From minichaperone to GroEL 3: properties of an active single-ring mutant of GroEL

The next step in our reductional analysis of GroEL was to study the activity of an isolated single seven-membered ring of the 14-mer. A known single-ring mutant, GroEL(SR1), contains four point mutations that prevent the formation of double-rings. That heptameric complex is functionally inactive because it is unable to release GroES. We found that the mutation E191G, which is responsible for the temperature sensitive (ts) Escherichia coli allele groEL44 and is located in the hinge region between the intermediate and apical domains of GroEL, appears to function by weakening the binding of GroES, without destabilizing the overall structure of GroEL44 mutant. We introduced, therefore, the mutation E191G into GroEL(SR1) in order to generate a single-ring mutant that may have weaker binding of GroES and hence be active. The new single-ring mutant, GroEL(SR44), was indeed effective in refolding both heat and dithiothreitol-denatured mitochondrial malate dehydrogenase with great efficiency. Further, unlike all smaller constructs of GroEL, the expression of GroEL(SR44) in E. coli that contained no endogenous GroEL restored biological viability, but not as efficiently as does wild-type GroEL. We envisage the notional evolution of the structure and properties of GroEL. The minichaperone core acts as a primitive chaperone by providing a binding surface for denatured states that prevents their self-aggregation. The assembly of seven minichaperones into a ring then enhances substrate binding by introducing avidity. The acquisition of binding sites for ATP then allows the modulation of substrate binding by introducing the allosteric mechanism that causes cycling between strong and weak binding sites. This is accompanied by the acquisition by the heptamer of the binding of GroES, which functions as a lid to the central cavity and competes for peptide binding sites. Finally, dimerization of the heptamer enhances its biological activity.     

Interaction of GroEL and GroEL/GroES complexes with a nonnative subtilisin variant: a small-angle neutron scattering study

Small-angle neutron scattering and contrast variation were used to study the solution structure of GroEL and GroEL/GroES chaperonins complexed with a nonnative variant of the polypeptide substrate, subtilisin (PJ9). The subtilisin was 86% deuterated (dPJ9) so that it contrasted sufficiently with the chaperonin, allowing the contrast variation technique to be used to separate the scattering from the two components bound in the complex. Both the native double-ring GroEL and a single-ring mutant were used with dPJ9 bound in a 1:1 stoichiometry per GroEL toroid. This allowed both the position and the shape of dPJ9 in the GroEL/dPJ9 complexes to be determined. A single-ring GroEL/GroES variant complexed with one dPJ9 molecule was used to study the structural changes of dPJ9 in GroEL/GroES/dPJ9 complexes formed with ADP and with ATP. It was found that both the shape and the position of the bound dPJ9 in the GroEL/GroES/dPJ9 complex with ADP were the same as those in the GroEL/dPJ9 complex. However, dPJ9 assumed a more symmetric shape when bound in the GroEL/GroES/dPJ9 complex with ATP. This important observation reflects the relative ability of ATP to promote refolding of protein substrates relative to that of ADP.     

Characterisation of a GroEL Single-Ring Mutant that Supports Growth of Escherichia coli and Has GroES-Dependent ATPase Activity

Binding and folding of substrate proteins by the molecular chaperone GroEL alternates between its two seven-membered rings in an ATP-regulated manner. The association of ATP and GroES to a polypeptide-bound ring of GroEL encapsulates the folding proteins in the central cavity of that ring (cis ring) and allows it to fold in a protected environment where the risk of aggregation is reduced. ATP hydrolysis in the cis ring changes the potentials within the system such that ATP binding to the opposite (trans) ring triggers the release of all ligands from the cis ring of GroEL through a complex network of allosteric communication between the rings. Inter-ring allosteric communication thus appears indispensable for the function of GroEL, and an engineered single-ring version (SR1) cannot substitute for GroEL in vivo. We describe here the isolation and characterisation of an active single-ring form of the GroEL protein (SR-A92T), which has an exceptionally low ATPase activity that is strongly stimulated by the addition of GroES. Dissection of the kinetic pathway of the ATP-induced structural changes in this active single ring can be explained by the fact that the mutation effectively blocks progression through the full allosteric pathway of the GroEL reaction cycle, thus trapping an early allosteric intermediate. Addition of GroES is able to overcome this block by binding this intermediate and pulling the allosteric pathway to completion via mass action, explaining how bacterial cells expressing this protein as their only chaperonin are viable.     

Co-translational binding of GroEL to nascent polypeptides is followed by post-translational encapsulation by GroES to mediate protein folding

The eubacterial chaperonins GroEL and GroES are essential chaperones and primarily assist protein folding in the cell. Although the molecular mechanism of the GroEL system has been examined previously, the mechanism by which GroEL and GroES assist folding of nascent polypeptides during translation is still poorly understood. We previously demonstrated a co-translational involvement of the Escherichia coli GroEL in folding of newly synthesized polypeptides using a reconstituted cell-free translation system (Ying, B. W., Taguchi, H., Kondo, M., and Ueda, T. (2005) J. Biol. Chem. 280, 12035-12040). Employing the same system here, we further characterized the mechanism by which GroEL assists folding of translated proteins via encapsulation into the GroEL-GroES cavity. The stable co-translational association between GroEL and the newly synthesized polypeptide is dependent on the length of the nascent chain. Furthermore, GroES is capable of interacting with the GroEL-nascent peptide-ribosome complex, and experiments using a single-ring variant of GroEL clearly indicate that GroES association occurs only at the trans-ring, not the cis-ring, of GroEL. GroEL holds the nascent chain on the ribosome in a polypeptide length-dependent manner and post-translationally encapsulates the polypeptide using the GroES cap to accomplish the chaperonin-mediated folding process.     

Chaperonins GroEL and GroES: views from atomic force microscopy.

The Escherichia coli chaperonins, GroEL and GroES, as well as their complexes in the presence of a nonhydrolyzable nucleotide AMP-PNP, have been imaged with the atomic force microscope (AFM). We demonstrate that both GroEL and GroES that have been adsorbed to a mica surface can be resolved directly by the AFM in aqueous solution at room temperature. However, with glutaraldehyde fixation of already adsorbed molecules, the resolution of both GroEL and GroES was further improved, as all seven subunits were well resolved without any image processing. We also found that chemical fixation was necessary for the contact mode AFM to image GroEL/ES complexes, and in the AFM images. GroEL with GroES bound can be clearly distinguished from those without. The GroEL/ES complex was about 5 nm higher than GroEL alone, indicating a 2 nm upward movement of the apical domains of GroEL. Using a slightly larger probe force, unfixed GroEL could be dissected: the upper heptamer was removed to expose the contact surface of the two heptamers. These results clearly demonstrate the usefulness of cross-linking agents for the determination of molecular structures with the AFM. They also pave the way for using the AFM to study the structural basis for the function of GroE system and other molecular chaperones.     

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.     

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.     

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.     

Chaperones GroEL/GroES Accelerate the Refolding of a Multidomain Protein through Modulating On-pathway Intermediates

Despite a vast amount information on the interplay of GroEL, GroES, and ATP in chaperone-assisted folding, the molecular details on the conformational dynamics of folding polypeptide during its GroEL/GroES-assisted folding cycle is quite limited. Practically no such studies have been reported to date on large proteins, which often have difficulty folding in vitro. The effect of the GroEL/GroES chaperonin system on the folding pathway of an 82-kDa slow folding protein, malate synthase G (MSG), was investigated. GroEL bound to the burst phase intermediate of MSG and accelerated the slowest kinetic phase associated with the formation of native topology in the spontaneous folding pathway. GroEL slowly induced conformational changes on the bound burst phase intermediate, which was then transformed into a more folding-compatible form. Subsequent addition of ATP or GroES/ATP to the GroEL-MSG complex led to the formation of the native state via a compact intermediate with the rate several times faster than that of spontaneous refolding. The presence of GroES doubled the ATP-dependent reactivation rate of bound MSG by preventing multiple cycles of its GroEL binding and release. Because GroES bound to the trans side of GroEL-MSG complex, it may be anticipated that confinement of the substrate underneath the co-chaperone is not required for accelerating the rate in the assisted folding pathway. The potential role of GroEL/GroES in assisted folding is most likely to modulate the conformation of MSG intermediates that can fold faster and thereby eliminate the possibility of partial aggregation caused by the slow folding intermediates during its spontaneous refolding pathway.     

Isolation and characterisation of mutants of GroEL that are fully functional as single rings

A key aspect of the reaction mechanism for the molecular chaperone GroEL is the transmission of an allosteric signal between the two rings of the GroEL complex. Thus, the single-ring mutant SR1 is unable to act as a chaperone as it cannot release bound substrate or GroES. We used a simple selection procedure to identify mutants of SR1 that restored chaperone activity in vivo. A large number of single amino acid changes, mapping at diverse positions throughout the protein, enabled SR1 to regain its ability to act as a chaperone while remaining as a single ring. In vivo assays were used to identify the proteins that had regained maximal activity. In some cases, no difference could be detected between strains expressing wild-type GroEL and those expressing the mutated proteins. Three of the most active proteins where the mutations were in distinct parts of the protein were purified to homogeneity and characterised in vitro. All were capable of acting efficiently as chaperones for two different GroES-dependent substrates. All three proteins bound nucleotide as effectively as did GroEL, but the binding of GroES in the presence of ATP or ADP was reduced significantly relative to the wild-type. These active single rings should provide a useful tool for studying the nature of the allosteric changes that occur in the GroEL reaction cycle.     

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.     

Functional characterization of an archaeal GroEL/GroES chaperonin system: significance of substrate encapsulation

In all three kingdoms of life chaperonins assist the folding of a range of newly synthesized proteins. As shown recently, Archaea of the genus Methanosarcina contain both group I (GroEL/GroES) and group II (thermosome) chaperonins in the cytosol. Here we report on a detailed functional analysis of the archaeal GroEL/GroES system of Methanosarcina mazei (Mm) in comparison to its bacterial counterpart from Escherichia coli (Ec). We find that the groESgroEL operon of M. mazei is unable to functionally replace groESgroEL in E. coli. However, the MmGroES protein can largely complement a mutant EcGroES protein in vivo. The ATPase rate of MmGroEL is very low and the dissociation of MmGroES from MmGroEL is 15 times slower than for the EcGroEL/GroES system. This slow ATPase cycle results in a prolonged enclosure time for model substrate proteins, such as rhodanese, in the MmGroEL:GroES folding cage before their release into the medium. Interestingly, optimal functionality of MmGroEL/GroES and its ability to encapsulate larger proteins, such as malate dehydrogenase, requires the presence of ammonium sulfate in vitro. In the absence of ammonium sulfate, malate dehydrogenase fails to be encapsulated by GroES and rather cycles on and off the GroEL trans ring in a non-productive reaction. These results indicate that the archaeal GroEL/GroES system has preserved the basic encapsulation mechanism of bacterial GroEL and suggest that it has adjusted the length of its reaction cycle to the slower growth rates of Archaea. Additionally, the release of only the folded protein from the GroEL/GroES cage may prevent adverse interactions of the GroEL substrates with the thermosome, which is not normally located within the same compartment.     

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.     

Chaperonin-encapsulation of proteins for NMR

A novel chaperonin-encapsulation system for NMR measurements has been designed. The single-ring variant SR398 with an ATPase deficient mutation of GroEL, also known as chaperonin, bound co-chaperonin GroES irreversibly, forming a stable cage to encapsulate a target protein. A small GroEL-binding tag made it possible to perform all steps of the encapsulation under near physiological conditions while retaining the native conformation of the target protein. About half of the SR398/GroES cages encapsulated target protein molecules. As binding only depends on the 12-residue tag sequence, this encapsulation method is applicable to a large number of proteins. Isolation of the target proteins in the molecular cage of chaperonin will allow the study of highly aggregation-prone proteins by solution NMR.     

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.     

Molecular chaperone GroEL/ES: Unfolding and refolding processes

Molecular chaperones are a special class of heat shock proteins (Hsp) that assist the folding and formation of the quaternary structure of other proteins both in vivo and in vitro. However, some chaperones are complex oligomeric proteins, and one of the intriguing questions is how the chaperones fold. The representatives of the Escherichia coli chaperone system GroEL (Hsp60) and GroES (Hsp10) have been studied most intensively. GroEL consists of 14 identical subunits combined into two interacting ring-like structures of seven subunits each, while the co-chaperone GroES interacting with GroEL consists of seven identical subunits combined into a dome-like oligomeric structure. In spite of their complex quaternary structure, GroEL and GroES fold well both in vivo and in vitro. However, the specific oligomerization of GroEL subunits is dependent on ligands and external conditions. This review analyzes the literature and our own data on the study of unfolding (denaturation) and refolding (renaturation) processes of these molecular chaperones and the effect of ligands and solvent composition. Such analysis seems to be useful for understanding the folding mechanism not only of the GroEL/GroES complex, but also of other oligomeric protein complexes.     

Requirement for GroEL/GroES-dependent protein folding under nonpermissive conditions of macromolecular crowding

Macromolecular crowding is a critical parameter affecting the efficiency of cellular protein folding. Here we show that the proteins dihydrofolate reductase, enolase, and green fluorescent protein, which can fold spontaneously in diluted buffer, lose this ability in a crowded environment. Instead, they accumulate as soluble, protease-sensitive non-native species. Their folding becomes dependent on the complete GroEL/GroES chaperonin system and is not affected by trap-GroEL, indicating that folding has to occur in the chaperonin cavity with release of nativelike proteins into the bulk solution. In addition, we demonstrate that efficient folding in the chaperonin cavity requires ATP hydrolysis, as formation of ternary GroEL/GroES complexes with substrate proteins in the presence of ADP results only in very inefficient reactivation. However, protein refolding reactions using ADP-fluoroaluminate complexes, or single-ring GroEL and GroES under conditions where only a single round of ATP hydrolysis occurs, yield large amounts of refolded enzymes. Thus, the mode of initial ternary complex formation appears to be critical for subsequent productive release of substrate into the cavity under certain crowding conditions, and is only efficient when triggered by ATP hydrolysis. Our data indicate that stringent conditions of crowding can impart a stronger dependence of folding proteins on the assistance by chaperonins.     

Characterisation of mutations in GroES that allow GroEL to function as a single ring

The chaperonin GroEL contains two seven-subunit rings, and allosteric signals between them are required to complete the GroEL reaction cycle. For this reason SR1, a mutant of GroEL that forms only single rings, cannot function as a chaperone. Mutations in SR1 that restore chaperone function weaken its interaction with the cochaperonin GroES. We predicted that GroES mutants with reduced affinity for GroEL would also restore function to SR1. To test this, we mutated residues in GroES in and near its contact site with GroEL. Nearly half of the mutants showed partial function with SR1. Two mutants were confirmed to have reduced affinity for GroEL. Intriguingly, some GroES mutants were able to function with active single ring mutants of GroEL.     

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.     

Kinetic analysis of ATP-dependent inter-ring communication in GroEL

Different concentrations of ATP were mixed rapidly with single-ring or double-ring forms of GroEL containing the Phe44-->Trp mutation and the time-resolved changes in fluorescence emission, upon excitation at 295 nm, were followed. Two kinetic phases that were previously found for double-ring GroEL are also observed in the case of the single-ring version: (i) a fast phase with a relatively large amplitude that is associated with the T-->R allosteric transition; (ii) and a slow phase with a smaller amplitude that is associated with ATP hydrolysis. In the case of weak intra-ring positive cooperativity, the rate constant corresponding to the T-->R allosteric switch of single-ring GroEL displays a bi-sigmoidal dependence on ATP concentration that may reflect parallel pathways of the allosteric transition. The slow phase is absent when double-ring or single-ring forms of GroEL are mixed with ADP or ATP without K(+), and it has a rate constant that is independent of ATP concentration. A third fast phase that is still unassigned is observed for both single-ring and double-ring GroEL when a large amount of data is collected. Finally, a fourth phase is observed in the case of double-ring GroEL that is found to be absent in the case of single-ring GroEL. This phase is here assigned to inter-ring communication by (i) determining its dependence on ATP concentration and (ii) based on its absence from single-ring GroEL and the Arg13-->Gly, Ala126-->Val GroEL mutant, which is defective in inter-ring negative cooperativity. The value of the rate constant corresponding to this phase is found to increase with increasing intra-ring positive cooperativity, with respect to ATP. This is the first report of the rate of ATP-mediated inter-ring communication in GroEL, in the presence of ATP alone, which is crucial for the cycling of this molecular machine between different functional states.