Domain motions in GroEL upon binding of an oligopeptide

GroEL assists protein folding by preventing the interaction of partially folded molecules with other non-native proteins. It binds them, sequesters them, and then releases them so that they can fold in an ATP-driven cycle. Previous studies have also shown that protein substrates, GroES, and oligopeptides bind to partially overlapped sites on the apical domain surfaces of GroEL. In this study, we have determined the crystal structure at 3.0A resolution of a symmetric (GroEL-peptide)(14) complex. The binding of each of these small 12 amino acid residue peptides to GroEL involves interactions between three adjacent apical domains of GroEL. Each peptide interacts primarily with a single GroEL subunit. Residues R231 and R268 from adjacent subunits isolate each substrate-binding pocket, and prevent bound substrates from sliding into adjacent binding pockets. As a consequence of peptide binding, domains rotate and inter-domain interactions are greatly enhanced. The direction of rotation of the apical domain of each GroEL subunit is opposite to that of its intermediate domain. Viewed from outside, the apical domains rotate clockwise within one GroEL ring, while the ATP-induced apical domain rotation is counter-clockwise.     

GroEL-substrate-GroES ternary complexes are an important transient intermediate of the chaperonin cycle

GroEL C138W is a mutant form of Escherichia coli GroEL, which forms an arrested ternary complex composed of GroEL, the co-chaperonin GroES and the refolding protein molecule rhodanese at 25 degrees C. This state of arrest could be reversed with a simple increase in temperature. In this study, we found that GroEL C138W formed both stable trans- and cis-ternary complexes with a number of refolding proteins in addition to bovine rhodanese. These complexes could be reactivated by a temperature shift to obtain active refolded protein. The simultaneous binding of GroES and substrate to the cis ring suggested that an efficient transfer of substrate protein into the GroEL central cavity was assured by the binding of GroES prior to complete substrate release from the apical domain. Stopped-flow fluorescence spectroscopy of the mutant chaperonin revealed a temperature-dependent conformational change in GroEL C138W that acts as a trigger for complete protein release. The behavior of GroEL C138W was reflected closely in its in vivo characteristics, demonstrating the importance of this conformational change to the overall activity of GroEL.     

Analysis of peptides and proteins in their binding to GroEL

The GroEL–GroES is an essential molecular chaperon system that assists protein folding in cell. Binding of various substrate proteins to GroEL is one of the key aspects in GroEL‐assisted protein folding. Small peptides may mimic segments of the substrate proteins in contact with GroEL and allow detailed structural analysis of the interactions. A model peptide SBP has been shown to bind to a region in GroEL that is important for binding of substrate proteins. Here, we investigated whether the observed GroEL–SBP interaction represented those of GroEL–substrate proteins, and whether SBP was able to mimic various aspects of substrate proteins in GroE‐assisted protein folding cycle. We found that SBP competed with substrate proteins, including α‐lactalbumin, rhodanese, and malate dehydrogenase, in binding to GroEL. SBP stimulated GroEL ATP hydrolysis rate in a manner similar to that of α‐lactalbumin. SBP did not prevent GroES from binding to GroEL, and GroES association reduced the ATPase rates of GroEL/SBP and GroEL/α‐lactalbumin to a comparable extent. Binding of both SBP and α‐lactalbumin to apo GroEL was dominated by hydrophobic interaction. Interestingly, association of α‐lactalbumin to GroEL/GroES was thermodynamically distinct from that to GroEL with reduced affinity and decreased contribution from hydrophobic interaction. However, SBP did not display such differential binding behaviors to apo GroEL and GroEL/GroES, likely due to the lack of a contiguous polypeptide chain that links all of the bound peptide fragments. Nevertheless, studies using peptides provide valuable information on the nature of GroEL–substrate protein interaction, which is central to understand the mechanism of GroEL‐assisted protein folding. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

The crystal structure of a GroEL/peptide complex: plasticity as a basis for substrate diversity

The chaperonin GroEL is a double toriodal assembly that with its cochaperonin GroES facilitates protein folding with an ATP-dependent mechanism. Nonnative conformations of diverse protein substrates bind to the apical domains surrounding the opening of the double toroid's central cavity. Using phage display, we have selected peptides with high affinity for the isolated apical domain. We have determined the crystal structures of the complexes formed by the most strongly bound peptide with the isolated apical domain, and with GroEL. The peptide interacts with the groove between paired alpha helices in a manner similar to that of the GroES mobile loop. Our structural analysis, combined with other results, suggests that various modes of molecular plasticity are responsible for tight promiscuous binding of nonnative substrates and their release into the shielded cis assembly.     

Rhodanese can partially refold in its GroEL-GroES-ADP complex and can be released to give a homogeneous product

Molecular chaperones GroEL and GroES facilitate reactivation of denatured rhodanese which folds poorly unless the process is assisted. The present work tests the hypothesis that more extensively unfolded forms of rhodanese bind tighter than those forms that appear later in the folding pathway. The study of the interaction of different urea-induced forms of rhodanese with GroEL suggests that species preceding the domain folded form bind directly and productively to GroEL. Rhodanese partially folds while in the GroEL-GroES-ADP complex, but it does not significantly reach an active state. Partially folded rhodanese can be released from the GroEL-GroES-ADP complex by subdenaturing concentrations of urea as a homogeneous species that is committed to fold to the native conformation with little or no partitioning to the aggregated state. Dilution of denatured rhodanese to the same final concentration gives less active enzyme and significant aggregation. Urea denaturation studies show that active rhodanese released from complexes behaves identically to native enzyme, while spontaneously folded rhodanese has a different stability. These results are interpreted using a previously proposed model based on studies of unassisted rhodanese folding [Gorovits, B. M., McGee, W. A., and Horowitz, P. M. (1998) Biochim. Biophys. Acta 1382, 120-128. Panda, M., Gorovits, B. M., and Horowitz, P. M. (2000) J. Biol. Chem. 275, 63-70].     

Hydrophilic residues at the apical domain of GroEL contribute to GroES binding but attenuate polypeptide binding

The GroES binding site at the apical domain of GroEL, mostly consisting of hydrophobic residues, overlaps largely with the substrate polypeptide binding site. Essential contribution of hydrophobic interaction to the binding of both GroES and polypeptide was exemplified by the mutant GroEL(L237Q) which lost the ability to bind either of them. The binding site, however, contains three hydrophilic residues, E238, T261, and N265. For GroES binding, N265 is essential since GroEL(N265A) is unable to bind GroES. E238 contributes to rapid GroES binding to GroEL because GroEL(E238A) is extremely sluggish in GroES binding. Polypeptide binding was not impaired by any mutations of E238A, T261A, and N265A. Rather, these mutants, especially GroEL(N265A), showed stronger polypeptide binding affinity than wild-type GroEL. Thus, these hydrophilic residues have a dual role; they help GroES binding on one hand but attenuate polypeptide binding on the other hand.     

Multivalent binding of nonnative substrate proteins by the chaperonin GroEL

The chaperonin GroEL binds nonnative substrate protein in the central cavity of an open ring through exposed hydrophobic residues at the inside aspect of the apical domains and then mediates productive folding upon binding ATP and the cochaperonin GroES. Whether nonnative proteins bind to more than one of the seven apical domains of a GroEL ring is unknown. We have addressed this using rings with various combinations of wild-type and binding-defective mutant apical domains, enabled by their production as single polypeptides. A wild-type extent of binary complex formation with two stringent substrate proteins, malate dehydrogenase or Rubisco, required a minimum of three consecutive binding-proficient apical domains. Rhodanese, a less-stringent substrate, required only two wild-type domains and was insensitive to their arrangement. As a physical correlate, multivalent binding of Rubisco was directly observed in an oxidative cross-linking experiment.     

"Half of the sites" binding of D-glyceraldehyde-3-phosphate dehydrogenase folding intermediate with GroEL

Two D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) folding intermediate subunits bind with chaperonin 60 (GroEL) to form a stable complex, which can no longer bind with additional GAPDH intermediate subunits, but does bind with one more lysozyme folding intermediate or one chaperonin 10 (GroES) molecule, suggesting that the two GAPDH subunits bind at one end of the GroEL molecule displaying a "half of the sites" binding profile. For lysozyme, GroEL binds with either one or two folding intermediates to form a stable 1:1 or 1:2 complex with one substrate on each end of the GroEL double ring for the latter. The 1:1 complex of GroEL.GroES binds with one lysozyme or one dimeric GAPDH folding intermediate to form a stable ternary complex. Both complexes of GroEL.lysozyme1 and GroEL.GAPDH2 bind with one GroES molecule only at the other end of the GroEL molecule forming a trans ternary complex. According to the stoichiometry of GroEL binding with the GAPDH folding intermediate and the formation of ternary complexes containing GroEL.GAPDH2, it is suggested that the folding intermediate of GAPDH binds, very likely in the dimeric form, with GroEL at one end only.     

GroEL Recognizes an Amphipathic Helix and Binds to the Hydrophobic Side

GroEL is an essential Escherichia coli molecular chaperon that uses ATP to facilitate correct folding of a range of proteins in a cell. Central to the GroEL substrate diversity is how GroEL recognizes the substrates. The interaction between GroEL and substrate has been proposed to be largely hydrophobic because GroEL interacts with proteins in non-native conformations but not in native forms. Analysis of GroEL substrate proteins reveals that one of its main substrates are proteins with αβ folding domains, suggesting that GroEL may stabilize the collapsed αβ core by binding to hydrophobic surfaces that are usually buried between the α and β elements. In this study, we characterize the interaction between GroEL and a peptide derived from our previous selection via a phage display method. NMR studies map the peptide-binding site to the region containing Helices H and I, which is consistent with evidence that this region comprises the primary substrate-binding site. The peptide is largely unstructured in solution but adopts a helical conformation when bound to the GroEL apical domain with a moderate affinity (K_d = 17.1 ± 2.5 μm). The helical conformation aligns residues to form an amphipathic structure, and the hydrophobic side of this amphipathic helix interacts with GroEL as suggested by fluorescence quenching studies. Together with previous structural studies on the GroEL-peptide complexes, our work supports the notion that the amphipathic secondary elements in the substrate proteins may be the structural motif recognized by GroEL.The bacterial chaperonin GroEL and its co-chaperonin GroES are essential for cell viability by assisting folding of a wide range of proteins via an ATP-dependent mechanism (1–3). Structurally, fourteen 57-kDa GroEL subunits assemble into two back-to-back stacking heptameric rings, giving rise to two functionally independent central cavities (4). Each GroEL subunit folds into three distinctive domains: equatorial domain, intermediate domain, and apical domain. The equatorial domains contain the ATP-binding sites and provide most of the intra-ring interactions and all the inter-ring interactions. The apical domains form the rims of the central cavities and contain the binding sites for the substrate proteins and GroES. The intermediate domains link the apical domains and the equatorial domains. For the co-chaperonin GroES, seven GroES subunits, of 10 kDa each, assemble into a heptamer ring (5, 6). In forming the GroEL-GroES complex, GroES caps one end of GroEL, and large structural changes are observed in both GroEL and GroES (7). In GroEL, the apical domain is rotated 90° along its axis and 60° upwards, and the intermediate domain is closed down ∼25° to the equatorial domain. A loop in GroES (residues 17–33) that is unstructured in the isolated GroES adopts a β-turn structure and forms contact with the GroEL apical domain. Compared with the unliganded GroEL, the volume of the enclosed GroEL-GroES cavity is doubled, and the surface lining the wall of the GroEL cavity changes from hydrophobic to hydrophilic.A wealth of information derived from both intensive biochemical and structural characterizations has revealed a general role of GroEL-GroES in assisting protein folding (see reviews in Refs. 3, 8, and 9). Briefly, GroEL binds the substrate proteins in their aggregation-prone non-native states, preventing them from aggregating. Binding of ATP to the substrate occupied GroEL ring (cis-ring) presumably induces large conformational change in GroEL that promotes binding of GroES to the cis-ring. As a result of ATP and GroES binding, the substrate protein is displaced into the GroEL central cavity, initiating the folding process. Both hydrolysis of ATP in the cis-ring and binding of ATP to the substrate unoccupied ring (trans ring) weaken the GroES-GroEL interaction, and ATP binding to the trans ring results in the dissociation of GroES from GroEL, releasing substrate from the central cavity of GroEL. The released substrate may continue folding into the native state if in a folding competent state or may rebind to GroEL if it is still misfolded.One of the most intriguing aspects of the GroE-assisted folding is the substrate promiscuity. It has been shown that about 300 Escherichia coli proteins can interact with GroEL, and these proteins are diverse in terms of both structures and functions (10). A range of techniques have been applied to investigate this important yet complex aspect, and salient features regarding GroEL-substrate interactions have emerged. The apical domains, on the rim of the GroEL central cavity, contain the main substrate-binding site (11–13). Structural flexibility, reflected by both high temperature factors of the apical domain in the crystal structure of tetradecameric GroEL (14) and conformational multiplicity around Helix H and I (15), is proposed to account for the diverse spectrum of GroEL substrates. Mutational studies on GroEL suggest that the GroEL-substrate interactions are largely hydrophobic (16). Structural study on GroEL-substrate interaction, however, is hindered mainly because of the multiple conformations of the bound substrate protein. Very recently, NMR techniques have been used to directly investigate the bound conformations of the substrate (17, 18); yet the nature of GroEL-substrate interaction is not revealed. Peptides may mimic segments of substrate proteins, and studies of GroEL-peptide interactions have uncovered detailed intermolecular interactions and provided insights into principles of substrate recognition by GroEL. The bound peptides may adopt α-helix (19–23), β-hairpin (15), or extended conformations (24), and despite different conformations, they all appear to bind to Helix H and I of GroEL. Hydrophobic interaction dominates the interface between GroEL and peptides in either β-hairpin or extended structures and is proposed so between GroEL and α-helical peptides. These detailed structural characterizations on GroEL-peptide interactions have contributed to dissecting the complex nature of the substrate recognition by GroEL (25).We previously identified a high affinity peptide (strong binding peptide (SBP))² for GroEL using a phage display method and found that SBP adopts a β-hairpin structure bound to GroEL (15, 26). To investigate the contribution of the β-turn in SBP to the GroEL-SBP interaction, we have created various SBP variants with the intension to disrupt the β-turn structure and have studied their binding to GroEL. One of the peptides (termed SBP-W2DP6V), however, adopts a helical conformation when bound to GroEL by NMR analysis. NMR results also map the peptide-binding site on GroEL to be a region formed by Helix H and I. The helical peptide has an amphipathic feature, and fluorescence studies provide direct evidence that the hydrophobic face is involved in the interaction with GroEL. Our structural analysis, combined with previous studies, suggests that GroEL recognizes the amphipathic property in the secondary structures of the substrate protein and binds preferably to the hydrophobic side of these structural elements to stabilize and preserve their structures.     

Role of Denatured-State Properties in Chaperonin Action Probed by Single-Molecule Spectroscopy

The bacterial chaperonin GroEL/GroES assists folding of a broad spectrum of denatured and misfolded proteins. Here, we explore the limits of this remarkable promiscuity by mapping two denatured proteins with very different conformational properties, rhodanese and cyclophilin A, during binding and encapsulation by GroEL/GroES with single-molecule spectroscopy, microfluidic mixing, and ensemble kinetics. We find that both proteins bind to GroEL with high affinity in a reaction involving substantial conformational adaptation. However, whereas the compact denatured state of rhodanese is encapsulated efficiently upon addition of GroES and ATP, the more expanded and unstructured denatured cyclophilin A is not encapsulated but is expelled into solution. The origin of this surprising disparity is the weaker interactions of cyclophilin A with a transiently formed GroEL-GroES complex, which may serve as a crucial checkpoint for substrate discrimination.     

Elucidation of steps in the capture of a protein substrate for efficient encapsulation by GroE

We have identified five structural rearrangements in GroEL induced by the ordered binding of ATP and GroES. The first discernable rearrangement (designated T --> R(1)) is a rapid, cooperative transition that appears not to be functionally communicated to the apical domain. In the second (R(1) --> R(2)) step, a state is formed that binds GroES weakly in a rapid, diffusion-limited process. However, a second optical signal, carried by a protein substrate bound to GroEL, responds neither to formation of the R(2) state nor to the binding of GroES. This result strongly implies that the substrate protein remains bound to the inner walls of the initially formed GroEL.GroES cavity, and is not yet displaced from its sites of interaction with GroEL. In the next rearrangement (R(2).GroES --> R(3).GroES) the strength of interaction between GroEL and GroES is greatly enhanced, and there is a large and coincident loss of fluorescence-signal intensity in the labeled protein substrate, indicating that there is either a displacement from its binding sites on GroEL or at least a significant change of environment. These results are consistent with a mechanism in which the shift in orientation of GroEL apical domains between that seen in the apo-protein and stable GroEL.GroES complexes is highly ordered, and transient conformational intermediates permit the association of GroES before the displacement of bound polypeptide. This ensures efficient encapsulation of the polypeptide within the GroEL central cavity underneath GroES.     

Minichaperone (GroEL191-345) mediated folding of MalZ proceeds by binding and release of native and functional intermediates

The isolated apical domain of GroEL consisting of residues 191–345 (known as “minichaperone”) binds and assists the folding of a wide variety of client proteins without GroES and ATP, but the mechanism of its action is still unknown. In order to probe into the matter, we have examined minichaperone-mediated folding of a large aggregation prone protein Maltodextrin-glucosidase (MalZ). The key objective was to identify whether MalZ exists free in solution, or remains bound to, or cycling on and off the minichaperone during the refolding process. When GroES was introduced during refolding process, production of the native MalZ was inhibited. We also observed the same findings with a trap mutant of GroEL, which stably captures a predominantly non-native MalZ released from minichaperone during refolding process, but does not release it. Tryptophan and ANS fluorescence measurements indicated that refolded MalZ has the same structure as the native MalZ, but that its structure when bound to minichaperone is different. Surface plasmon resonance measurements provide an estimate for the equilibrium dissociation constant KD for the MalZ-minichaperone complex of 0.21 ± 0.04 μM, which are significantly higher than for most GroEL clients. This showed that minichaperone interacts loosely with MalZ to allow the protein to change its conformation and fold while bound during the refolding process. These observations suggest that the minichaperone works by carrying out repeated cycles of binding aggregation-prone protein MalZ in a relatively compact conformation and in a partially folded but active state, and releasing them to attempt to fold in solution.     

Triggering protein folding within the GroEL-GroES complex

The folding of many proteins depends on the assistance of chaperonins like GroEL and GroES and involves the enclosure of substrate proteins inside an internal cavity that is formed when GroES binds to GroEL in the presence of ATP. Precisely how assembly of the GroEL-GroES complex leads to substrate protein encapsulation and folding remains poorly understood. Here we use a chemically modified mutant of GroEL (EL43Py) to uncouple substrate protein encapsulation from release and folding. Although EL43Py correctly initiates a substrate protein encapsulation reaction, this mutant stalls in an intermediate allosteric state of the GroEL ring, which is essential for both GroES binding and the forced unfolding of the substrate protein. This intermediate conformation of the GroEL ring possesses simultaneously high affinity for both GroES and non-native substrate protein, thus preventing escape of the substrate protein while GroES binding and substrate protein compaction takes place. Strikingly, assembly of the folding-active GroEL-GroES complex appears to involve a strategic delay in ATP hydrolysis that is coupled to disassembly of the old, ADP-bound GroEL-GroES complex on the opposite ring.     

Interaction of oxidized chaperonin GroEL with an unfolded protein at low temperatures

The chaperonin GroEL binds to non-native substrate proteins via hydrophobic interactions, preventing their aggregation, which is minimized at low temperatures. In the present study, we investigated the refolding of urea-denatured rhodanese at low temperatures, in the presence of ox-GroEL (oxidized GroEL), which contains increased exposed hydrophobic surfaces and retains its ability to hydrolyse ATP. We found that ox-GroEL could efficiently bind the urea-unfolded rhodanese at 4°C, without requiring excess amount of chaperonin relative to normal GroEL (i.e. non-oxidized). The release/reactivation of rhodanese from GroEL was minimal at 4°C, but was found to be optimal between 22 and 37°C. It was found that the loss of the ATPase activity of ox-GroEL at 4°C prevented the release of rhodanese from the GroEL-rhodanese complex. Thus ox-GroEL has the potential to efficiently trap recombinant or non-native proteins at 4°C and release them at higher temperatures under appropriate conditions.     

GroEL recognises sequential and non-sequential linear structural motifs compatible with extended beta-strands and alpha-helices.

The chaperonin GroEL binds a variety of polypeptides that share no obvious sequence similarity. The precise structural, chemical and dynamic features that are recognised remain largely unknown. Structural models of the complex between GroEL and its co-chaperonin GroES, and of the isolated apical domain of GroEL (minichaperone; residues 191-376) with a 17 residue N-terminal tag show that a linear sequential sequence (extended beta-strand) can be bound. We have analysed characteristics of the motifs that bind to GroEL by using affinity panning of immobilised GroEL minichaperones for a library of bacteriophages that display the fungal cellulose-binding domain of the enzyme cellobiohydrolase I. This protein has seven non-sequential residues in its binding site that form a linear binding motif with similar dimensions and characteristics to the peptide tag that was bound to the minichaperone GroEL(191-376). The seven residues thus form a constrained scaffold. We find that GroEL does bind suitable mutants of these seven residues. The side-chains recognised do not have to be totally hydrophobic, but polar and positively charged chains can be accommodated. Further, the spatial distribution of the side-chains is also compatible with those in an alpha-helix. This implies that GroEL can bind a wide range of structures, from extended beta-strands and alpha-helices to folded states, with exposed side-chains. The binding site can accommodate substrates of approximately 18 residues when in a helical or seven when in an extended conformation. The data support two activities of GroEL: the ability to act as a temporary parking spot for sticky intermediates by binding many motifs; and an unfolding activity of GroEL by binding an extended sequential conformation of the substrate.     

Structural changes in GroEL effected by binding a denatured protein substrate

In the absence of nucleotides or cofactors, the Escherichia coli chaperonin GroEL binds select proteins in non-native conformations, such as denatured glutamine synthetase (GS) monomers, preventing their aggregation and spontaneous renaturation. The nature of the GroEL-GS complexes thus formed, specifically the effect on the conformation of the GroEL tetradecamer, has been examined by electron microscopy. We find that specimens of GroEL-GS are visibly heterogeneous, due to incomplete loading of GroEL with GS. Images contain particles indistinguishable from GroEL alone, and also those with consistent identifiable differences. Side-views of the modified particles reveal additional protein density at one end of the GroEL-GS complex, and end-views display chirality in the heptameric projection not seen in the unliganded GroEL. The coordinate appearance of these two projection differences suggests that binding of GS, as representative of a class of protein substrates, induces or stabilizes a conformation of GroEL that differs from the unliganded chaperonin. Three-dimensional reconstruction of the GroEL-GS complex reveals the location of the bound protein substrate, as well as complex conformational changes in GroEL itself, both cis and trans with respect to the bound GS. The most apparent structural alterations are inward movements of the apical domains of both GroEL heptamers, protrusion of the substrate protein from the cavity of the cis ring, and a narrowing of the unoccupied opening of the trans ring.     

Determination of the number of active GroES subunits in the fused heptamer GroES required for interactions with GroEL

A double-heptamer ring chaperonin GroEL binds denatured substrate protein, ATP, and GroES to the same heptamer ring and encapsulates substrate into the central cavity underneath GroES where productive folding occurs. GroES is a disk-shaped heptamer, and each subunit has a GroEL-binding loop. The residues of the GroEL subunit responsible for GroES binding largely overlap those involved in substrate binding, and the mechanism by which GroES can replace the substrate when GroES binds to GroEL/substrate complex remains to be clarified. To address this question, we generated single polypeptide GroES by fusing seven subunits with various combinations of active and GroEL binding-defective subunits. Functional tests of the fused GroES variants indicated that four active GroES subunits were required for efficient formation of the stable GroEL/GroES complex and five subunits were required for the productive GroEL/substrate/GroES complex. An increase in the number of defective GroES subunits resulted in a slowing of encapsulation and folding. These results indicate the presence of an intermediate GroEL/substrate/GroES complex in which the substrate and GroES bind to GroEL by sharing seven common binding sites.     

Chaperonin-mediated protein folding: fate of substrate polypeptide

Chaperonins are megadalton ring assemblies that mediate essential ATP-dependent assistance of protein folding to the native state in a variety of cellular compartments, including the mitochondrial matrix, the eukaryotic cytosol, and the bacterial cytoplasm. Structural studies of the bacterial chaperonin, GroEL, both alone and in complex with its co-chaperonin, GroES, have resolved the states of chaperonin that bind and fold non-native polypeptides. Functional studies have resolved the action of ATP binding and hydrolysis in driving the GroEL-GroES machine through its folding-active and binding-active states, respectively. Yet the exact fate of substrate polypeptide during these steps is only poorly understood. For example, while binding involves multivalent interactions between hydrophobic side-chains facing the central cavity of GroEL and exposed hydrophobic surfaces of the non-native protein, the structure of any polypeptide substrate while bound to GroEL remains unknown. It is also unclear whether binding to an open GroEL ring is accompanied by structural changes in the non-native substrate, in particular whether there is an unfolding action. As a polypeptide-bound ring becomes associated with GroES, do the large rigid-body movements of the GroEL apical domains serve as another source of a potential unfolding action? Regarding the encapsulated folding-active state, how does the central cavity itself influence the folding trajectory of a substrate? Finally, how do GroEL and GroES serve, as recently recognized, to assist the folding of substrates too large to be encapsulated inside the machine? Here, such questions are addressed with the findings available to date, and means of further resolving the states of chaperonin-associated polypeptide are discussed.     

Crystal structure of the CCTgamma apical domain: implications for substrate binding to the eukaryotic cytosolic chaperonin

The chaperonin containing TCP-1 (CCT, also known as TRiC) is the only member of the chaperonin family found in the cytosol of eukaryotes. Like other chaperonins, it assists the folding of newly synthesised proteins. It is, however, unique in its specificity towards only a small subset of non-native proteins. We determined two crystal structures of mouse CCTgamma apical domain at 2.2 A and 2.8 A resolution. They reveal a surface patch facing the inside of the torus that is highly evolutionarily conserved and specific for the CCTgamma apical domain. This putative substrate-binding region consists of predominantly positively charged side-chains. It suggests that the specificity of this apical domain towards its substrate, partially folded tubulin, is conferred by polar and electrostatic interactions. The site and nature of substrate interaction are thus profoundly different between CCT and its eubacterial homologue GroEL, consistent with their different functions in general versus specific protein folding assistance.     

GroEL binds artificial proteins with random sequences

Chaperonin GroEL from Escherichia coli binds to the non-native states of many unrelated proteins, and GroEL-recognizable structural features have been argued. As model substrate proteins of GroEL, we used seven artificial proteins (138 approximately 141 residues), each of which has a unique but randomly chosen amino acid sequence and no propensity to fold into a certain structure. Two of them were water-soluble, and the rest were soluble in 3 m urea. The soluble ones interacted with GroEL in a manner similar to that of a natural substrate; they stimulated the ATPase cycle of GroEL and GroEL/GroES and inhibited GroEL-assisted folding of other protein. All seven artificial proteins were able to bind to GroEL. The results suggest that the secondary structure as well as the specific sequence motif of the substrate proteins are not necessary to be recognized by GroEL.