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.     

Sequential ATP-induced allosteric transitions of the cytoplasmic chaperonin containing TCP-1 revealed by EM analysis

The eukaryotic cytoplasmic chaperonin containing TCP-1 (CCT) is a hetero-oligomeric complex that assists the folding of actins, tubulins and other proteins in an ATP-dependent manner. To understand the allosteric transitions that occur during the functional cycle of CCT, we imaged the chaperonin complex in the presence of different ATP concentrations. Labeling by monoclonal antibodies that bind specifically to the CCTalpha and CCTdelta subunits enabled alignment of all the CCT subunits of a given type in different particles. The analysis shows that the apo state of CCT has considerable apparent conformational heterogeneity that decreases with increasing ATP concentration. In contrast with the concerted allosteric switch of GroEL, ATP-induced conformational changes in CCT are found to spread around the ring in a sequential fashion that may facilitate domain-by-domain substrate folding. The approach described here can be used to unravel the allosteric mechanisms of other ring-shaped molecular machines.     

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.     

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.     

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.     

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.     

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.     

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.     

PhLP3 modulates CCT-mediated actin and tubulin folding via ternary complexes with substrates

Many ATP-dependent molecular chaperones, including Hsp70, Hsp90, and the chaperonins GroEL/Hsp60, require cofactor proteins to regulate their ATPase activities and thus folding functions in vivo. One conspicuous exception has been the eukaryotic chaperonin CCT, for which no regulator of its ATPase activity, other than non-native substrate proteins, is known. We identify the evolutionarily conserved PhLP3 (phosducin-like protein 3) as a modulator of CCT function in vitro and in vivo. PhLP3 binds CCT, spanning the cylindrical chaperonin cavity and contacting at least two subunits. When present in a ternary complex with CCT and an actin or tubulin substrate, PhLP3 significantly diminishes the chaperonin ATPase activity, and accordingly, excess PhLP3 perturbs actin or tubulin folding in vitro. Most interestingly, however, the Saccharomyces cerevisiae PhLP3 homologue is required for proper actin and tubulin function. This cellular role of PhLP3 is most apparent in a strain that also lacks prefoldin, a chaperone that facilitates CCT-mediated actin and tubulin folding. We propose that the antagonistic actions of PhLP3 and prefoldin serve to modulate CCT activity and play a key role in establishing a functional cytoskeleton in vivo.     

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.     

Interactions between the actin filament capping and severing protein gelsolin and the molecular chaperone CCT: evidence for nonclassical substrate interactions

CCT is a member of the chaperonin family of molecular chaperones and consists of eight distinct subunit species which occupy fixed positions within the chaperonin rings. The activity of CCT is closely linked to the integrity of the cytoskeleton as newly synthesized actin and tubulin monomers are dependent upon CCT to reach their native conformations. Furthermore, an additional role for CCT involving interactions with assembling/assembled microfilaments and microtubules is emerging. CCT is also known to interact with other proteins, only some of which will be genuine folding substrates. Here, we identify the actin filament remodeling protein gelsolin as a CCT-binding partner, and although it does not behave as a classical folding substrate, gelsolin binds to CCT with a degree of specificity. In cultured cells, the levels of CCT monomers affect levels of gelsolin, suggesting an additional link between CCT and the actin cytoskeleton that is mediated via the actin filament severing and capping protein gelsolin.     

Overexpressed ribosomal proteins suppress defective chaperonins in Saccharomyces cerevisiae

The chaperonin Cct complex of the yeast Saccharomyces cerevisiae is composed of eight different subunits encoded by eight essential genes, CCT1-CCT8. This Cct complex is responsible for the folding of a number of proteins including actin and tubulin. We have isolated and characterized 22 multicopy suppressors of the temperature-sensitive allele, cct4-1, which encodes an altered protein with a G345D replacement that diminishes ATP hydrolysis. Fourteen of the suppressors encode ribosomal proteins, four have roles in ribosome biogenesis, two have phosphatase activities, one is involved in protein synthesis and one of the suppressors corresponded to Cct4p. Some of the suppressors also acted on certain cct1, cct2, cct3 and cct6 mutations. We suggest that certain overexpressed ribosomal and other proteins can act as weak chaperones, phenotypically alleviating the partial defects of mutationally altered Cct subunits.     

Cystosolic chaperonin subunits have a conserved ATPase domain but diverged polypeptide-binding domains

CCT (also called the TCP-1 complex or TriC) is a chaperonin found in the eukaryotic cytosol, and has unique structural and functional features. Unlike homo-oligomeric chaperonins, CCT comprises at least eight different subunits, and appears to have a limited range of physiological substrates. We have analysed CCT sequences in light of the recent determination of the crystal structure and mutational identification of the functional domains of the bacterial chaperonin GroEL. A high level of identity among all chaperonin subunits is observed in those regions that correspond to the ATP-binding site of GroEL. By contrast, no significant identity is shared in the region corresponding to the polypeptide-binding region of GroEL, either between CCT subunits or between CCT subunits and GroEL. This suggests that the polypeptide-binding sites of CCT subunits have diverged both from each other and from GroEL, which may explain the apparently different range of substrates recognized by CCT.     

Characterization of the cytoplasmic chaperonin containing TCP-1 from the Antarctic fish Notothenia coriiceps

The cytoplasmic chaperonin containing TCP-1 (CCT) plays a critically important role in the folding and biogenesis of many cytoskeletal proteins, including tubulin and actin. For marine ectotherms, the chronically cold Southern Ocean (−2 to +2°C) poses energetic challenges to protein folding, both at the level of substrate proteins and with respect to the chaperonin/chaperone folding system. Here we report the partial functional and structural characterization of CCT from an Antarctic notothenioid fish, Notothenia coriiceps. We find that the mechanism of folding by the Antarctic fish CCT differed from that of mammalian CCT: (1) the former complex was able to bind denatured β-tubulin but (2) when reconstituted with rabbit Cofactor A, failed to release the protein to yield the tubulin/cofactor intermediate. Moreover, the amino acid sequences of the N. coriiceps CCT β and θ chains contained residue substitutions in the equatorial, apical, and intermediate domains that would be expected to increase the flexibility of the subunits, thus facilitating function of the chaperonin in an energy poor environment. Our work contributes to the growing realization that protein function in cold-adapted organisms reflects a delicate balance between the necessity of structural flexibility for catalytic activity and the concomitant hazard of cold-induced denaturation.     

Upregulation of cytosolic chaperonin CCT subunits during recovery from chemical stress that causes accumulation of unfolded proteins.

The chaperonin containing TCP-1 (CCT) is a molecular chaperone consisting of eight subunit species and assists in the folding of actin, tubulin and some other cytosolic proteins. We examined the stress response of CCT subunit proteins in mammalian cultured cells using chemical stressors that cause accumulation of unfolded proteins. Levels of CCT subunit proteins in HeLa cells were coordinately and transiently upregulated under continuous chemical stress with sodium arsenite. CCT subunit levels in several mammalian cell lines were also upregulated during recovery from chemical stress caused by sodium arsenite or a proline analogue, L-azetidine-2-carboxylic acid. Several unidentified proteins that were newly synthesized and associated with CCT were found to increase concomitantly with CCT subunits themselves and known substrates during recovery from the stress. These results suggest that CCT plays important roles in the recovery of cells from protein damage by assisting in the folding of proteins that are actively synthesized and/or renatured during this period.     

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.     

A Two-step Mechanism for the Folding of Actin by the Yeast Cytosolic Chaperonin

Actin requires the chaperonin containing TCP1 (CCT), a hexadecameric ATPase essential for cell viability in eukaryotes, to fold to its native state. Following binding of unfolded actin to CCT, the cavity of the chaperone closes and actin is folded and released in an ATP-dependent folding cycle. In yeast, CCT forms a ternary complex with the phosducin-like protein PLP2p to fold actin, and together they can return nascent or chemically denatured actin to its native state in a pure in vitro folding assay. The complexity of the CCT-actin system makes the study of the actin folding mechanism technically challenging. We have established a novel spectroscopic assay through selectively labeling the C terminus of yeast actin with acrylodan and observe significant changes in the acrylodan fluorescence emission spectrum as actin is chemically unfolded and then refolded by the chaperonin. The variation in the polarity of the environment surrounding the fluorescent probe during the unfolding/folding processes has allowed us to monitor actin as it folds on CCT. The rate of actin folding at a range of temperatures and ATP concentrations has been determined for both wild type CCT and a mutant CCT, CCT4anc2, defective in folding actin in vivo. Binding of the non-hydrolysable ATP analog adenosine 5′-(β,γ-imino)triphosphate to the ternary complex leads to 3-fold faster release of actin from CCT following addition of ATP, suggesting a two-step folding process with a conformational change occurring upon closure of the cavity and a subsequent final folding step involving packing of the C terminus to the native-like state.     

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.     

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.