein-substrate, one can suggest that the diverse proteins that bind to GroEL represent a spectrum of binding affinities for GroEL relative to the binding affinity for GroES. Further, a single protein may fold (or misfold) through multiple states that have different affinities for GroEL. In the present study, possible differences in the mechanisms of chaperonin interactions for weak and tight binding proteins have been explored by comparing interactions of urea- and heat-unfolded bovine dihydrofolate reductase (DHFR).


MATERIALS AND METHODS

Reagents and Proteins

GroEL was expressed in E. coli and purified as described previously (12). Bovine DHFR, Proteinase K, dihydrofolate (H2F), NADPH, ATP, and ADP were purchased from Sigma.

Preparation of Urea-unfolded DHFR

DHFR was unfolded by dialyzing against 8 M urea (50 mM Tris, 1 mM EDTA, 10 mM beta ME) for at least 2 h at room temperature. Protein concentration of DHFR was determined spectrophotometrically based on the extinction coefficient at 280 nm reported as 27,280 cm-1 M-1 (13).

DHFR Refolding Assay

Urea-unfolded DHFR was rapidly diluted into buffer A (50 mM Tris, 10 mM MgCl2, 10 mM beta ME, 0.1 mM H2F, and 0.075 mM NADPH, pH 7.8) with or without GroEL and nucleotide as described in the individual figure legends. Final concentrations of the enzyme varied from 0.24 to 0.3 µM. The concentration of the GroEL was varied from 0 to 1.2 µM, and the final urea concentration did not exceed 0.5 M. The solution was mixed, and, after a 1-min incubation at room temperature, enzyme activity was analyzed by the decrease in the NADPH concentration detected spectrophotometrically at 340 nm. In some experiments, GroES (final concentration varied from 0 to 1.2 µM) was added to the refolding solution after 30 s of incubation. The enzyme activity was detected as above after an additional 30-s incubation. Resulting data were analyzed in terms of the initial rate of the reaction.

Proteinase K Digestion Assay

Urea-unfolded DHFR was rapidly diluted to buffer A (50 mM Tris, 50 mM KCl, 10 mM MgCl2, 10 mM beta ME, pH 7.8) with or without GroEL. Final concentrations of the proteins were: 0.24 µM DHFR and 0.277 µM GroEL. The final concentration of urea did not exceed 0.6 M.

After a 5-min incubation at room temperature, the mixture was applied on a Superose 12 gel filtration column (1.5 × 30 cm, flow rate 1 ml/min) equilibrated with buffer A without beta ME. Fractions containing GroEL were collected, pooled, and concentrated using Ultrafree-MC concentration units with 30,000 cutoff (Millipore). SDS Laemmli gel electrophoresis was used to confirm the presence of DHFR in these fractions. GroES was added to some solutions to a final concentration of 0.7 µM. ADP was added to all solutions to give a final concentration of 2 mM. Proteinase K was then added to 0.2% of the total protein (w/w), and the solution was incubated for 10 min at room temperature, after which the reaction was stopped by adding phenylmethylsulfonyl fluoride to a final concentration of 3 mM. Solutions were incubated for 10 min at room temperature, boiled, and analyzed on the 12% SDS Laemmli gel system. Protein was visualized by silver staining (14). The density of the protein bands was analyzed using NIH Image software.


RESULTS

GroEL Can Arrest Spontaneous Refolding of Urea-unfolded Bovine DHFR

Bovine DHFR was unfolded in 8 M urea. Dilution of the unfolded material to the buffer, containing no denaturant, results in fast unassisted refolding, detected as an appearance of enzymatic activity of the native protein. The kinetics of NADPH oxidation by dihydrofolate, catalyzed by native DHFR, have been used in this study to analyze the amount of successfully folded enzyme in solution. As shown in Fig. 1, introduction of GroEL to the refolding solution results in a decrease of the enzyme activity. A similar result has been reported for the mouse DHFR whose refolding can be completely arrested upon binding to GroEL (8). Fig. 1 demonstrates that almost 80% of DHFR can be captured at 1:1 ratio of GroEL to the unfolded DHFR whether or not ADP is present in solution.


Fig. 1. GroEL arrests refolding of the urea-unfolded DHFR. DHFR was unfolded in 8 M urea for 24 h. Unfolded protein was diluted into buffer A (see "Materials and Methods") containing 0.1 mM H2F, 0.075 mM NADPH, and various concentrations of GroEL in the absence (open squares) or presence (closed squares) of 2 mM ADP. Final DHFR concentration was 0.24 µM. The mixture was incubated for 1 min after which enzymatic activity of refolded DHFR was analyzed. The initial slopes of the progress curves were used to determine the amount of active protein. The activity of the spontaneously refolded DHFR in the absence of GroEL (corresponding to 94 ± 5% of the activity of the native enzyme) is taken as 100%. The abscissa shows the ratio of the concentration of GroEL oligomer to the final DHFR concentration.
[View Larger Version of this Image (14K GIF file)]


The GroEL-DHFR complex slowly dissociates (data not shown and Ref. 8). Under conditions when enzyme substrates (NADPH and dihydrofolate) are present in the solution, considerable dissociation was detected after 1 h of incubation (data not shown). The mouse DHFR-GroEL complex has been reported to completely dissociate after 3 h of incubation under similar conditions (8). These data suggest that the enzyme exists in dynamic equilibrium between free and bound states.

As with several other proteins, the addition of ATP alone can lead to the release of fully active bovine DHFR from its complex with GroEL. These weakly bound proteins contrast with the class of tightly bound proteins, like rhodanese (2), 6-hydroxy-D-nicotine oxidase (15), and ornithine transcarbamylase (16). These latter proteins require the presence of the complete chaperonin system, including the co-chaperonin GroES, in order to be successfully released. The presence of GroES, although not required, does facilitate dissociation of DHFR from the GroEL-DHFR complex (8).

GroES Can Partially Discharge DHFR from the Complex with GroEL in the Presence of ADP

Fig. 2 shows that GroEL can arrest approximately 72% of the spontaneous folding of DHFR (open squares, Fig. 2). Addition of GroES to the preformed GroEL-DHFR complex results in partial discharge of the active enzyme (closed squares, Fig. 2). The amount of the released protein increases up to about 1:1 the ratio of GroES to GroEL-DHFR complex. Subsequent addition of GroES does not result in any considerable change of the free enzyme concentration. The maximum level of DHFR released (65%) represents about 50% of the total protein that had been captured by GroEL (Fig. 2). The remaining half of the captured enzyme could be released upon addition of ATP and KCl, producing a 95% yield of active protein. As a control, a similar titration of GroES to the GroEL-DHFR complex with no nucleotide present in the solution was carried out. Since the GroEL-GroES interaction is not possible under this condition (data not shown and Ref. 4), no significant discharge of the DHFR over a control sample was observed within the time of this experiment.


Fig. 2. DHFR can be partially discharged from the complex with GroEL by GroES in the presence of ADP. Unfolded protein was diluted into buffer A containing 0.1 mM H2F, 0.075 mM NADPH, and 0.3 µM GroEL 14-mer in the absence (open squares) or presence (closed squares) of 2 mM ADP. Final concentration of the enzyme was 0.3 µM. After 30 s of incubation, an aliquot of GroES was added. Enzyme activity of refolded DHFR was analyzed after a 30-s incubation as above. The abscissa shows the ratio of GroES 7-mer concentration to the concentration of the GroEL-DHFR complex.
[View Larger Version of this Image (14K GIF file)]


Digestion Analysis of the GroEL-DHFR-GroES Complex

The presence of a nucleotide is not required for the binding of an unfolded protein-substrate to GroEL (Fig. 1; Ref. 2). Therefore, a random distribution of the bound nucleotide (ADP) relative to the site of the unfolded polypeptide binding on two GroEL toroids can result in two distinct types of GroES-GroEL interactions (4). Thus, GroES binding can occur on the same toroid (cis) where protein-substrate is bound or on the opposite site (trans) of the GroEL oligomer. These two complexes can be distinguished by using protease digestion analysis as was reported earlier (4). In the present study, two different methods were used to form the GroEL-DHFR complex. In the first case, DHFR was unfolded in 8 M urea for 15 h before dilution in the presence of GroEL. In the second case, the complex was formed by heating together GroEL and native DHFR at 45 °C for 10 min as reported earlier (7). Complexes were purified using size exclusion chromatography, and fractions containing GroEL were collected and concentrated. Solutions were supplemented with ADP and GroES (when needed). Digestion analysis was performed using Proteinase K (final concentration 0.2% w/w). Two different results were observed. While unfolded DHFR can be partially (42 ± 5%) protected by GroEL-GroES interaction if the GroEL-DHFR complex was preformed by heat denaturation (Fig. 3, lane 7), no protection could be detected if the complex was formed by addition of urea-unfolded protein (Fig. 3, lane 3). The positive controls, containing GroEL-DHFR complexes, formed by using urea or heat-denatured enzyme and consequently treated with ADP and GroES, but not Proteinase K, are shown in lanes 1 and 6, respectively. These results suggest that, due to the considerable differences in the protein conformation between chemically and thermally unfolded protein, the resulting GroEL-protein complexes have different properties, leading to the different mechanism of the GroES-facilitated protein release.


Fig. 3. Proteolytic analysis of the DHFR-GroEL complexes. Complexes between DHFR and GroEL were formed with urea-denatured (lanes 1, 2, and 3) and heat-denatured DHFR (lanes 4, 5, 6, and 7). Complexes were purified on a Superose 12 gel filtration column. Fractions containing GroEL were collected and concentrated to the final GroEL concentration of 0.35 µM. All samples were supplied with 2 mM ADP, and 0.7 µM GroES was added if necessary (lanes 1, 3, 6, and 7). Proteinase K was added (lanes 2, 3, 5, and 7) to a final concentration of 0.2% of the total protein (w/w), and the solution was incubated for 10 min at room temperature. The reaction was stopped by adding phenylmethylsulfonyl fluoride to 3 mM. Samples were analyzed on the 12% SDS Laemmli gel system followed by silver staining. The arrows show GroEL (1) and DHFR (2).
[View Larger Version of this Image (27K GIF file)]



DISCUSSION

Several different mechanisms have been suggested for the function of the GroEL-GroES system (4, 6). The release of the bound polypeptide from its complex with GroEL is one of the central steps that such a mechanism must address. Several aspects of these mechanisms are based on the ability to form relatively stable complexes involving GroEL in the presence of ADP. It was demonstrated (7) that ternary complexes containing DHFR, GroEL, and GroES could be formed by thermal denaturation and that a significant fraction of the bound DHFR was protected against Proteinase K digestion. This was taken to imply that GroES could bind in one of two ways to the complex. First, GroES could bind over the central cavity of GroEL on the same toroid as DHFR (cis), thus capping the complex and ejecting the DHFR into the chamber formed from the central cavity in the GroEL toroid and the overlying GroES dome; or second, GroES could bind on the toroid opposite to the bound DHFR (trans) leaving the DHFR on the surface. In this model, the DHFR in the cis complex is protected from Proteinase K, while that in the trans complex can be proteolyzed.

The present results are consistent with the observations for the thermally denatured protein, but the results are quite different for the urea-unfolded DHFR. While nearly 50% of DHFR can be protected by GroEL-GroES interaction in the former case, no protection was detected in the case of urea-unfolded protein-substrate (Fig. 3). Thus, it is clear that the complexes formed in these different ways have different characteristics. In contrast to the thermally produced complex, the urea complex can be partially discharged by GroES, and the undischarged part of the bound DHFR is sensitive to Proteinase K digestion. The considerable conformational differences between heat- and chemically unfolded protein can explain this phenomenon. DHFR in 8 M urea is likely to be fully unfolded, and the collapsed states that form on dilution could bind extensively to the binding sites on one of the two GroEL toroids. These extensive surfaces and the rapid collapse of the DHFR on dilution would favor binding close to the surface of the chaperonin. This surface location would be favored by the fact that the protein is introduced to a relatively unperturbed GroEL molecule. Since protein binding and GroES binding appear to involve the same or similar sites, peripheral binding of DHFR would lead to a weak complex in which cis GroES binding could release DHFR into the bulk solution by direct displacement. The trans ternary complex would be relatively stable in the presence of ADP. Thus, urea-unfolded DHFR would form a complex with GroEL, and GroES would release 50% by direct displacement and leave 50% as a trans complex that is susceptible to Proteinase K.

Thermally produced DHFR-GroEL complexes are different from complexes formed using urea-denatured DHFR. Thermal experiments that demonstrate the complex are performed by heating DHFR together with GroEL at 45 °C, so that thermal effects on both DHFR and GroEL must be considered. Bovine DHFR has a thermal transition with a midpoint at ~50 °C (17), so the 45 °C incubation does not lead to fully heat-denatured protein. In addition, many proteins, even after complete thermal denaturation, still contain significant ordered structures that can be unfolded further (18). In addition, GroEL itself has been reported to undergo two thermal transitions between 25 and 35 °C (19), so that temperature-induced formation of complexes influences the structure of GroEL. This suggestion is supported by the observation that there are proteolytic bands that must come from GroEL after proteolytic treatment of the thermally induced complexes (Fig. 3). It is possible, then, that heating DHFR in the presence of GroEL results in small, partially unfolded structures that can bind inside of the perturbed GroEL cavity. Then, the subsequent addition of GroES can result in forming a capped cis complex as described previously. The heating together of DHFR and GroEL is important, since the control experiments that have been carried out in the present study showed that no complexes between GroEL and DHFR can be formed when GroEL is preheated at 45 °C for 10 min and then added to native DHFR at 25 °C (data not shown). When DHFR was heated in the absence of GroEL, a precipitate formed, and the protein that remained in the solution could not be captured by GroEL at 25 °C.

The present data support the concept that the ability of the unfolded polypeptide to form a stable complex with GroEL at different steps of its folding pathway can influence the detailed mechanism of its assisted refolding. This would be in keeping with suggestions that chaperonin-facilitated folding can be modulated by the properties of both the protein-substrate and the GroEL (20, 21, 22).


FOOTNOTES

*   This research was supported by National Institutes of Health Research Grants GM25177 and ES05729 and Welch Grant AQ 723 (to P. M. H.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Dept. of Biochemistry, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78284. Tel.: 210-567-3737; Fax: 210-567-6595.
1    The abbreviations used are: GroEL, molecular chaperone 60; GroES, molecular chaperone 10; DHFR, bovine dihydrofolate reductase; H2F, dihydrofolate; beta ME, 2-mercaptoethanol.

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