Identification of the TRiC/CCT substrate binding sites uncovers the function of subunit diversity in eukaryotic chaperonins

The ring-shaped hetero-oligomeric chaperonin TRiC/CCT uses ATP to fold a diverse subset of eukaryotic proteins. To define the basis of TRiC/CCT substrate recognition, we mapped the chaperonin interactions with the VHL tumor suppressor. VHL has two well-defined TRiC binding determinants. Each determinant contacts a specific subset of chaperonin subunits, indicating that TRiC paralogs exhibit distinct but overlapping specificities. The substrate binding site in these subunits localizes to a helical region in the apical domains that is structurally equivalent to that of bacterial chaperonins. Transferring the distal portion of helix 11 between TRiC subunits suffices to transfer specificity for a given substrate motif. We conclude that the architecture of the substrate binding domain is evolutionarily conserved among eukaryotic and bacterial chaperonins. The unique combination of specificity and plasticity in TRiC substrate binding may diversify the range of motifs recognized by this chaperonin and contribute to its unique ability to fold eukaryotic proteins.     

Molecular determinants of the ATP hydrolysis asymmetry of the CCT chaperonin complex

The eukaryotic cytosolic chaperonin CCT is a molecular machine involved in assisting the folding of proteins involved in important cellular processes. Like other chaperonins, CCT is formed by a double‐ring structure but, unlike all of them, each ring is composed of eight different, albeit homologous subunits. This complexity has probably to do with the specificity in substrate interaction and with the mechanism of protein folding that takes place during the chaperonin functional cycle, but its detailed molecular basis remains unknown. We have analyzed the known proteomes in search of residues that are differentially conserved in the eight subunits, as predictors of functional specificity (specificity‐determining positions; SDPs). We have found that most of these SDPs are located near the ATP binding site, and that they define four CCT clusters, corresponding to subunits CCT3, CCT6, CCT8 and CCT1/2/4/5/7. Our results point to a spatial organisation of the CCT subunits in two opposite areas of the ring and provide a molecular explanation for the previously described asymmetry in the hydrolysis of ATP. Proteins 2014; 82:703–707. © 2014 Wiley Periodicals, Inc.     

The group II chaperonin Mm-Cpn binds and refolds human γD crystallin

Chaperonins assist in the folding of nascent and misfolded proteins, though the mechanism of folding within the lumen of the chaperonin remains poorly understood. The archeal chaperonin from Methanococcus marapaludis, Mm-Cpn, shares the eightfold double barrel structure with other group II chaperonins, including the eukaryotic TRiC/CCT, required for actin and tubulin folding. However, Mm-Cpn is composed of a single species subunit, similar to group I chaperonin GroEL, rather than the eight subunit species needed for TRiC/CCT. Features of the β-sheet fold have been identified as sites of recognition by group II chaperonins. The crystallins, the major components of the vertebrate eye lens, are β-sheet proteins with two homologous Greek key domains. During refolding in vitro a partially folded intermediate is populated, and partitions between productive folding and off-pathway aggregation. We report here that in the presence of physiological concentrations of ATP, Mm-Cpn suppressed the aggregation of HγD-Crys by binding the partially folded intermediate. The complex was sufficiently stable to permit recovery by size exclusion chromatography. In the presence of ATP, Mm-Cpn promoted the refolding of the HγD-Crys intermediates to the native state. The ability of Mm-Cpn to bind and refold a human β-sheet protein suggests that Mm-Cpn may be useful as a simplified model for the substrate recognition mechanism of TRiC/CCT.     

The interaction network of the chaperonin CCT

The eukaryotic cytosolic chaperonin containing TCP-1 (CCT) has an important function in maintaining cellular homoeostasis by assisting the folding of many proteins, including the cytoskeletal components actin and tubulin. Yet the nature of the proteins and cellular pathways dependent on CCT function has not been established globally. Here, we use proteomic and genomic approaches to define CCT interaction networks involving 136 proteins/genes that include links to the nuclear pore complex, chromatin remodelling, and protein degradation. Our study also identifies a third eukaryotic cytoskeletal system connected with CCT: the septin ring complex, which is essential for cytokinesis. CCT interactions with septins are ATP dependent, and disrupting the function of the chaperonin in yeast leads to loss of CCT-septin interaction and aberrant septin ring assembly. Our results therefore provide a rich framework for understanding the function of CCT in several essential cellular processes, including epigenetics and cell division.     

Cryo-EM reveals an asymmetry in a novel single-ring viral chaperonin

Chaperonins are ubiquitously present protein complexes, which assist the proper folding of newly synthesized proteins and prevent aggregation of denatured proteins in an ATP-dependent manner. They are classified into group I (bacterial, mitochondrial, chloroplast chaperonins) and group II (archaeal and eukaryotic cytosolic variants). However, both of these groups do not include recently discovered viral chaperonins. Here, we solved the symmetry-free cryo-EM structures of a single-ring chaperonin encoded by the gene 246 of bacteriophage OBP Pseudomonas fluorescens, in the nucleotide-free, ATPγS-, and ADP-bound states, with resolutions of 4.3 Å, 5.0 Å, and 6 Å, respectively. The structure of OBP chaperonin reveals a unique subunit arrangement, with three pairs of subunits and one unpaired subunit. Each pair combines subunits in two possible conformations, differing in nucleotide-binding affinity. The binding of nucleotides results in the increase of subunits’ conformational variability. Due to its unique structural and functional features, OBP chaperonin can represent a new group.     

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.     

Positive selection and subfunctionalization of duplicated CCT chaperonin subunits

To reach a functional and energetically stable conformation, many proteins need molecular helpers called chaperonins. Among the group II chaperonins, CCT proteins provide crucial machinery for the stabilization and proper folding of several proteins in the cytosol of eukaryotic cells through interactions that are subunit-specific and geometry-dependent. CCT proteins are made up of eight different subunits, all with similar sequences, positioned in a precise arrangement. Each subunit has been proposed to have a specialized function during the binding and folding of the CCT protein substrate. Here, we demonstrate that functional divergence occurred after several CCT duplication events due to the fixation of amino acid substitutions by positive selection. Sites critical for ATP binding and substrate binding were found to have undergone positive selection and functional divergence predominantly in subunits that bind tubulin but not actin. Furthermore, we show clear functional divergence between CCT subunits that bind the C-terminal domains of actin and tubulin and those that bind the N-terminal domains. Phylogenetic analyses could not resolve the deep relationships between most subunits, except for the groups alpha/beta/eta and delta/epsilon, suggesting several almost simultaneous ancient duplication events. Together, the results support the idea that, in contrast to homo-oligomeric chaperonins such as GroEL, the high divergence level between CCT subunits is the result of positive selection after each duplication event to provide a specialized role for each CCT subunit in the different steps of protein folding.     

Partial occlusion of both cavities of the eukaryotic chaperonin with antibody has no effect upon the rates of beta-actin or alpha-tubulin folding

The eukaryotic chaperonin containing T-complex polypeptide 1 (CCT) is required in vivo for the production of native actin and tubulin. It is a 900-kDa oligomer formed from two back-to-back rings, each containing eight different subunits surrounding a central cavity in which interactions with substrates are thought to occur. Here, we show that a monoclonal antibody recognizing the C terminus of the CCTalpha subunit can bind inside, and partially occlude, both cavities of apo-CCT. Rabbit reticulocyte lysate was programmed to synthesize beta-actin and alpha-tubulin in the presence and absence of anti-CCTalpha antibody. The binding of the antibody inside the cavity and its occupancy of a large part of it does not prevent the folding of beta-actin and alpha-tubulin by CCT, despite the fact that all the CCT in the in vitro translation reactions was continuously bound by two antibody molecules. Furthermore, no differences in the protease susceptibility of actin bound to CCT in the presence and absence of the monoclonal antibody were detected, indicating that the antibody molecules do not perturb the conformation of actin folding intermediates substantially. These data indicate that complete sequestration of substrate by CCT may not be required for productive folding, suggesting that there are differences in its folding mechanism compared with the Group I chaperonins.     

Quantitative actin folding reactions using yeast CCT purified via an internal tag in the CCT3/gamma subunit

The eukaryotic cytosolic chaperonin CCT is an essential ATP-dependent protein folding machine whose action is required for folding the cytoskeletal proteins actin and tubulin, and a small number of other substrates, including members of the WD40-propellor repeat-containing protein family. An efficient purification protocol for CCT from Saccharomyces cerevisiae has been developed. It uses the calmodulin binding peptide as an affinity tag in an internal loop in the apical domain of the CCT3 subunit, which is predicted to be located on the outside of the double-ring assembly. This purified yeast CCT was used for a novel quantitative actin-folding assay with human beta-actin or yeast ACT1p protein folding intermediates, Ac(I), pre-synthesised in an Escherichia coli translation system. The formation of native actin follows approximately a first-order reaction with a rate constant of about 0.03 min(-1). Yeast CCT catalyses the folding of yeast ACT1p and human beta-actin with nearly identical rate constants and yields. The results from this controlled CCT-actin folding assay are consistent with a model where CCT and Ac(I) are in a binding pre-equilibrium with a rate-limiting binding step, followed by a faster ATP-driven processing to native actin. In this pure in vitro system, the human beta-actin mutants, D244S and G150P, show impaired folding behaviour in the manner predicted by our sequence-specific recognition model for CCT-actin interaction.     

Essential function of the built-in lid in the allosteric regulation of eukaryotic and archaeal chaperonins

Chaperonins are allosteric double-ring ATPases that mediate cellular protein folding. ATP binding and hydrolysis control opening and closing of the central chaperonin chamber, which transiently provides a protected environment for protein folding. During evolution, two strategies to close the chaperonin chamber have emerged. Archaeal and eukaryotic group II chaperonins contain a built-in lid, whereas bacterial chaperonins use a ring-shaped cofactor as a detachable lid. Here we show that the built-in lid is an allosteric regulator of group II chaperonins, which helps synchronize the subunits within one ring and, to our surprise, also influences inter-ring communication. The lid is dispensable for substrate binding and ATP hydrolysis, but is required for productive substrate folding. These regulatory functions of the lid may serve to allow the symmetrical chaperonins to function as 'two-stroke' motors and may also provide a timer for substrate encapsulation within the closed chamber.     

Versatile substrate protein recognition mechanism of the eukaryotic chaperonin CCT

Group II chaperonins, found in eukaryotic and archaeal organisms, recognize substrate proteins through diverse mechanisms that involve either hydrophobic‐ or electrostatic‐dominated interactions. This action is distinct from the universal substrate recognition mechanism of group I chaperonins, which bind a wide spectrum of non‐native proteins primarily through hydrophobic interactions. We use computational approaches to pinpoint the substrate protein binding sites of the γ‐subunit of the eukaryotic chaperonin CCT and to identify its interactions with the stringent substrate β‐tubulin. Protein–protein docking methods reveal intrinsic binding sites of CCT comprising a helical (HL) region, homologous to the GroEL‐binding site, and the helical protrusion (HP) region. We performed molecular dynamics simulations of the solvated CCTγ apical domain, β‐tubulin peptide‐CCTγ complexes, and isolated β‐tubulin peptides. We find that tubulin binds to CCTγ through an extensive interface that spans both the HL region and the HP region. HL interactions involve both hydrophobic and electrostatic contacts, while binding to the HP region is stabilized almost exclusively by a salt bridge network. On the basis of additional simulations of a β‐tubulin‐CCTγ complex that involves a reduced interface, centered onto the HP region, we conclude that this salt bridge network is the minimal stabilizing interaction required. Strong conservation of the charged amino acids that participate in the salt bridge network, Arg306 and Glu271, indicates a general mechanism across the nonidentical CCT subunits and group II chaperonins. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Analysis of the interaction between the eukaryotic chaperonin CCT and its substrates actin and tubulin

Two mechanisms have thus far been characterized for the assistance by chaperonins of the folding of other proteins. The first and best described is that of the prokaryotic chaperonin GroEL, which interacts with a large spectrum of proteins. GroEL uses a nonspecific mechanism by which any conformation of practically any unfolded polypeptide interacts with it through exposed, hydrophobic residues. ATP binding liberates the substrate in the GroEL cavity where it is given a chance to fold. A second mechanism has been described for the eukaryotic chaperonin CCT, which interacts mainly with the cytoskeletal proteins actin and tubulin. Cryoelectron microscopy and biochemical studies have revealed that both of these proteins interact with CCT in quasi-native, defined conformations. Here we have performed a detailed study of the docking of the actin and tubulin molecules extracted from their corresponding CCT:substrate complexes obtained from cryoelectron microscopy and image processing to localize certain regions in actin and tubulin that are involved in the interaction with CCT. These regions of actin and tubulin, which are not present in their prokaryotic counterparts FtsA and FtsZ, are involved in the polymerization of the two cytoskeletal proteins. These findings suggest coevolution of CCT with actin and tubulin in order to counteract the folding problems associated with the generation in these two cytoskeletal protein families of new domains involved in their polymerization.     

Eukaryotic chaperonin CCT stabilizes actin and tubulin folding intermediates in open quasi-native conformations

Three-dimensional reconstruction from cryoelectron micrographs of the eukaryotic cytosolic chaperonin CCT complexed to tubulin shows that CCT interacts with tubulin (both the alpha and beta isoforms) using five specific CCT subunits. The CCT-tubulin interaction has a different geometry to the CCT-actin interaction, and a mixture of shared and unique CCT subunits is used in binding the two substrates. Docking of the atomic structures of both actin and tubulin to their CCT-bound conformation suggests a common mode of chaperonin-substrate interaction. CCT stabilizes quasi-native structures in both proteins that are open through their domain-connecting hinge regions, suggesting a novel mechanism and function of CCT in assisted protein folding.     

MgATP binding to the nucleotide-binding domains of the eukaryotic cytoplasmic chaperonin induces conformational changes in the putative substrate-binding domains.

The eukaryotic cytosolic chaperonins are large heterooligomeric complexes with a cylindrical shape, resembling that of the homooligomeric bacterial counterpart, GroEL. In analogy to GroEL, changes in shape of the cytosolic chaperonin have been detected in the presence of MgATP using electron microscopy but, in contrast to the nucleotide-induced conformational changes in GroEL, no details are available about the specific nature of these changes. The present study identifies the structural regions of the cytosolic chaperonin that undergo conformational changes when MgATP binds to the nucleotide binding domains. It is shown that limited proteolysis with trypsin in the absence of MgATP cleaves each of the eight subunits approximately in half, generating two fragments of approximately 30 kDa. Using mass spectrometry (MS) and N-terminal sequence analysis, the cleavage is found to occur in a narrow span of the amino acid sequence, corresponding to the peptide binding regions of GroEL and to the helical protrusion, recently identified in the structure of the substrate binding domain of the archeal group II chaperonin. This proteolytic cleavage is prevented by MgATP but not by ATP in the absence of magnesium, ATP analogs (MgATPyS and MgAMP-PNP) or MgADP. These results suggest that, in analogy to GroEL, binding of MgATP to the nucleotide binding domains of the cytosolic chaperonin induces long range conformational changes in the polypeptide binding domains. It is postulated that despite their different subunit composition and substrate specificity, group I and group II chaperonins may share similar, functionally-important, conformational changes. Additional conformational changes are likely to involve a flexible helix-loop-helix motif, which is characteristic for all group II chaperonins.     

Conformational rearrangements of tail-less complex polypeptide 1 (TCP-1) ring complex (TRiC)-bound actin

The mechanism of chaperonins is still under intense investigation. Earlier studies by others and us on the bacterial chaperonin GroEL points to an active role of chaperonins in unfolding the target protein during initial binding. Here, a natural eukaryotic chaperonin system [tail-less complex polypeptide 1 (TCP-1) ring complex (TRiC) and its target protein actin] was investigated to determine if the active participation of the chaperonin in the folding process is evolutionary-conserved. Using fluorescence resonance energy transfer (FRET) measurements on four distinct doubly fluorescein-labeled variants of actin, we have obtained a fairly detailed map of the structural rearrangements that occur during the TRiC-actin interaction. The results clearly show that TRiC has an active role in rearranging the bound actin molecule. The target is stretched as a consequence of binding to TRiC and further rearranged in a second step as a consequence of ATP binding; i.e., the mechanism of chaperonins is conserved during evolution.     

ATP-induced structural change of the thermosome is temperature-dependent

Protein folding by chaperonins is powered by ATP binding and hydrolysis. ATPase activity drives the folding machine through a series of conformational rearrangements, extensively described for the group I chaperonin GroEL from Escherichia coli but still poorly understood for the group II chaperonins. The latter--archaeal thermosome and eukaryotic TRiC/CCT--function independently of a GroES-like cochaperonin and are proposed to rely on protrusions of their own apical domains for opening and closure in an ATP-controlled fashion. Here we use small-angle neutron scattering to analyze structural changes of the recombinant alpha-only and the native alphabeta-thermosome from Thermoplasma acidophilum upon their ATPase cycling in solution. We show that specific high-salt conditions, but not the presence of MgATP alone, induce formation of higher order thermosome aggregates. The mechanism of the open-closed transition of the thermosome is strongly temperature-dependent. ATP binding to the chaperonin appears to be a two-step process: at lower temperatures an open state of the ATP-thermosome is predominant, whereas heating to physiological temperatures induces its switching to a closed state. Our data reveal an analogy between the ATPase cycles of the two groups of chaperonins and enable us to put forward a model of thermosome action.     

The 'sequential allosteric ring' mechanism in the eukaryotic chaperonin-assisted folding of actin and tubulin

Folding to completion of actin and tubulin in the eukaryotic cytosol requires their interaction with cytosolic chaperonin CCT [chaperonin containing tailless complex polypeptide 1 (TCP-1)]. Three-dimensional reconstructions of nucleotide-free CCT complexed to either actin or tubulin show that CCT stabilizes both cytoskeletal proteins in open and quasi-folded conformations mediated through interactions that are both subunit specific and geometry dependent. Here we find that upon ATP binding, mimicked by the non-hydrolysable analog AMP-PNP (5'-adenylyl-imido-diphosphate), to both CCT-alpha-actin and CCT- beta-tubulin complexes, the chaperonin component undergoes concerted movements of the apical domains, resulting in the cavity being closed off by the helical protrusions of the eight apical domains. However, in contrast to the GroE system, generation of this closed state does not induce the release of the substrate into the chaperonin cavity, and both cytoskeletal proteins remain bound to the chaperonin apical domains. Docking of the AMP-PNP-CCT-bound conformations of alpha-actin and beta-tubulin to their respective native atomic structures suggests that both proteins have progressed towards their native states.     

Symmetry-free cryo-EM structures of the chaperonin TRiC along its ATPase-driven conformational cycle

The eukaryotic group II chaperonin TRiC/CCT is a 16-subunit complex with eight distinct but similar subunits arranged in two stacked rings. Substrate folding inside the central chamber is triggered by ATP hydrolysis. We present five cryo-EM structures of TRiC in apo and nucleotide-induced states without imposing symmetry during the 3D reconstruction. These structures reveal the intra- and inter-ring subunit interaction pattern changes during the ATPase cycle. In the apo state, the subunit arrangement in each ring is highly asymmetric, whereas all nucleotide-containing states tend to be more symmetrical. We identify and structurally characterize an one-ring closed intermediate induced by ATP hydrolysis wherein the closed TRiC ring exhibits an observable chamber expansion. This likely represents the physiological substrate folding state. Our structural results suggest mechanisms for inter-ring-negative cooperativity, intra-ring-positive cooperativity, and protein-folding chamber closure of TRiC. Intriguingly, these mechanisms are different from other group I and II chaperonins despite their similar architecture.     

Chaperonins: two rings for folding

Chaperonins are ubiquitous chaperones found in Eubacteria, eukaryotic organelles (group I), Archaea and the eukaryotic cytosol (group II). They all share a common structure and a basic functional mechanism. Although a large amount of information has been gathered for the simpler group I, much less is known about group II chaperonins. Recent crystallographic and electron microscopy structures have provided new insights into the mechanism of these chaperonins and revealed important differences between group I and II chaperonins, mainly in the molecular rearrangements that take place during the functional cycle. These differences are evident for the most complex chaperonin, the eukaryotic cytosolic CCT, which highlights the uniqueness of this important molecular machine.