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.     

A newly synthesized protein interacts with GroES on the surface of chaperonin GroEL.

To facilitate folding and assembly of different proteins, chaperonin GroEL requires the presence of its helper protein GroES. Using a photochemical cross-linking approach, we show that GroES and newly synthesized pre-beta-lactamase (pre-beta lac) contact with each other only within the ternary complex with GroEL. Possibly owing to this contact GroES is able to directly influence the pre-beta lac/GroEL interaction. Furthermore, the cross-linking of pre-beta lac to GroES suggests that the binding of the protein ligands to GroEL occurs near the GroES binding site, known to be in the central hole space of GroEL.     

Mechanism of chaperonin action: GroES binding and release can drive GroEL-mediated protein folding in the absence of ATP hydrolysis.

As a basic principle, assisted protein folding by GroEL has been proposed to involve the disruption of misfolded protein structures through ATP hydrolysis and interaction with the cofactor GroES. Here, we describe chaperonin subreactions that prompt a re-examination of this view. We find that GroEL-bound substrate polypeptide can induce GroES cycling on and off GroEL in the presence of ADP. This mechanism promotes efficient folding of the model protein rhodanese, although at a slower rate than in the presence of ATP. Folding occurs when GroES displaces the bound protein into the sequestered volume of the GroEL cavity. Resulting native protein leaves GroEL upon GroES release. A single-ring variant of GroEL is also fully functional in supporting this reaction cycle. We conclude that neither the energy of ATP hydrolysis nor the allosteric coupling of the two GroEL rings is directly required for GroEL/GroES-mediated protein folding. The minimal mechanism of the reaction is the binding and release of GroES to a polypeptide-containing ring of GroEL, thereby closing and opening the GroEL folding cage. The role of ATP hydrolysis is mainly to induce conformational changes in GroEL that result in GroES cycling at a physiologically relevant rate.     

On the role of symmetrical and asymmetrical chaperonin complexes in assisted protein folding.

The cylindrical chaperonin GroEL of E. coli and its ring-shaped cofactor GroES cooperate in mediating the ATP-dependent folding of a wide range of polypeptides in vivo and in vitro. By binding to the ends of the GroEL cylinder, GroES displaces GroEL-bound polypeptide into an enclosed folding cage, thereby preventing protein aggregation during folding. The dynamic interaction of GroEL and GroES is regulated by the GroEL ATPase and involves the formation of asymmetrical GroEL:GroES1 and symmetrical GroEL: GroES2 complexes. The proposed role of the symmetrical complex as a catalytic intermediate of the chaperonin mechanism has been controversial. It has also been suggested that the formation of GroEL:GroES2 complexes allows the folding of two polypeptide molecules per GroEL reaction cycle, one in each ring of GroEL. By making use of a procedure to stabilize chaperonin complexes by rapid crosslinking for subsequent analysis by native PAGE, we have quantified the occurrence of GroEL:GroES1 and GroEL:GroES2 complexes in active refolding reactions under a variety of conditions using mitochondrial malate dehydrogenase (mMDH) as a substrate. Our results show that the symmetrical complexes are neither required for chaperonin function nor does their presence significantly increase the rate of mMDH refolding. In contrast, chaperonin-assisted folding is strictly dependent on the formation of asymmetrical GroEL:GroES1 complexes. These findings support the view that GroEL:GroES2 complexes have no essential role in the chaperonin mechanism.     

The affinity of the GroEL/GroES complex for peptides under conditions of protein folding

The affinity of four short peptides for the Escherichia coli molecular chaperone GroEL was studied in the presence of the co-chaperone GroES and nucleotides. Our data show that binding of GroES to one ring enhances the interaction of the peptides with the opposite GroEL ring, a finding that was related to the structural readjustments in GroEL following GroES binding. We further report that the GroEL/GroES complex has a high affinity for peptides during ATP hydrolysis when protein substrates would undergo repeated cycles of assisted folding. Although we could not determine at which step(s) during the cycle our peptides interacted with GroEL, we propose that successive state changes in GroEL during ATP hydrolysis may create high affinity complexes and ensure maximum efficiency of the chaperone machinery under conditions of protein folding.     

GroEL mediates protein folding with a two successive timer mechanism

GroEL encapsulates nonnative substrate proteins in a central cavity capped by GroES, providing a safe folding cage. Conventional models assume that a single timer lasting approximately 8 s governs the ATP hydrolysis-driven GroEL chaperonin cycle. We examine single molecule imaging of GFP folding within the cavity, binding release dynamics of GroEL-GroES, ensemble measurements of GroEL/substrate FRET, and the initial kinetics of GroEL ATPase activity. We conclude that the cycle consists of two successive timers of approximately 3 s and approximately 5 s duration. During the first timer, GroEL is bound to ATP, substrate protein, and GroES. When the first timer ends, the substrate protein is released into the central cavity and folding begins. ATP hydrolysis and phosphate release immediately follow this transition. ADP, GroES, and substrate depart GroEL after the second timer is complete. This mechanism explains how GroES binding to a GroEL-substrate complex encapsulates the substrate rather than allowing it to escape into solution.     

Chaperone activity of a chimeric GroEL protein that can exist in a single or double ring form.

The molecular chaperone GroEL is a protein complex consisting of two rings each of seven identical subunits. It is thought to act by providing a cavity in which a protein substrate can fold into a form that has no propensity to aggregate. Substrate proteins are sequestered in the cavity while they fold, and prevented from diffusion out of the cavity by the action of the GroES complex, that caps the open end of the cavity. A key step in the mechanism of action of GroEL is the transmission of a conformational change between the two rings, induced by the binding of nucleotides to the GroEL ring opposite to the one containing the polypeptide substrate. This conformational change then leads to the discharge of GroES from GroEL, enabling polypeptide release. Single ring forms of GroEL are thus predicted to be unable to chaperone the folding of GroES-dependent substrates efficiently, since they are unable to discharge the bound GroES and unable to release folded protein. We describe here a detailed functional analysis of a chimeric GroEL protein, which we show to exist in solution in equilibrium between single and double ring forms. We demonstrate that whereas the double ring form of the GroEL chimera functions effectively in refolding of a GroES-dependent substrate, the single ring form does not. The single ring form of the chimera, however, is able to chaperone the folding of a substrate that does not require GroES for its efficient folding. We further demonstrate that the double ring structure of GroEL is likely to be required for its activity in vivo.     

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.     

Encapsulation of an 86-kDa assembly intermediate inside the cavities of GroEL and its single-ring variant SR1 by GroES

We described previously that during the assembly of the alpha(2)beta(2) heterotetramer of human mitochondrial branched-chain alpha-ketoacid dehydrogenase (BCKD), chaperonins GroEL/GroES interact with the kinetically trapped heterodimeric (alphabeta) intermediate to facilitate conversion of the latter to the native BCKD heterotetramer. Here, we show that the 86-kDa heterodimeric intermediate possesses a native-like conformation as judged by its binding to a fluorescent probe 1-anilino-8-naphthalenesulfonate. This large heterodimeric intermediate is accommodated as an entity inside cavities of GroEL and its single-ring variant SR1 and is encapsulated by GroES as indicated by the resistance of the heterodimer to tryptic digestion. The SR1-alphabeta-GroES complex is isolated as a stable single species by gel filtration in the presence of Mg-ATP. In contrast, an unfolded BCKD fusion protein of similar size, which also resides in the GroEL or SR1 cavity, is too large to be capped by GroES. The cis-capping mechanism is consistent with the high level of BCKD activity recovered with the GroEL-alphabeta complex, GroES, and Mg-ATP. The 86-kDa native-like heterodimeric intermediate in the BCKD assembly pathway represents the largest protein substrate known to fit inside the GroEL cis cavity underneath GroES, which significantly exceeds the current size limit of 57 kDa established for unfolded proteins.     

GroEL/GroES-mediated folding of a protein too large to be encapsulated.

The chaperonin GroEL binds nonnative proteins too large to fit inside the productive GroEL-GroES cis cavity, but whether and how it assists their folding has remained unanswered. We have examined yeast mitochondrial aconitase, an 82 kDa monomeric Fe(4)S(4) cluster-containing enzyme, observed to aggregate in chaperonin-deficient mitochondria. We observed that aconitase folding both in vivo and in vitro requires both GroEL and GroES, and proceeds via multiple rounds of binding and release. Unlike the folding of smaller substrates, however, this mechanism does not involve cis encapsulation but, rather, requires GroES binding to the trans ring to release nonnative substrate, which likely folds in solution. Following the phase of ATP/GroES-dependent refolding, GroEL stably bound apoaconitase, releasing active holoenzyme upon Fe(4)S(4) cofactor formation, independent of ATP and GroES.     

Design of a molecular chaperone-assisted protein folding bioreactor

Escherichia coli molecular chaperone GroEL and co-chaperone GroES are well known to assist the folding/refolding of a diverse range of substrate proteins. Despite this, there have been relatively few reports of the GroEL/GroES molecular chaperone system being used as a biotechnology tool for protein folding/refolding. In this paper, a solution-phase protein folding bioreactor is described that involves the complete GroEL/GroES system. The main features of this bioreactor are the use of a stirred-cell concentrator fitted with a 100 kDa molecular weight cutoff membrane and an attached buffer reservoir. This bioreactor system was used successfully for assisted-batch refolding of guanidinium chloride (Gu-HCl) unfolded mitochondrial malate dehydrogenase (mMDH). We believe that protein folding bioreactor systems of this type could have wide potential utility for the folding/refolding of unfolded protein substrates.     

Football- and bullet-shaped GroEL-GroES complexes coexist during the reaction cycle

GroEL is an Escherichia coli chaperonin that is composed of two heptameric rings stacked back-to-back. GroEL assists protein folding with its cochaperonin GroES in an ATP-dependent manner in vitro and in vivo. However, it is still unclear whether GroES binds to both rings of GroEL simultaneously under physiological conditions. In this study, we monitored the GroEL-GroES interaction in the reaction cycle using fluorescence resonance energy transfer. We found that nearly equivalent amounts of symmetric GroEL-(GroES)(2) (football-shaped) complex and asymmetric GroEL-GroES (bullet-shaped) complex coexist during the functional reaction cycle. We also found that D398A, an ATP hydrolysis defective mutant of GroEL, forms a football-shaped complex with ATP bound to the two rings. Furthermore, we showed that ADP prevents the association of ATP to the trans-ring of GroEL, and as a consequence, the second GroES cannot bind to GroEL. Considering the concentrations of ADP and ATP in E. coli, ADP is expected to have a small effect on the inhibition of GroES binding to the trans-ring of GroEL in vivo. These results suggest that we should reconsider the chaperonin-mediated protein-folding mechanism that involves the football-shaped complex.     

"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.     

Mimicking the action of GroEL in molecular dynamics simulations: application to the refinement of protein structures

Bacterial chaperonin, GroEL, together with its co-chaperonin, GroES, facilitates the folding of a variety of polypeptides. Experiments suggest that GroEL stimulates protein folding by multiple cycles of binding and release. Misfolded proteins first bind to an exposed hydrophobic surface on GroEL. GroES then encapsulates the substrate and triggers its release into the central cavity of the GroEL/ES complex for folding. In this work, we investigate the possibility to facilitate protein folding in molecular dynamics simulations by mimicking the effects of GroEL/ES namely, repeated binding and release, together with spatial confinement. During the binding stage, the (metastable) partially folded proteins are allowed to attach spontaneously to a hydrophobic surface within the simulation box. This destabilizes the structures, which are then transferred into a spatially confined cavity for folding. The approach has been tested by attempting to refine protein structural models generated using the ROSETTA procedure for ab initio structure prediction. Dramatic improvements in regard to the deviation of protein models from the corresponding experimental structures were observed. The results suggest that the primary effects of the GroEL/ES system can be mimicked in a simple coarse-grained manner and be used to facilitate protein folding in molecular dynamics simulations. Furthermore, the results support the assumption that the spatial confinement in GroEL/ES assists the folding of encapsulated proteins.     

Trigger factor is involved in GroEL-dependent protein degradation in Escherichia coli and promotes binding of GroEL to unfolded proteins.

In Escherichia coli, the molecular chaperones of hsp60/hsp10 (GroEL/GroES) families are required not only for protein folding but also for the rapid degradation of certain abnormal proteins. The rate-limiting step in the degradation of the fusion protein CRAG by protease ClpP appears to be the formation of a complex with GroEL. We have isolated these complexes and found that each GroEL 14mer contained a short-lived fragment of CRAG plus a 50 kDa polypeptide, which we identified by sequencing and immunological methods as Trigger Factor (TF). Upon ATP addition, GroEL and TF dissociated together from CRAG but remained tightly associated with each other even upon gel filtration. TF was originally proposed to function in protein translocation across membranes but altering cellular content of TF did not affect this process in vivo. By contrast, low levels of TF expression markedly reduced CRAG degradation, while an overproduction of TF greatly stimulated this process. Furthermore, in extracts of cells expressing high levels of TF, the capacity of GroEL to bind to CRAG is greatly increased. Overproduction of TF also stimulated GroEL's ability to bind to other unfolded proteins (fetuin and histone). Thus, TF is a rate-limiting factor for CRAG degradation; it appears to regulate GroEL function and to promote the formation of TF-GroEL-CRAG complexes which are critical for proteolysis.     

Leu309 plays a critical role in the encapsulation of substrate protein into the internal cavity of GroEL

In the crystal structure of the native GroEL.GroES.substrate protein complex from Thermus thermophilus, one GroEL subunit makes contact with two GroES subunits. One contact is through the H-I helices, and the other is through a novel GXXLE region. The side chain of Leu, in the GXXLE region, forms a hydrophobic cluster with residues of the H helix (Shimamura, T., Koike-Takeshita, A., Yokoyama, K., Masui, R., Murai, N., Yoshida, M., Taguchi, H., and Iwata, S. (2004) Structure (Camb.) 12, 1471-1480). Here, we investigated the functional role of Leu in the GXXLE region, using Escherichia coli GroEL. The results are as follows: (i) cross-linking between introduced cysteines confirmed that the GXXLE region in the E. coli GroEL.GroES complex is also in contact with GroES; (ii) when Leu was replaced by Lys (GroEL(L309K)) or other charged residues, chaperone activity was largely lost; (iii) the GroEL(L309K).substrate complex failed to bind GroES to produce a stable GroEL(L309K).GroES.substrate complex, whereas free GroEL(L309K) bound GroES normally; (iv) the GroEL(L309K).GroES.substrate complex was stabilized with BeF(x), but the substrate protein in the complex was readily digested by protease, indicating that it was not properly encapsulated into the internal cavity of the complex. Thus, conformational communication between the two GroES contact sites, the H helix and the GXXLE region (through Leu(309)), appears to play a critical role in encapsulation of the substrate.     

New aspects on the mechanism of GroEL-assisted protein folding

The mechanism of assisted protein folding by the chaperonin GroEL alone or in complex with the co-chaperonin GroES and in the presence or absence of nucleotides has been subject to extensive investigations during the last years. In this paper we present data where we have inactivated GroEL by stepwise blocking the nucleotide binding sites using the non-hydrolyzable ATP analogue, (Cr(H2O)4)3+ATP. We correlated the amount of accessible nucleotide binding sites with the residual ATP hydrolysis activity of GroEL as well as the residual refolding activity for two different model substrates. Under the conditions used, folding of the substrate proteins and ATP hydrolysis were directly proportional to the residual, accessible nucleotide binding sites. In the presence of GroES, 50% of the nucleotide binding sites were protected from inactivation by CrATP and the resulting protein retains 50% of both ATPase and refolding activity. The results strongly suggest that under the conditions used in our experiments, the nucleotide binding sites are additive in character and that by blocking of a certain number of binding sites a proportional amount of ATP hydrolysis and refolding activities are inactivated. The experiments including GroES suggest that full catalytic activity of GroEL requires both rings of the chaperonin. Blocking of the nucleotide binding sites of one ring still allows function of the second ring.     

Function in protein folding of TRiC, a cytosolic ring complex containing TCP-1 and structurally related subunits.

T-complex polypeptide 1 (TCP-1) was analyzed as a potential chaperonin (GroEL/Hsp60) equivalent of the eukaryotic cytosol. We found TCP-1 to be part of a hetero-oligomeric 970 kDa complex containing several structurally related subunits of 52-65 kDa. These members of a new protein family are assembled into a TCP-1 ring complex (TRiC) which resembles the GroEL double ring. The main function of TRiC appears to be in chaperoning monomeric protein folding: TRiC binds unfolded polypeptides, thereby preventing their aggregation, and mediates the ATP-dependent renaturation of unfolded firefly luciferase and tubulin. At least in vitro, TRiC appears to function independently of a small co-chaperonin protein such as GroES. Folding of luciferase is mediated by TRiC but not by GroEL/ES. This suggests that the range of substrate proteins interacting productively with TRiC may differ from that of GroEL. We propose that TRiC mediates the folding of cytosolic proteins by a mechanism distinct from that of the chaperonins in specific aspects.     

Single-molecule observation of protein-protein interactions in the chaperonin system

We have analyzed the dynamics of the chaperonin (GroEL)-cochaperonin (GroES) interaction at the single-molecule level. In the presence of ATP and non-native protein, binding of GroES to the immobilized GroEL occurred at a rate that is consistent with bulk kinetics measurements. However, the release of GroES from GroEL occurred after a lag period ( approximately 3 s) that was not recognized in earlier bulk-phase studies. This observation suggests a new kinetic intermediate in the GroEL-GroES reaction pathway.     

BeF(x) stops the chaperonin cycle of GroEL-GroES and generates a complex with double folding chambers

Coupling with ATP hydrolysis and cooperating with GroES, the double ring chaperonin GroEL assists the folding of other proteins. Here we report novel GroEL-GroES complexes formed in fluoroberyllate (BeF(x)) that can mimic the phosphate part of the enzyme-bound nucleotides. In ATP, BeF(x) stops the functional turnover of GroEL by preventing GroES release and produces a symmetric 1:2 GroEL-GroES complex in which both GroEL rings contain ADP.BeF(x) and an encapsulated substrate protein. In ADP, the substrate protein-loaded GroEL cannot bind GroES. In ADP plus BeF(x), however, it can bind GroES to form a stable 1:1 GroEL-GroES complex in which one of GroEL rings contains ADP.BeF(x) and an encapsulated substrate protein. This 1:1 GroEL-GroES complex is converted into the symmetric 1:2 GroEL-GroES complex when GroES is supplied in ATP plus BeF(x). Thus, BeF(x) stabilizes two GroEL-GroES complexes; one with a single folding chamber and the other with double folding chambers. These results shed light on the intermediate ADP.P(i) nucleotide states in the functional cycle of GroEL.