Cryo-EM structure of a group II chaperonin in the prehydrolysis ATP-bound state leading to lid closure

Chaperonins are large ATP-driven molecular machines that mediate cellular protein folding. Group II chaperonins use their "built-in lid" to close their central folding chamber. Here we report the structure of an archaeal group II chaperonin in its prehydrolysis ATP-bound state at subnanometer resolution using single particle cryo-electron microscopy (cryo-EM). Structural comparison of Mm-cpn in ATP-free, ATP-bound, and ATP-hydrolysis states reveals that ATP binding alone causes the chaperonin to close slightly with a ～45° counterclockwise rotation of the apical domain. The subsequent ATP hydrolysis drives each subunit to rock toward the folding chamber and to close the lid completely. These motions are attributable to the local interactions of specific active site residues with the nucleotide, the tight couplings between the apical and intermediate domains within the subunit, and the aligned interactions between two subunits across the rings. This mechanism of structural changes in response to ATP is entirely different from those found in group I chaperonins.     

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.     

Crystal structure of wild-type chaperonin GroEL

The 2.9A resolution crystal structure of apo wild-type GroEL was determined for the first time and represents the reference structure, facilitating the study of structural and functional differences observed in GroEL variants. Until now the crystal structure of the mutant Arg13Gly, Ala126Val GroEL was used for this purpose. We show that, due to the mutations as well as to the presence of a crystallographic symmetry, the ring-ring interface was inaccurately described. Analysis of the present structure allowed the definition of structural elements at this interface, essential for understanding the inter-ring allosteric signal transmission. We also show unambiguously that there is no ATP-induced 102 degrees rotation of the apical domain helix I around its helical axis, as previously assumed in the crystal structure of the (GroEL-KMgATP)(14) complex, and analyze the apical domain movements. These results enabled us to compare our structure with other GroEL crystal structures already published, allowing us to suggest a new route through which the allosteric signal for negative cooperativity propagates within the molecule. The proposed mechanism, supported by known mutagenesis data, underlines the importance of the switching of salt bridges.     

Crystal structure of the temperature-sensitive and allosteric-defective chaperonin GroELE461K

The chaperonin GroEL adopts a double-ring structure with various modes of allosteric communication. The simultaneous positive intra-ring and negative inter-ring co-operativities alternate the functionality of the folding cavities in both protein rings. Negative inter-ring co-operativity is maintained through different inter-ring interactions, including a salt bridge involving Glu 461. Replacement of this residue by Lys modifies the temperature sensitivity of the substrate-folding activity of this protein, most likely as a result of the loss of inter-ring co-operativity. The crystal structure of the mutant chaperonin GroELE461K has been determined at 3.3A and compared with other structures: the wild-type GroEL, an allosteric defective GroEL double mutant and the GroEL-GroES-(ADP)7 complex. The inter-ring region of the mutant exhibits the following characteristics: (i) no salt-bridge stabilizes the inter-ring interface; (ii) the mutated residue plays a central role in defining the relative ring rotation (of about 22 degrees) around the 7-fold axis; (iii) an increase in the inter-ring distance and solvent accessibility of the inter-ring interface; and (iv) a 2-fold reduction in the stabilization energy of the inter-ring interface, due to the modification of inter-ring interactions. These characteristics explain how the thermal sensitivity of the protein's fundamental properties permits GroEL to distinguish physiological (37 degrees C) from stress (42 degrees C) temperatures.     

Functional characterization of the recombinant group II chaperonin alpha from Thermoplasma acidophilum

The functional characteristics of group II chaperonins, especially those from archaea, have not been elucidated extensively. Here, we performed a detailed functional characterization of recombinant chaperonin alpha subunits (16-mer) (Ta-cpn alpha) from the thermophilic archaea Thermoplasma acidophilum as a model protein of archaeal group II chaperonins. Recombinant Ta-cpn alpha formed an oligomeric ring structure similar to that of native protein, and displayed an ATP hydrolysis activity (optimal temperature: 60 degrees C) in the presence of either magnesium, manganese or cobalt ions. Ta-cpn alpha was able to bind refolding intermediates of Thermus MDH and GFP in the absence of ATP, and to promote the refolding of Thermus MDH at 50 degrees C in the presence of Mg2+-, Mn2+-, or Co2+-ATP. Ta-cpn alpha also prevented thermal aggregation of rhodanese and luciferase at 50 degrees C. Interestingly, Ta-cpn alpha in the presence of Mn2+ ion showed an increased hydrophobicity, which correlated with an increased efficiency in substrate protein binding. Our finding that Ta-cpn alpha chaperonin system displays folding assistance ability with ATP-dependent substrate release may provide a detailed look at the potential functional capabilities of archaeal chaperonins.     

The composition,structure and stability of a group II chaperonin are temperature regulated in a hyperthermophilic archaeon

The hyperthermoacidophilic archaeon Sulfolobus shibatae contains group II chaperonins, known as rosettasomes, which are two nine-membered rings composed of three different 60 kDa subunits (TF55 alpha, beta and gamma). We sequenced the gene for the gamma subunit and studied the temperature-dependent changes in alpha, beta and gamma expression, their association into rosettasomes and their phylogenetic relationships. Alpha and beta gene expression was increased by heat shock (30 min, 86 degrees C) and decreased by cold shock (30 min, 60 degrees C). Gamma expression was undetectable at heat shock temperatures and low at normal temperatures (75-79 degrees C), but induced by cold shock. Polyacrylamide gel electrophoresis indicated that in vitro alpha and beta subunits form homo-oligomeric rosettasomes, and mixtures of alpha, beta and gamma form hetero-oligomeric rosettasomes. Transmission electron microscopy revealed that beta homo-oligomeric rosettasomes and all hetero-oligomeric rosettasomes associate into filaments. In vivo rosettasomes were hetero-oligomeric with an average subunit ratio of 1alpha:1beta:0.1gamma in cultures grown at 75 degrees C, a ratio of 1alpha:3beta:1gamma in cultures grown at 60 degrees C and a ratio of 2alpha:3beta:0gamma after 86 degrees C heat shock. Using differential scanning calorimetry, we determined denaturation temperatures (Tm) for alpha, beta and gamma subunits of 95.7 degrees C, 96.7 degrees C and 80.5 degrees C, respectively, and observed that rosettasomes containing gamma were relatively less stable than those with alpha and/or beta only. We propose that, in vivo, the rosettasome structure is determined by the relative abundance of subunits and not by a fixed geometry. Furthermore, phylogenetic analyses indicate that archaeal chaperonin subunits underwent multiple duplication events within species (paralogy). The independent evolution of these paralogues raises the possibility that chaperonins have functionally diversified between species.     

Single-molecule fluorescence polarization study of conformational change in archaeal group II chaperonin

Group II chaperonins found in archaea and in eukaryotic cytosol mediate protein folding without a GroES-like cofactor. The function of the cofactor is substituted by the helical protrusion at the tip of the apical domain, which forms a built-in lid on the central cavity. Although many studies on the change in lid conformation coupled to the binding and hydrolysis of nucleotides have been conducted, the molecular mechanism of lid closure remains poorly understood. Here, we performed a single-molecule polarization modulation to probe the rotation of the helical protrusion of a chaperonin from a hyperthermophilic archaeum, Thermococcus sp. strain KS-1. We detected approximately 35° rotation of the helical protrusion immediately after photorelease of ATP. The result suggests that the conformational change from the open lid to the closed lid state is responsible for the approximately 35° rotation of the helical protrusion.     

On the oligomeric state of chloroplast chaperonin 10 and chaperonin 20

Type I chaperonins are fundamental protein folding machineries that function in eubacteria, mitochondria and chloroplasts. Eubacteria and mitochondria contain chaperonin systems comprised of homo-oligomeric chaperonin 60 tetradecamers and co-chaperonin 10 heptamers. In contrast, the chloroplast chaperonins are heterooligomeric tetradecamers that are composed of two subunit types, alpha and beta. Additionally, chloroplasts contain two structurally distinct co-chaperonins. One, ch-cpn10, is probably similar to the mitochondrial and bacterial co-chaperonins, and is composed of 10 kDa subunits. The other, termed ch-cpn20 is composed of two cpn10-like domains that are held together by a short linker. While the oligomeric structure of ch-cpn10 remains to be elucidated, it was previously suggested that ch-cpn20 forms tetramers in solution, and that this is the functional oligomer. In the present study, we investigated the properties of purified ch-cpn10 and ch-cpn20. Using bifunctional cross-linking reagents, gel filtration chromatography and analytical ultracentrifugation, we show that ch-cpn10 is a heptamer in solution. In contrast, ch-cpn20 forms multiple oligomers that are in dynamic equilibrium with each other and cover a broad spectrum of molecular weights in a concentration-dependent manner. However, upon association with GroEL, only one type of co-chaperonin-GroEL complex is formed.     

Molecular cloning and characterization of a group II chaperonin delta-subunit from soybean

Molecular characterization of plant group II chaperonin (CCT, c-cpn, or TriC) still remains elusive. By PCR-based cloning techniques using soybeans, we have made a successful attempt to clone a delta-subunit homologue of CCT (CCTdelta). This subunit is responsible for the binding of an in vivo substrate, alpha-actin, by assisting the correct folding of the cytoskeletal protein in mouse, and the occurrence of the subunit homologue in plant CCT was unclear. As the cloning strategy, a putative amino acid segment, NH(2)-Gly-Gly-Gly-Ala-Pro-Glu-COOH, which is tightly conserved in all known animal and yeast CCTdelta subunits, was chosen for designing a degenerate primer of the PCR-cloning. The resultant 1881-bp cDNA was found to have an open-reading frame of 533 amino acids with a calculated molecular mass of 57,677 Da and to share about 58-65% identity overall at the amino acid level with the corresponding subunits known to date. Using antibodies raised against Escherichia coli-produced soybean insoluble CCTdelta as a monitoring tool, we purified soybean CCT from the extract of its immature seeds. STEM images demonstrated that the molecular shape of soybean CCT is a double eight-membered ring, which resembles the known group II chaperonins. The CCT also reactivated a denatured firefly luciferase with a significant, but limited level of the native enzymic activity in an in vitro system. Northern blot analysis showed that soybean CCTdelta gene, which is intronless and composed of a small family, was only expressed at a very early stage of seed development of soybean.     

Molecular characterization of the group II chaperonin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3

The group II chaperonin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 (PhCPN) and its functional cooperation with the cognate prefoldin were investigated. PhCPN existed as a homo-oligomer in a double-ring structure, which protected the citrate synthase of a porcine heart from thermal aggregation at 45°C, and did the same on the isopropylmalate dehydrogenase (IPMDH) of a thermophilic bacterium, Thermus thermophilus HB8, at 90°C. PhCPN also enhanced the refolding of green fluorescent protein (GFP), which had been unfolded by low pH, in an ATP-dependent manner. Unexpectedly, functional cooperation between PhCPN and Pyrococcus prefoldin (PhPFD) in the refolding of GFP was not observed. Instead, cooperation between PhCPN and PhPFD was observed in the refolding of IPMDH unfolded with guanidine hydrochloride. Although PhCPN alone was not effective in the refolding of IPMDH, the refolding efficiency was enhanced by the cooperation of PhCPN with PhPFD.     

Oligomerization of an archaeal group II chaperonin is mediated by N-terminal salt bridges

Group II chaperonins (Cpns) are essential mediators of cellular protein folding in eukaryotes and archaea. They consist of two back-to-back rings forming symmetrical cavities in which non-native substrates undergo appropriate folding, but the primary structural basis for the double ring formation remains unclear. To address this, we carried out systematic mutagenesis on the Cpn from the hyperthermophilic archaeon Pyrococcus furiosus, which is assembled from identical subunits. In our study, ²¹GRDAQRMNIL³⁰ was found to be a critical domain for double ring formation. Deletion of this section stepwise beyond residue 20 resulted in failure to assemble double-ring oligomers and the progressive loss of chaperone function. A key domain spanning the residues 21–50 that is essential for the formation of tetramers that appear to be the intermediates for double ring assembly. Mutation of either Arg22 to Ala22 or Glu37 to Ala37 resulted in similar defects in double-ring assembly and functional deficits. A mutant with Arg22 and Glu37 switched assembled double rings efficiently and exhibited chaperone functions similar to the wild-type. Therefore, Arg22 and Glu37 could form inter-ring salt bridges critical for double ring formation. In addition, Asn28 and Ile29 were found to contribute significantly to ring formation. Sequence alignment revealed that these four residues are highly conserved among group II Cpns. This is the first report of a comprehensive N-terminal mutational analysis for elucidating the oligomerization of group II Cpns.     

Dynamics of group II chaperonin and prefoldin probed by 13C NMR spectroscopy

Group II chaperonin (CPN) cooperates with prefoldin (PFD), which forms a jellyfish-shaped heterohexameric complex with a molecular mass of 87 kDa. PFD captures an unfolded protein with the tentacles and transfers it to the cavity of CPN. Although X-ray crystal structures of CPN and PFD have been reported, no structural information has been so far available for the terminal regions of the PFD tentacles nor for the C-terminal segments of CPNs, which were regarded to be functionally significant in the previous studies. Here we report 13C NMR analyses on archaeal PFD, CPN, and their complex, focusing on those structurally uncharacterized regions. The PFD and CPN complexes selectively labeled with 13C at methionyl carbonyl carbons were separately and jointly subjected to NMR measurements. 13C NMR spectral data demonstrated that the N-terminal segment of the alpha and beta subunits of PFD as well as the C-terminal segments of the CPN hexadecamer retain significant degrees of freedom in internal motion even in the complex with a molecular mass of 1.1 MDa.     

Crystal structure of human mitochondrial PheRS complexed with tRNA(Phe) in the active "open" state

Monomeric human mitochondrial phenylalanyl-tRNA synthetase (PheRS), or hmPheRS, is the smallest known enzyme exhibiting aminoacylation activity. HmPheRS consists of only two structural domains and differs markedly from heterodimeric eukaryotic cytosolic and bacterial analogs both in the domain organization and in the mode of tRNA binding. Here, we describe the first crystal structure of mitochondrial aminoacyl-tRNA synthetase (aaRS) complexed with tRNA at a resolution of 3.0 Å. Unlike bacterial PheRSs, the hmPheRS recognizes C74, the G1–C72 base pair, and the “discriminator” base A73, proposed to contribute to tRNA^Phe identity in the yeast mitochondrial enzyme. An interaction of the tRNA acceptor stem with the signature motif 2 residues of hmPheRS is of critical importance for the stabilization of the CCA-extended conformation and its correct placement in the synthetic site of the enzyme. The crystal structure of hmPheRS–tRNA^Phe provides direct evidence that the formation of the complex with tRNA requires a significant rearrangement of the anticodon-binding domain from the “closed” to the productive “open” state. Global repositioning of the domain is tRNA modulated and governed by long-range electrostatic interactions.     

Crystal structure of the native chaperonin complex from Thermus thermophilus revealed unexpected asymmetry at the cis-cavity

The chaperonins GroEL and GroES are essential mediators of protein folding. GroEL binds nonnative protein, ATP, and GroES, generating a ternary complex in which protein folding occurs within the cavity capped by GroES (cis-cavity). We determined the crystal structure of the native GroEL-GroES-ADP homolog from Thermus thermophilus, with substrate proteins in the cis-cavity, at 2.8 A resolution. Twenty-four in vivo substrate proteins within the cis-cavity were identified from the crystals. The structure around the cis-cavity, which encapsulates substrate proteins, shows significant differences from that observed for the substrate-free Escherichia coli GroEL-GroES complex. The apical domain around the cis-cavity of the Thermus GroEL-GroES complex exhibits a large deviation from the 7-fold symmetry. As a result, the GroEL-GroES interface differs considerably from the previously reported E. coli GroEL-GroES complex, including a previously unknown contact between GroEL and GroES.     

Crystal structure of Thermus caldophilus phosphoglycerate kinase in the open conformation

Phosphoglycerate kinase (PGK) is a key glycolytic enzyme that catalyzes the reversible transfer of a phosphate from 1,3-bisphosphoglycerate to ADP to form 3-phosphoglycerate and ATP in the presence of magnesium. During catalysis, a conformational change occurs that brings the N- and C-domains of PGK closer together. Here we present the 1.8A crystal structure of unliganded PGK from Thermus caldophilus (Tca). Comparison of the structure of TcaPGK (open conformation) with that of Thermotoga maritima (Tma) PGK (closed conformation) revealed that the conformational change reflects a change in the interaction between the domains. We identified Arg148 as a key residue involved in open-to-closed transition. The open conformation of TcaPGK is stabilized by an interdomain salt bridge between Arg148 and Glu375. The binding of 3-PG (or maybe 1,3-BPG) disrupts this salt bridge and, in ternary complex, the formation of new salt bridge between Arg60 and Asp197 stabilizes the closed conformation.     

Analysis of the interaction mode between hyperthermophilic archaeal group II chaperonin and prefoldin using a platform of chaperonin oligomers of various subunit arrangements

Prefoldin is a co-chaperone that captures an unfolded protein substrate and transfers it to the group II chaperonin for completion of protein folding. Group II chaperonin of a hyperthermophilic archaeon, Thermococcus strain KS-1, interacts and cooperates with archaeal prefoldins. Although the interaction sites within chaperonin and prefoldin have been analyzed, the binding mode between jellyfish-like hexameric prefoldin and the double octameric ring group II chaperonin remains unclear. As prefoldin binds the chaperonin β subunit more strongly than the α subunit, we analyzed the binding mode between prefoldin and chaperonin in the context of Thermococcus group II chaperonin complexes of various subunit compositions and arrangements. The oligomers exhibited various affinities for prefoldins according to the number and order of subunits. Binding affinity increased with the number of Cpnβ subunits. Interestingly, chaperonin complexes containing two β subunits adjacently exhibited stronger affinities than other chaperonin complexes containing the same number of β subunits. The result suggests that all four β tentacles of prefoldin interact with the helical protrusions of CPN in the PFD–CPN complex as the previously proposed model that two adjacent PFD β subunits seem to interact with two CPN adjacent subunits.     

Archaeal group II chaperonin mediates protein folding in the cis-cavity without a detachable GroES-like co-chaperonin.

Group II chaperonins of archaea and eukaryotes are distinct from group I chaperonins of bacteria. Whereas group I chaperonins require the co-chaperonin Cpn-10 or GroES for protein folding, no co-chaperonin has been known for group II. The protein folding mechanism of group II chaperonins is not yet clear. To understand this mechanism, we examined protein refolding by the recombinant alpha or beta-subunit chaperonin homo-oligomer (alpha16mer and beta16mer) from a hyperthermoplilic archaeum, Thermococcus strain KS-1, using a model substrate, green fluorescent protein (GFP). The alpha16mer and beta16mer captured the non-native GFP and promoted its refolding without any co-chaperonin in an ATP dependent manner. A non-hydrolyzable ATP analog, AMP-PNP, induced the GFP refolding mediated by beta16mer but not by the alpha16mer. A mutant alpha-subunit chaperonin homo-oligomer (trap-alpha) could capture the non-native protein but lacked the ability to refold it. Although trap-alpha suppressed ATP-dependent refolding of GFP mediated by alpha16mer or beta16mer, it did not affect the AMP-PNP-dependent refolding. This indicated that the GFP refolding mediated by beta16mer with AMP-PNP was not accessible to the trap-alpha. Gel filtration chromatography and a protease protection experiment revealed that this refolded GFP, in the presence of AMP-PNP, was associated with beta16mer. After the completion of GFP refolding mediated by beta16mer with AMP-PNP, addition of ATP induced an additional refolding of GFP. Furthermore, the beta16mer preincubated with AMP-PNP showed the ability to capture the non-native GFP. These suggest that AMP-PNP induced one of two chaperonin rings (cis-ring) to close and induced protein refolding in this ring, and that the other ring (trans-ring) could capture the unfolded GFP which was refolded by adding ATP. The present data indicate that, in the group II chaperonin of Thermococcus strain KS-1, the protein folding proceeds in its cis-ring in an ATP-dependent fashion without any co-chaperonin.     

Crystal structure of the 65-kilodalton heat shock protein, chaperonin 60.2, of Mycobacterium tuberculosis

Chaperonin 60s are a ubiquitous class of proteins that promote folding and assembly of other cellular polypeptides in an ATP-dependent manner. The oligomeric state of chaperonin 60s has been shown to be crucial to their role as molecular chaperones. Chaperonin 60s are also known to be important stimulators of the immune system. Mycobacterium tuberculosis possesses a duplicate set of chaperonin 60s, both of which have been shown to be potent cytokine stimulators. The M. tuberculosis chaperonin 60s are present in the extracellular milieu at concentrations that are extremely low for the formation of an oligomer. Here we present the crystal structure of one of the chaperonin 60s of M. tuberculosis, also called Hsp65 or chaperonin 60.2, at 3.2-A resolution. We were able to crystallize the protein in its dimeric state. The unusual dimerization of the protein leads to exposure of certain hydrophobic patches on the surface of the protein, and we hypothesize that this might have relevance in binding to immunogenic peptides, as it does in the eukaryotic homologs.     

Kinetics and binding sites for interaction of the prefoldin with a group II chaperonin: contiguous non-native substrate and chaperonin binding sites in the archaeal prefoldin

Prefoldin is a jellyfish-shaped hexameric co-chaperone of the group II chaperonins. It captures a protein folding intermediate and transfers it to a group II chaperonin for completion of folding. The manner in which prefoldin interacts with its substrates and cooperates with the chaperonin is poorly understood. In this study, we have examined the interaction between a prefoldin and a chaperonin from hyperthermophilic archaea by immunoprecipitation, single molecule observation, and surface plasmon resonance. We demonstrate that Pyrococcus prefoldin interacts most tightly with its cognate chaperonin, and vice versa, suggesting species specificity in the interaction. Using truncation mutants, we uncovered by kinetic analyses that this interaction is multivalent in nature, consistent with multiple binding sites between the two chaperones. We present evidence that both N- and C-terminal regions of the prefoldin beta sub-unit are important for molecular chaperone activity and for the interaction with a chaperonin. Our data are consistent with substrate and chaperonin binding sites on prefoldin that are different but in close proximity, which suggests a possible handover mechanism of prefoldin substrates to the chaperonin.     

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.