Human CCT4 and CCT5 Chaperonin Subunits Expressed in Escherichia coli Form Biologically Active Homo-oligomers

Chaperonins are a family of chaperones that encapsulate their substrates and assist their folding in an ATP-dependent manner. The ubiquitous eukaryotic chaperonin, TCP-1 ring complex (TRiC), is a hetero-oligomeric complex composed of two rings, each formed from eight different CCT (chaperonin containing TCP-1) subunits. Each CCT subunit may have distinct substrate recognition and ATP hydrolysis properties. We have expressed each human CCT subunit individually in Escherichia coli to investigate whether they form chaperonin-like double ring complexes. CCT4 and CCT5, but not the other six CCT subunits, formed high molecular weight complexes within the E. coli cells that sedimented about 20S in sucrose gradients. When CCT4 and CCT5 were purified, they were both organized as two back-to-back rings of eight subunits each, as seen by negative stain and cryo-electron microscopy. This morphology is consistent with that of the hetero-oligomeric double-ring TRiC purified from bovine testes and HeLa cells. Both CCT4 and CCT5 homo-oligomers hydrolyzed ATP at a rate similar to human TRiC and were active as assayed by luciferase refolding and human γD-crystallin aggregation suppression and refolding. Thus, both CCT4 and CCT5 homo-oligomers have the property of forming 8-fold double rings absent the other subunits, and these complexes carry out chaperonin reactions without other partner subunits.     

The inter-ring arrangement of the cytosolic chaperonin CCT

The eukaryotic cytosolic chaperonin CCT (chaperonin containing TCP-1) is the most complex of all chaperonins-an oligomeric structure built from two identical rings, each composed of single copies of eight different subunits. The arrangement of the eight subunits within each ring has been characterised for some time, but the phasing between the two rings remains unknown. Here, three-dimensional reconstructions generated by cryoelectron microscopy of complexes between CCT and either of two different monoclonal antibodies that react specifically with the CCTepsilon and CCTdelta subunits have been used to determine the phasing between the two chaperonin rings. The inter-ring arrangement is such that up/down inter-ring communication always involves two different CCT subunits in all eight positions, and the group of subunits concerned with the initiation and completion of the folding cycle cluster together both in the intra- and inter-ring arrangement. This supports a sequential mechanism of conformational changes between the two interacting rings.     

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.     

Modeling of possible subunit arrangements in the eukaryotic chaperonin TRiC

The eukaryotic cytosolic chaperonin TRiC (TCP-1 Ring Complex), also known as CCT (Cytosolic Chaperonin containing TCP-1), is a hetero-oligomeric complex consisting of two back-to-back rings of eight different subunits each. The general architecture of the complex has been determined, but the arrangement of the subunits within the complex remains an open question. By assuming that the subunits have a defined arrangement within each ring, we constructed a simple model of TRiC that analyzes the possible arrangements of individual subunits in the complex. By applying the model to existing data, we find that there are only four subunit arrangements consistent with previous observations. Our analysis provides a framework for the interpretation and design of experiments to elucidate the quaternary structure of TRiC/CCT. This in turn will aid in the understanding of substrate binding and allosteric properties of this chaperonin.     

Quantitative analysis of the impact of a human pathogenic mutation on the CCT5 chaperonin subunit using a proxy archaeal ortholog

The human chaperonin complex is a ~ 1 MDa nanomachine composed of two octameric rings formed from eight similar but non-identical subunits called CCT. Here, we are elucidating the mechanism of a heritable CCT5 subunit mutation that causes profound neuropathy in humans. In previous work, we introduced an equivalent mutation in an archaeal chaperonin that assembles into two octameric rings like in humans but in which all subunits are identical. We reported that the hexadecamer formed by the mutant subunit is unstable with impaired chaperoning functions. This study quantifies the loss of structural stability in the hexadecamer due to the pathogenic mutation, using differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). The disassembly of the wild type complex, which is tightly coupled with subunit denaturation, was decoupled by the mutation without affecting the stability of individual subunits. Our results verify the effectiveness of the homo-hexadecameric archaeal chaperonin as a proxy to assess the impact of subtle defects in heterologous systems with mutations in a single subunit.     

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.     

ATP-induced allostery in the eukaryotic chaperonin CCT is abolished by the mutation G345D in CCT4 that renders yeast temperature-sensitive for growth

Saccharomyces cerevisiae yeast cells containing the chaperonin CCT (chaperonin-containing t-complex polypeptide 1 (TCP-1)) with the G345D mutation in subunit CCT4 (anc2-1) are temperature-sensitive for growth and display defects in organization of actin structure, budding and cell shape. In this first structure-function analysis of CCT, we show that this mutation abolishes both intra- and inter-ring cooperativity in ATP binding by CCT. The finding that a single mutation in only one subunit in each CCT ring has such drastic effects highlights the importance of allostery for its in vivo function. These results, together with other kinetic data for wild-type CCT reported in this study, provide support for the sequential model for ATP-dependent allosteric transitions in CCT.     

Equivalent Mutations in the Eight Subunits of the Chaperonin CCT Produce Dramatically Different Cellular and Gene Expression Phenotypes

The eukaryotic cytoplasmic chaperonin-containing TCP-1 (CCT) is a complex formed by two back-to-back stacked hetero-octameric rings that assists the folding of actins, tubulins, and other proteins in an ATP-dependent manner. Here, we tested the significance of the hetero-oligomeric nature of CCT in its function by introducing, in each of the eight subunits in turn, an identical mutation at a position that is conserved in all the subunits and is involved in ATP hydrolysis, in order to establish the extent of ‘individuality’ of the various subunits. Our results show that these identical mutations lead to dramatically different phenotypes. For example, Saccharomyces cerevisiae yeast cells with the mutation in subunit CCT2 display heat sensitivity and cold sensitivity for growth, have an excess of actin patches, and are the only strain here generated that is pseudo-diploid. By contrast, cells with the mutation in subunit CCT7 are the only ones to accumulate juxtanuclear protein aggregates that may reflect an impaired stress response in this strain. System-level analysis of the strains using RNA microarrays reveals connections between CCT and several cellular networks, including ribosome biogenesis and TOR2, that help to explain the phenotypic variability observed.     

Testing the neutral fixation of hetero-oligomerism in the archaeal chaperonin CCT

The evolutionary transition from homo-oligomerism to hetero-oligomerism in multimeric proteins and its contribution to function innovation and organism complexity remain to be investigated. Here, we undertake the challenge of contributing to this theoretical ground by investigating the hetero-oligomerism in the molecular chaperonin cytosolic chaperonin containing tailless complex polypeptide 1 (CCT) from archaea. CCT is amenable to this study because, in contrast to eukaryotic CCTs where sub-functionalization after gene duplication has been taken to completion, archaeal CCTs present no evidence for subunit functional specialization. Our analyses yield additional information to previous reports on archaeal CCT paralogy by identifying new duplication events. Analyses of selective constraints show that amino acid sites from 1 subunit have fixed slightly deleterious mutations at inter-subunit interfaces after gene duplication. These mutations have been followed by compensatory mutations in nearby regions of the same subunit and in the interface contact regions of its paralogous subunit. The strong selective constraints in these regions after speciation support the evolutionary entrapment of CCTs as hetero-oligomers. In addition, our results unveil different evolutionary dynamics depending on the degree of CCT hetero-oligomerism. Archaeal CCT protein complexes comprising 3 distinct classes of subunits present 2 evolutionary processes. First, slightly deleterious and compensatory mutations were fixed neutrally at inter-subunit regions. Second, sub-functionalization may have occurred at substrate-binding and adenosine triphosphate-binding regions after the 2nd gene duplication event took place. CCTs with 2 distinct types of subunits did not present evidence of sub-functionalization. Our results provide the 1st in silico evidence for the neutral fixation of hetero-oligomerism in archaeal CCTs and provide information on the evolution of hetero-oligomerism toward sub-functionalization in archaeal CCTs.     

Reconstitution of the human chaperonin CCT by co-expression of the eight distinct subunits in mammalian cells

The eukaryotic cytosolic chaperonin CCT (chaperonin-containing TCP-1) assists folding of newly synthesized polypeptides. The fully functional CCT is built from two identical rings, each composed of single copies of eight distinct subunits. To study the structure and function of the CCT complex and the role of each subunit, a rapid and efficient method for preparing a recombinant CCT complex is needed. In this work, we established an efficient expression and purification method to obtain human recombinant CCT. BHK-21 cells were infected with a vaccinia virus expressing T7 RNA polymerase and transfected with eight plasmids, each encoding any one of the eight CCT subunits in the T7 RNA polymerase promoter/terminator unit. The CCT1 subunit was engineered to carry a hexa-histidine tag or FLAG tag in the internal loop region. Three days later, cells were harvested for purification of the CCT complex through tag-dependent affinity chromatography and gel filtration. The purified recombinant CCT complexes were indistinguishable from the endogenous CCT purified from HeLa cells in terms of morphology and function. In conclusion, the co-expression system established in this study should be a simple and powerful tool for reconstitution of a large multi-subunit complex.     

Human TRiC complex purified from HeLa cells contains all eight CCT subunits and is active in vitro

Archaeal and eukaryotic cytosols contain group II chaperonins, which have a double-barrel structure and fold proteins inside a cavity in an ATP-dependent manner. The most complex of the chaperonins, the eukaryotic TCP-1 ring complex (TRiC), has eight different subunits, chaperone containing TCP-1 (CCT1–8), that are arranged so that there is one of each subunit per ring. Aspects of the structure and function of the bovine and yeast TRiC have been characterized, but studies of human TRiC have been limited. We have isolated and purified endogenous human TRiC from HeLa suspension cells. This purified human TRiC contained all eight CCT subunits organized into double-barrel rings, consistent with what has been found for bovine and yeast TRiC. The purified human TRiC is active as demonstrated by the luciferase refolding assay. As a more stringent test, the ability of human TRiC to suppress the aggregation of human γD-crystallin was examined. In addition to suppressing off-pathway aggregation, TRiC was able to assist the refolding of the crystallin molecules, an activity not found with the lens chaperone, α-crystallin. Additionally, we show that human TRiC from HeLa cell lysate is associated with the heat shock protein 70 and heat shock protein 90 chaperones. Purification of human endogenous TRiC from HeLa cells will enable further characterization of this key chaperonin, required for the reproduction of all human cells.     

Subunits of the chaperonin CCT interact with F-actin and influence cell shape and cytoskeletal assembly

The integrity of the cytoskeleton is closely linked to the oligomeric chaperonin containing TCP-1 (CCT) via the folding requirements of actin and tubulin, but the role of CCT in cytoskeletal organization remains unclear. We address this issue by analyzing the effects of targeting CCT subunits via siRNA and assessing their location/assembly state in cultured mammalian cells. Reducing levels of individual CCT subunits implicates CCT? in influencing cell shape and reduced levels of this subunit limit the cells' ability to recover from microfilament depolymerization. Conversely, cells displayed enhanced microtubule regrowth when CCT subunit levels were altered by siRNA. Some CCT subunits co-localize with F-actin, whilst all are predominantly monomeric in extracts enriched for the cytoskeleton. This provides compelling evidence that some CCT subunits as monomers can influence cytoskeletal organization/polymerization. Therefore the activity of CCT may well extend beyond the folding of newly synthesized polypeptides, representing a novel function for CCT subunits distinct from their role in the CCT oligomer.     

3D reconstruction of the ATP-bound form of CCT reveals the asymmetric folding conformation of a type II chaperonin.

The type II chaperonin CCT (chaperonin containing Tcp-1) of eukaryotic cytosol is a heteromeric 16-mer particle composed of eight different subunits. Three-dimensional reconstructions of apo-CCT and ATP-CCT have been obtained at 28 A resolution by cryo-electron microscopy. Binding of ATP generates an asymmetric particle; one ring has a slightly different conformation from the apo-CCT ring, while the other has undergone substantial movements in the apical domains. Upon ATP binding the apical domains rotate and point towards the cylinder axis, so that the helical protrusions present at their tips could act as a lid closing the ring cavity.     

Chaperonin containing T-complex polypeptide (CCT) subunit expression in oral mucosal wounds and fibroblasts

Mucosal wound healing in adults has been reported to feature diminished scar formation compared to healing skin wounds. We sought to determine if the expression pattern of chaperonin containing T-complex polypeptide (CCT) subunits in mucosal wounds and fibroblasts is different from that observed in skin wounds and fibroblasts. We found that CCT-beta is the only subunit message to be reduced in wounded mucosa versus unwounded control, and this reduction was confirmed at the protein level. In contrast, mRNA levels of CCT-zeta, -delta, -eta, and -epsilon were significantly increased in mucosal wounds. The increase in CCT-eta was also confirmed at the protein level. Expression levels of CCT-alpha, -beta, -delta; -epsilon, and -theta mRNAs were significantly increased in adult mucosal fibroblasts in culture compared to skin-derived fibroblasts. Western blot analyses confirmed a modest increase in CCT-beta in adult mucosal fibroblasts relative to skin fibroblasts, but CCT-eta protein was unaffected. These differences may contribute to the reported difference in healing outcomes between these two tissue types.     

The molecular architecture of the eukaryotic chaperonin TRiC/CCT

TRiC/CCT is a highly conserved and essential chaperonin that uses ATP cycling to facilitate folding of approximately 10% of the eukaryotic proteome. This 1 MDa hetero-oligomeric complex consists of two stacked rings of eight paralogous subunits each. Previously proposed TRiC models differ substantially in their subunit arrangements and ring register. Here, we integrate chemical crosslinking, mass spectrometry, and combinatorial modeling to reveal the definitive subunit arrangement of TRiC. In vivo disulfide mapping provided additional validation for the crosslinking-derived arrangement as the definitive TRiC topology. This subunit arrangement allowed the refinement of a structural model using existing X-ray diffraction data. The structure described here explains all available crosslink experiments, provides a rationale for previously unexplained structural features, and reveals a surprising asymmetry of charges within the chaperonin folding chamber.     

Origin and evolution of eukaryotic chaperonins: phylogenetic evidence for ancient duplications in CCT genes

Chaperonins are oligomeric protein-folding complexes which are divided into two distantly related structural classes. Group I chaperonins (called GroEL/cpn60/hsp60) are found in bacteria and eukaryotic organelles, while group II chaperonins are present in archaea and the cytoplasm of eukaryotes (called CCT/TriC). While archaea possess one to three chaperonin subunit-encoding genes, eight distinct CCT gene families (paralogs) have been characterized in eukaryotes. We are interested in determining when during eukaryotic evolution the multiple gene duplications producing the CCT subunits occurred. We describe the sequence and phylogenetic analysis of five CCT genes from TRICHOMONAS: vaginalis and seven from GIARDIA: lamblia, representatives of amitochondriate protist lineages thought to have diverged early from other eukaryotes. Our data show that the gene duplications producing the eight CCT paralogs took place prior to the organismal divergence of TRICHOMONAS: and GIARDIA: from other eukaryotes. Thus, these divergent protists likely possess completely hetero-oligomeric CCT complexes like those in yeast and mammalian cells. No close phylogenetic relationship between the archaeal chaperonins and specific CCT subunits was observed, suggesting that none of the CCT gene duplications predate the divergence of archaea and eukaryotes. The duplications producing the CCTdelta and CCTepsilon subunits, as well as CCTalpha, CCTbeta, and CCTeta, are the most recent in the CCT gene family. Our analyses show significant differences in the rates of evolution of archaeal chaperonins compared with the eukaryotic CCTs, as well as among the different CCT subunits themselves. We discuss these results in light of current views on the origin, evolution, and function of CCT complexes.     

The crystal structure of yeast CCT reveals intrinsic asymmetry of eukaryotic cytosolic chaperonins

The cytosolic chaperonin CCT is a 1‐MDa protein‐folding machine essential for eukaryotic life. The CCT interactome shows involvement in folding and assembly of a small range of proteins linked to essential cellular processes such as cytoskeleton assembly and cell‐cycle regulation. CCT has a classic chaperonin architecture, with two heterogeneous 8‐membered rings stacked back‐to‐back, enclosing a folding cavity. However, the mechanism by which CCT assists folding is distinct from other chaperonins, with no hydrophobic wall lining a potential Anfinsen cage, and a sequential rather than concerted ATP hydrolysis mechanism. We have solved the crystal structure of yeast CCT in complex with actin at 3.8 Å resolution, revealing the subunit organisation and the location of discrete patches of co‐evolving ‘signature residues’ that mediate specific interactions between CCT and its substrates. The intrinsic asymmetry is revealed by the structural individuality of the CCT subunits, which display unique configurations, substrate binding properties, ATP‐binding heterogeneity and subunit–subunit interactions. The location of the evolutionarily conserved N‐terminus of Cct5 on the outside of the barrel, confirmed by mutational studies, is unique to eukaryotic cytosolic chaperonins.     

Point mutations in a hinge linking the small and large domains of beta-actin result in trapped folding intermediates bound to cytosolic chaperonin CCT

The 30-A cryo-EM-derived structure of apo-CCT-alpha-actin shows actin opened up across its nucleotide-binding cleft and binding to either of two CCT subunit pairs, CCTbeta-CCTdelta or CCTepsilon-CCTdelta, in a similar 1:4 arrangement. The two main duplicated domains of native actin are linked twice, topologically, by the connecting residues, Q137-S145 and P333-S338, and are tightly held together by hydrogen bonding with bound adenine nucleotide. We carried out a mutational screen to find residues in actin that might be involved in the huge rotations observed in the CCT-bound folding intermediate. When two evolutionarily highly conserved glycine residues of beta-actin, G146 and G150, were changed to proline, both mutant actin proteins were poorly processed by CCT in in vitro translation assays; they become arrested on CCT. A three-dimensional reconstruction of the substrate-bound ring of the apo-CCT-beta-actin complex shows that beta-actin G150P is not able to bind across the chaperonin cavity to interact with the CCTdelta subunit. beta-actin G150P seems tightly packed and apparently bound only to the CCTbeta and CCTepsilon subunits, which further indicates that these CCT subunits drive the interaction between CCT and actin. Hinge opening seems to be critical for actin folding, and we suggest that residues G146 and G150 are important components of the hinge around which the rigid subdomains, presumably already present in early actin folding intermediates, rotate during CCT-assisted folding.