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.     

Stopped-flow fluorescence analysis of the conformational changes in the GroEL apical domain: relationships between movements in the apical domain and the quaternary structure of GroEL

GroEL undergoes numerous conformational alterations in the course of facilitating the folding of various proteins, and the specific movements of the GroEL apical domain are of particular importance in the molecular mechanism. In order to monitor in detail the numerous movements of the GroEL apical domain, we have constructed a mutant chaperonin (GroEL R231W) with wild type-like function and a fluorescent probe introduced into the apical domain. By monitoring the tryptophan fluorescence changes of GroEL R231W upon ATP addition in the presence and absence of the co-chaperonin GroES, we detected a total of four distinct kinetic phases that corresponded to conformational changes of the apical domain and GroES binding. By introducing this mutation into a single ring variant of GroEL (GroEL SR-1), we determined the extent of inter-ring cooperation that was involved in apical domain movements. Surprisingly, we found that the apical domain movements of GroEL were affected only slightly by the change in quaternary structure. Our experiments provide a number of novel insights regarding the dynamic movements of this protein.     

Mechanisms of protein folding

The strong correlation between protein folding rates and the contact order suggests that folding rates are largely determined by the topology of the native structure. However, for a given topology, there may be several possible low free energy paths to the native state and the path that is chosen (the lowest free energy path) may depend on differences in interaction energies and local free energies of ordering in different parts of the structure. For larger proteins whose folding is assisted by chaperones, such as the Escherichia coli chaperonin GroEL, advances have been made in understanding both the aspects of an unfolded protein that GroEL recognizes and the mode of binding to the chaperonin. The possibility that GroEL can remove non-native proteins from kinetic traps by unfolding them either during polypeptide binding to the chaperonin or during the subsequent ATP-dependent formation of folding-active complexes with the co-chaperonin GroES has also been explored.     

The importance of a mobile loop in regulating chaperonin/ co-chaperonin interaction: humans versus Escherichia coli

Chaperonins are universally conserved proteins that nonspecifically facilitate the folding of a wide spectrum of proteins. While bacterial GroEL is functionally promiscuous with various co-chaperonin partners, its human homologue, Hsp60 functions specifically with its co-chaperonin partner, Hsp10, and not with other co-chaperonins, such as the bacterial GroES or bacteriophage T4-encoded Gp31. Co-chaperonin interaction with chaperonin is mediated by the co-chaperonin mobile loop that folds into a beta-hairpin conformation upon binding to the chaperonin. A delicate balance of flexibility and conformational preferences of the mobile loop determines co-chaperonin affinity for chaperonin. Here, we show that the ability of Hsp10, but not GroES, to interact specifically with Hsp60 lies within the mobile loop sequence. Using mutational analysis, we show that three substitutions in the GroES mobile loop are necessary and sufficient to acquire Hsp10-like specificity. Two of these substitutions are predicted to preorganize the beta-hairpin turn and one to increase the hydrophobicity of the GroEL-binding site. Together, they result in a GroES that binds chaperonins with higher affinity. It seems likely that the single ring mitochondrial Hsp60 exhibits intrinsically lower affinity for the co-chaperonin that can be compensated for by a higher affinity mobile loop.     

Role of the helical protrusion in the conformational change and molecular chaperone activity of the archaeal group II chaperonin

To elucidate the exact role of the helical protrusion of a group II chaperonin in its molecular chaperone function, three deletion mutants of the chaperonin from a hyperthermophilic archaeum (Thermococcus sp. strain KS-1) lacking one-third, two-thirds, and the whole of the helical protrusion were constructed. The helical protrusion is thought to be substituted for the co-chaperonin GroES of a group I chaperonin and to be important for binding to unfolded proteins. Protease sensitivity assays and small angle x-ray scattering experiments were performed to demonstrate the conformation change of the wild type protein and the deletion mutants by adenine nucleotides. Whereas the binding of ATP to the wild type protein induced a structural transition corresponding to the closure of the built-in lid, it did not cause significant structural changes in deletion mutants. Although the mutants effectively protected proteins from thermal aggregation, ATP-dependent protein folding ability was remarkably diminished. We conclude that the helical protrusion is not necessarily important for binding to unfolded proteins, but its ATP-dependent conformational change mediates folding of captured unfolded proteins.     

Fast-scanning atomic force microscopy reveals the ATP/ADP-dependent conformational changes of GroEL

In order to fold non-native proteins, chaperonin GroEL undergoes numerous conformational changes and GroES binding in the ATP-dependent reaction cycle. We constructed the real-time three-dimensional-observation system at high resolution using a newly developed fast-scanning atomic force microscope. Using this system, we visualized the GroES binding to and dissociation from individual GroEL with a lifetime of 6 s (k=0.17 s(-1)). We also caught ATP/ADP-induced open-closed conformational changes of individual GroEL in the absence of qGroES and substrate proteins. Namely, the ATP/ADP-bound GroEL can change its conformation 'from closed to open' without additional ATP hydrolysis. Furthermore, the lifetime of open conformation in the presence of ADP ( approximately 1.0 s) was apparently lower than those of ATP and ATP-analogs (2-3 s), meaning that ADP-bound open-form is structurally less stable than ATP-bound open-form. These results indicate that GroEL has at least two distinct open-conformations in the presence of nucleotide; ATP-bound prehydrolysis open-form and ADP-bound open-form, and the ATP hydrolysis in open-form destabilizes its open-conformation and induces the 'from open to closed' conformational change of GroEL.     

Probing the Dynamic Process of Encapsulation in Escherichia coli GroEL

Kinetic analyses of GroE-assisted folding provide a dynamic sequence of molecular events that underlie chaperonin function. We used stopped-flow analysis of various fluorescent GroEL mutants to obtain details regarding the sequence of events that transpire immediately after ATP binding to GroEL and GroEL with prebound unfolded proteins. Characterization of GroEL CP86, a circularly permuted GroEL with the polypeptide ends relocated to the vicinity of the ATP binding site, showed that GroES binding and protection of unfolded protein from solution is achieved surprisingly early in the functional cycle, and in spite of greatly reduced apical domain movement. Analysis of fluorescent GroEL SR-1 and GroEL D398A variants suggested that among other factors, the presence of two GroEL rings and a specific conformational rearrangement of Helix M in GroEL contribute significantly to the rapid release of unfolded protein from the GroEL apical domain.     

Mapping pathways of allosteric communication in GroEL by analysis of correlated mutations

An interesting example of an allosteric protein is the chaperonin GroEL. It undergoes adenosine 5'-triphosphate-induced conformational changes that are reflected in binding of adenosine 5'-triphosphate with positive cooperativity within rings and negative cooperativity between rings. Herein, correlated mutations in chaperonins are analyzed to unravel routes of allosteric communication in GroEL and in its complex with its co-chaperonin GroES. It is shown that analysis of correlated mutations in the chaperonin family can provide information about pathways of allosteric communication within GroEL and between GroEL and GroES. The results are discussed in the context of available structural, genetic, and biochemical data concerning short- and long-range interactions in the GroE system.     

Combined thermodynamic and kinetic analysis of GroEL interacting with CXCR4 transmembrane peptides

GroEL along with ATP and its co-chaperonin GroES has been demonstrated to significantly enhance the folding of newly translated G-protein-coupled receptors (GPCRs). This work extends the previous studies to explore the guest capture and release processes in GroEL-assisted folding of GPCRs, by the reduced approach of employing CXCR4 transmembrane peptides as model substrates. Each of the CXCR4-derived peptides exhibited high affinity for GroEL with a binding stoichiometry near seven. It is found that the peptides interact with the paired α helices in the apical domain of the chaperonin which are similar with the binding sites of SBP (strongly binding peptide: SWMTTPWGFLHP). Complementary binding study with a single-ring version of GroEL indicates that each of the two chaperonin rings is competent for accommodating all the seven CXCR4 peptides bound to GroEL under saturation condition. Meanwhile, the binding kinetics of CXCR4 peptides with GroEL was also examined; ATP alone, or in combination of GroES evidently promoted the release of the peptide substrates from the chaperonin. The results obtained would be beneficial to understand the thermodynamic and kinetic nature of GroEL-GPCRs interaction which is the central molecular event in the assisted folding process.     

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.     

Chaperonin-mediated protein folding: GroES binds to one end of the GroEL cylinder, which accommodates the protein substrate within its central cavity.

The mechanism of GroEL (chaperonin)-mediated protein folding is only partially understood. We have analysed structural and functional properties of the interaction between GroEL and the co-chaperonin GroES. The stoichiometry of the GroEL 14mer and the GroES 7mer in the functional holo-chaperonin is 1:1. GroES protects half of the GroEL subunits from proteolytic truncation of the approximately 50 C-terminal residues. Removal of this region results in an inhibition of the GroEL ATPase, mimicking the effect of GroES on full-length GroEL. Image analysis of electron micrographs revealed that GroES binding triggers conspicuous conformational changes both in the GroES adjacent end and at the opposite end of the GroEL cylinder. This apparently prohibits the association of a second GroES oligomer. Addition of denatured polypeptide leads to the appearance of irregularly shaped, stain-excluding masses within the GroEL double-ring, which are larger with bound alcohol oxidase (75 kDa) than with rhodanese (35 kDa). We conclude that the functional complex of GroEL and GroES is characterized by asymmetrical binding of GroES to one end of the GroEL cylinder and suggest that binding of the substrate protein occurs within the central cavity of GroEL.     

Identification of Elements That Dictate the Specificity of Mitochondrial Hsp60 for Its Co-Chaperonin

Type I chaperonins (cpn60/Hsp60) are essential proteins that mediate the folding of proteins in bacteria, chloroplast and mitochondria. Despite the high sequence homology among chaperonins, the mitochondrial chaperonin system has developed unique properties that distinguish it from the widely-studied bacterial system (GroEL and GroES). The most relevant difference to this study is that mitochondrial chaperonins are able to refold denatured proteins only with the assistance of the mitochondrial co-chaperonin. This is in contrast to the bacterial chaperonin, which is able to function with the help of co-chaperonin from any source. The goal of our work was to determine structural elements that govern the specificity between chaperonin and co-chaperonin pairs using mitochondrial Hsp60 as model system. We used a mutagenesis approach to obtain human mitochondrial Hsp60 mutants that are able to function with the bacterial co-chaperonin, GroES. We isolated two mutants, a single mutant (E321K) and a double mutant (R264K/E358K) that, together with GroES, were able to rescue an E. coli strain, in which the endogenous chaperonin system was silenced. Although the mutations are located in the apical domain of the chaperonin, where the interaction with co-chaperonin takes place, none of the residues are located in positions that are directly responsible for co-chaperonin binding. Moreover, while both mutants were able to function with GroES, they showed distinct functional and structural properties. Our results indicate that the phenotype of the E321K mutant is caused mainly by a profound increase in the binding affinity to all co-chaperonins, while the phenotype of R264K/E358K is caused by a slight increase in affinity toward co-chaperonins that is accompanied by an alteration in the allosteric signal transmitted upon nucleotide binding. The latter changes lead to a great increase in affinity for GroES, with only a minor increase in affinity toward the mammalian mitochondrial co-chaperonin.     

Closing the folding chamber of the eukaryotic chaperonin requires the transition state of ATP hydrolysis

Chaperonins use ATPase cycling to promote conformational changes leading to protein folding. The prokaryotic chaperonin GroEL requires a cofactor, GroES, which serves as a "lid" enclosing substrates in the central cavity and confers an asymmetry on GroEL required for cooperative transitions driving the reaction. The eukaryotic chaperonin TRiC/CCT does not have such a cofactor but appears to have a "built-in" lid. Whether this seemingly symmetric chaperonin also operates through an asymmetric cycle is unclear. We show that unlike GroEL, TRiC does not close its lid upon nucleotide binding, but instead responds to the trigonal-bipyramidal transition state of ATP hydrolysis. Further, nucleotide analogs inducing this transition state confer an asymmetric conformation on TRiC. Similar to GroEL, lid closure in TRiC confines the substrates in the cavity and is essential for folding. Understanding the distinct mechanisms governing eukaryotic and bacterial chaperonin function may reveal how TRiC has evolved to fold specific eukaryotic proteins.     

Ionic interactions at both inter-ring contact sites of GroEL are involved in transmission of the allosteric signal: a time-resolved infrared difference study

The biological activity of the double-ring chaperonin GroEL is regulated by complex allosteric interactions, which include positive intra-ring and negative inter-ring cooperativity. To further characterize inter-ring communication, the nucleotide-induced absorbance changes in the vibrational spectrum of the chaperonin GroEL, of two single-point mutants suppressing one inter-ring ionic contact (E461K and E434K) and of a single-ring version of this protein, were investigated by time-resolved infrared difference spectroscopy. Interaction of the nucleotide with the proteins was triggered by its photochemical release from a biologically inactive caged precursor [P3-1-(2-nitro) phenylethyl nucleotide]. The results indicate that (1) ATP binding to the protein induces a conformational change that affects concomitantly both intra-ring and inter-ring communication, and (2) the experimental absorbance changes are sensitive to the double-ring structure of the protein. The characterization of the single-point, inter-ring mutants demonstrates that ionic interactions at both contact sites are involved in the transmission of the allosteric signal. However, both mutations have different effects on the inter-ring interface. While that of E461K still retains ionic contacts sensitive to ATP binding, E434K shows spectroscopic features similar to those of the single-ring version of the protein, therefore suggesting that electrostatic interactions at these contact sites contribute differently to the stability of the inter-ring interface.     

Identification of a major inter-ring coupling step in the GroEL reaction cycle

It has been shown previously that the double-ring structure of GroEL can be converted to a single-ring species by site-directed amino acid replacements at the ring interface and that the resultant molecule retains many of the crucial chaperonin properties; it is structurally stable, hydrolytically active, and can bind both the co-chaperonin, GroES, and unfolded substrate proteins. By comparing the behavior of the double- and single-ring structures in response to nucleotide binding and hydrolysis, we elucidate steps in the ATP-driven reaction cycle at which there is conformational coupling between the rings. Remarkably, the parting of the rings has little effect either on the thermodynamic properties of ATP binding or on the ATP-induced conformational changes prior to hydrolysis. However, there is a marked effect on the rate-limiting process in the steady-state cycle; a step that is coincident with bond cleavage in ATP. The effect of the ring-ring interaction is to increase its activation enthalpy from 42.0 to 94.2 kJ/mol. These results show that the major conformational coupling step, where structural rearrangements in one ring are propagated to the other, is the slowest process the ATPase cycle of GroEL.     

Inter-ring communication allows the GroEL chaperonin complex to distinguish between different substrates

The productive folding of substrate proteins by the GroEL complex of Escherichia coli requires the activity of both the chaperonin rings. These heptameric rings were shown to regulate the chaperonins' affinity for substrates and co-chaperonin via inter-ring communications; however, the molecular details of the interactions are not well understood. We have investigated the effect of substrate binding on inter-ring communications of the chaperonin complex, both the double-ring GroEL as well as the single-ring SR1 chaperonin in complex with four different substrates by using mass spectrometry. This approach shows that whereas SR1 is unable to distinguish between Rubisco, gp23, gp5, and MDH, GroEL shows clear differences upon binding these substrates. The most distinctive binding behavior is observed for Rubisco, which only occupies one GroEL ring. Both bacteriophage capsid proteins (gp23 and gp5) as well as MDH are able to bind to the two GroEL rings simultaneously. Our data suggest that inter-ring communication allows the chaperonin complex to differentiate between substrates. Using collision induced dissociation in the gas phase, differences between the chaperonin(substrate) complexes are observed only when both rings are present. The data indicate that the size of the substrate is an important factor that determines the degree of stabilization of the chaperonin complex.     

Transient conformational remodeling of folding proteins by GroES—individually and in concert with GroEL

The commonly accepted dogma of the bacterial GroE chaperonin system entails protein folding mediated by cycles of several ATP-dependent sequential steps where GroEL interacts with the folding client protein. In contrast, we herein report GroES-mediated dynamic remodeling (expansion and compression) of two different protein substrates during folding: the endogenous substrate MreB and carbonic anhydrase (HCAII), a well-characterized protein folding model. GroES was also found to influence GroEL binding induced unfolding and compression of the client protein underlining the synergistic activity of both chaperonins, even in the absence of ATP. This previously unidentified activity by GroES should have important implications for understanding the chaperonin mechanism and cellular stress response. Our findings necessitate a revision of the GroEL/ES mechanism.     

Dissociation of the GroEL-GroES asymmetric complex is accelerated by increased cooperativity in ATP binding to the GroEL ring distal to GroES

A kinetic analysis of the ATP-dependent dissociation of wild-type GroEL and mutants from immobilized GroES was carried out using surface plasmon resonance. Excellent fits of the data were obtained using a double-exponential equation with a linear drift. Both the fast and slow observed dissociation rate constants are found to have a sigmoidal dependence on the concentration of ATP. The values of the Hill coefficients corresponding to the fast and slow observed rate constants of dissociation of wild-type GroEL and the Arg197-->Ala mutant are in good agreement with the respective values of the Hill coefficients previously determined for these proteins from plots of initial rates of ATP hydrolysis as a function of ATP concentration, in the presence of GroES. Our results are consistent with a kinetic mechanism for dissociation of the GroEL-GroES complex according to which GroES release takes place after an ATP-induced conformational change in the trans ring that is preceded by ATP hydrolysis and a subsequent conformational change in the cis ring. It is shown that the rate of complex dissociation increases with increasing positive cooperativity in ATP binding by the GroEL ring distal to GroES in the GroEL-GroES complex.     

ATP binding is critical for the conformational change from an open to closed state in archaeal group II chaperonin

Group II chaperonins, found in archaea and in eukaryotic cytosol, do not have a co-chaperonin corresponding to GroES. Instead, it is suggested that the helical protrusion extending from the apical domain acts as a built-in lid for the central cavity and that the opening and closing of the lid is regulated by ATP binding and hydrolysis. However, details of this conformational change remain unclear. To investigate the conformational change associated with the ATP-driven cycle, we conducted protease sensitivity analyses and tryptophan fluorescence spectroscopy of alpha-chaperonin from a hyperthermophilic archaeum, Thermococcus strain KS-1. In the nucleotide-free or ADP-bound state, the chaperonin, especially in the helical protrusion region, was highly sensitive to proteases. Addition of ATP and ammonium sulfate induced the transition to the relatively protease-resistant form. The fluorescence intensity of the tryptophan residue introduced at the tip of the helical protrusion was enhanced by the presence of ATP or ammonium sulfate. We conclude that ATP binding induces the conformational change from the lid-open to lid-closed form in archaeal group II chaperonin.     

Group II chaperonins: new TRiC(k)s and turns of a protein folding machine.

In the past decade, the eubacterial group I chaperonin GroEL became the paradigm of a protein folding machine. More recently, electron microscopy and X-ray crystallography offered insights into the structure of the thermosome, the archetype of the group II chaperonins which also comprise the chaperonin from the eukaryotic cytosol TRiC. Some structural differences from GroEL were revealed, namely the existence of a built-in lid provided by the helical protrusions of the apical domains instead of a GroES-like co-chaperonin. These structural studies provide a framework for understanding the differences in the mode of action between the group II and the group I chaperonins. In vitro analyses of the folding of non-native substrates coupled to ATP binding and hydrolysis are progressing towards establishing a functional cycle for group II chaperonins. A protein complex called GimC/prefoldin has recently been found to cooperate with TRiC in vivo, and its characterization is under way.