Molecular Dynamics Mappings of the CCT/TRiC Complex-Mediated Protein Folding Cycle Using Diffracted X-ray Tracking.

The CCT/TRiC complex is a type II chaperonin that undergoes ATP-driven conformational changes during its functional cycle. Structural studies have provided valuable insights into the mechanism of this process, but real-time dynamics analyses of mammalian type II chaperonins are still scarce. We used diffracted X-ray tracking (DXT) to investigate the intramolecular dynamics of the CCT complex. We focused on three surface-exposed loop regions of the CCT1 subunit: the loop regions of the equatorial domain (E domain), the E and intermediate domain (I domain) juncture near the ATP-binding region, and the apical domain (A domain). Our results showed that the CCT1 subunit predominantly displayed rotational motion, with larger mean square displacement (MSD) values for twist (χ) angles compared with tilt (θ) angles. Nucleotide binding had a significant impact on the dynamics. In the absence of nucleotides, the region between the E and I domain juncture could act as a pivotal axis, allowing for greater motion of the E domain and A domain. In the presence of nucleotides, the nucleotides could wedge into the ATP-binding region, weakening the role of the region between the E and I domain juncture as the rotational axis and causing the CCT complex to adopt a more compact structure. This led to less expanded MSD curves for the E domain and A domain compared with nucleotide-absent conditions. This change may help to stabilize the functional conformation during substrate binding. This study is the first to use DXT to probe the real-time molecular dynamics of mammalian type II chaperonins at the millisecond level. Our findings provide new insights into the complex dynamics of chaperonins and their role in the functional folding cycle.

Chaperonin: Co-chaperonin Interactions.

Co-chaperonins function together with chaperonins to mediate ATP-dependent protein folding in a variety of cellular compartments. Chaperonins are evolutionarily conserved and form two distinct classes, namely, group I and group II chaperonins. GroEL and its co-chaperonin GroES form part of group I and are the archetypal members of this family of protein folding machines. The unique mechanism used by GroEL and GroES to drive protein folding is embedded in the complex architecture of double-ringed complexes, forming two central chambers that undergo conformational rearrangements that enable protein folding to occur. GroES forms a lid over the chamber and in doing so dislodges bound substrate into the chamber, thereby allowing non-native proteins to fold in isolation. GroES also modulates allosteric transitions of GroEL. Group II chaperonins are functionally similar to group I chaperonins but differ in structure and do not require a co-chaperonin. A significant number of bacteria and eukaryotes house multiple chaperonin and co-chaperonin proteins, many of which have acquired additional intracellular and extracellular biological functions. In some instances, co-chaperonins display contrasting functions to those of chaperonins. Human HSP60 (HSPD) continues to play a key role in the pathogenesis of many human diseases, in particular autoimmune diseases and cancer. A greater understanding of the fascinating roles of both intracellular and extracellular Hsp10 on cellular processes will accelerate the development of techniques to treat diseases associated with the chaperonin family.

Progressive retinal degeneration of rods and cones in a Bardet-Biedl syndrome type 10 mouse model.

Bardet-Biedl syndrome (BBS) is a multi-organ autosomal-recessive disorder caused by mutations in at least 22 different genes. A constant feature is early-onset retinal degeneration leading to blindness. Among the most common forms is BBS type 10 (BBS10), which is caused by mutations in a gene encoding a chaperonin-like protein. To aid in developing treatments, we phenotyped a Bbs10 knockout (Bbs10-/-) mouse model. Analysis by optical coherence tomography (OCT), electroretinography (ERG) and a visually guided swim assay (VGSA) revealed a progressive degeneration (from P19 to 8 months of age) of the outer nuclear layer that is visible by OCT and histology. Cone ERG was absent from at least P30, at which time rod ERG was reduced to 74.4% of control levels; at 8 months, rod ERG was 2.3% of that of controls. VGSA demonstrated loss of functional vision at 9 months. These phenotypes progressed more rapidly than retinal degeneration in the Bbs1M390R/M390R knock-in mouse. This study defines endpoints for preclinical trials that can be utilized to detect a treatment effect in the Bbs10-/- mouse and extrapolated to human clinical trials.

Bardet-Biedl syndrome: The pleiotropic role of the chaperonin-like BBS6, 10, and 12 proteins.

Bardet-Biedl syndrome (BBS) is a rare pleiotropic disorder known as a ciliopathy. Despite significant genetic heterogeneity, BBS1 and BBS10 are responsible for major diagnosis in western countries. It is well established that eight BBS proteins, namely BBS1, 2, 4, 5, 7, 8, 9, and 18, form the BBSome, a multiprotein complex serving as a regulator of ciliary membrane protein composition. Less information is available for BBS6, BBS10, and BBS12, three proteins showing sequence homology with the CCT/TRiC family of group II chaperonins. Even though their chaperonin function is debated, scientific evidence demonstrated that they are required for initial BBSome assembly in vitro. Recent studies suggest that genotype may partially predict clinical outcomes. Indeed, patients carrying truncating mutations in any gene show the most severe phenotype; moreover, mutations in chaperonin-like BBS proteins correlated with severe kidney impairment. This study is a critical review of the literature on genetics, expression level, cellular localization and function of BBS proteins, focusing primarily on the chaperonin-like BBS proteins, and aiming to provide some clues to understand the pathomechanisms of disease in this setting.

CryoEM reveals the stochastic nature of individual ATP binding events in a group II chaperonin.

Chaperonins are homo- or hetero-oligomeric complexes that use ATP binding and hydrolysis to facilitate protein folding. ATP hydrolysis exhibits both positive and negative cooperativity. The mechanism by which chaperonins coordinate ATP utilization in their multiple subunits remains unclear. Here we use cryoEM to study ATP binding in the homo-oligomeric archaeal chaperonin from Methanococcus maripaludis (MmCpn), consisting of two stacked rings composed of eight identical subunits each. Using a series of image classification steps, we obtained different structural snapshots of individual chaperonins undergoing the nucleotide binding process. We identified nucleotide-bound and free states of individual subunits in each chaperonin, allowing us to determine the ATP occupancy state of each MmCpn particle. We observe distinctive tertiary and quaternary structures reflecting variations in nucleotide occupancy and subunit conformations in each chaperonin complex. Detailed analysis of the nucleotide distribution in each MmCpn complex indicates that individual ATP binding events occur in a statistically random manner for MmCpn, both within and across the rings. Our findings illustrate the power of cryoEM to characterize a biochemical property of multi-subunit ligand binding cooperativity at the individual particle level.

Functional analysis of new human Bardet-Biedl syndrome loci specific variants in the zebrafish model.

The multiple genetic approaches available for molecular diagnosis of human diseases have made possible to identify an increasing number of pathogenic genetic changes, particularly with the advent of next generation sequencing (NGS) technologies. However, the main challenge lies in the interpretation of their functional impact, which has resulted in the widespread use of animal models. We describe here the functional modelling of seven BBS loci variants, most of them novel, in zebrafish embryos to validate their in silico prediction of pathogenicity. We show that target knockdown (KD) of known BBS (BBS1, BB5 or BBS6) loci leads to developmental defects commonly associated with ciliopathies, as previously described. These KD pleiotropic phenotypes were rescued by co-injecting human wild type (WT) loci sequence but not with the equivalent mutated mRNAs, providing evidence of the pathogenic effect of these BBS changes. Furthermore, direct assessment of cilia located in Kupffer's vesicle (KV) showed a reduction of ciliary length associated with all the studied variants, thus confirming a deleterious effect. Taken together, our results seem to prove the pathogenicity of the already classified and unclassified new BBS variants, as well as highlight the usefulness of zebrafish as an animal model for in vivo assays in human ciliopathies.

Bardet-Biedl Syndrome proteins regulate cilia disassembly during tissue maturation.

Primary cilia are conserved organelles that mediate cellular communication crucial for organogenesis and homeostasis in numerous tissues. The retinal pigment epithelium (RPE) is a ciliated monolayer in the eye that borders the retina and is vital for visual function. Maturation of the RPE is absolutely critical for visual function and the role of the primary cilium in this process has been largely ignored to date. We show that primary cilia are transiently present during RPE development and that as the RPE matures, primary cilia retract, and gene expression of ciliary disassembly components decline. We observe that ciliary-associated BBS proteins protect against HDAC6-mediated ciliary disassembly via their recruitment of Inversin to the base of the primary cilium. Inhibition of ciliary disassembly components was able to rescue ciliary length defects in BBS deficient cells. This consequently affects ciliary regulation of Wnt signaling. Our results shed light onto the mechanisms by which cilia-mediated signaling facilitates tissue maturation.

Structural investigation of a chaperonin in action reveals how nucleotide binding regulates the functional cycle.

Chaperonins are ubiquitous protein assemblies present in bacteria, eukaryota, and archaea, facilitating the folding of proteins, preventing protein aggregation, and thus participating in maintaining protein homeostasis in the cell. During their functional cycle, they bind unfolded client proteins inside their double ring structure and promote protein folding by closing the ring chamber in an adenosine 5'-triphosphate (ATP)-dependent manner. Although the static structures of fully open and closed forms of chaperonins were solved by x-ray crystallography or electron microscopy, elucidating the mechanisms of such ATP-driven molecular events requires studying the proteins at the structural level under working conditions. We introduce an approach that combines site-specific nuclear magnetic resonance observation of very large proteins, enabled by advanced isotope labeling methods, with an in situ ATP regeneration system. Using this method, we provide functional insight into the 1-MDa large hsp60 chaperonin while processing client proteins and reveal how nucleotide binding, hydrolysis, and release control switching between closed and open states. While the open conformation stabilizes the unfolded state of client proteins, the internalization of the client protein inside the chaperonin cavity speeds up its functional cycle. This approach opens new perspectives to study structures and mechanisms of various ATP-driven biological machineries in the heat of action.

Clinical characteristics of a Japanese patient with Bardet-Biedl syndrome caused by BBS10 mutations.

PURPOSE: Bardet-Biedl syndrome (BBS) is a rare autosomal recessive disorder characterized by retinal dystrophy, renal dysfunction, central obesity, mental impairment, polydactyly, and hypogonadism. Only limited information on BBS is available from Japanese patients. In addition, there are currently no reports of Japanese patients with BBS caused by BBS10 mutations. The purpose of this study was to present the characteristics of a Japanese patient with BBS caused by BBS10 mutations. PATIENT AND METHODS: The patient was a 22-year-old Japanese woman. Comprehensive ophthalmic examinations, including visual acuity measurements, perimetry, electroretinography (ERG), fundus autofluorescence imaging, and optical coherence tomography, were performed. Trio-based whole-exome sequencing was performed to identify potential pathogenic mutations, confirmed by Sanger sequencing. RESULTS: The patient showed neither renal malformation nor dysfunction, and visual impairment seemed to be relatively mild for BBS. The fundus examination revealed diffuse retinal degeneration without pigmentary deposits, and ERG scans showed undetectable responses. She had a history of surgically corrected polydactyly, and displayed symptoms of obesity. There was also a menstrual irregularity that could require progestin administration. Genetic analysis revealed compound heterozygous BBS10 mutations in the patient: a novel missense mutation c.98G>A [p.(G33E)], and a novel nonsense mutation c.2125A>T [p.(R709*)]. CONCLUSION: To our knowledge, this is the first description of a Japanese patient with BBS caused by BBS10 mutations. The clinical characteristics of our patient were mild, as neither renal impairment nor legal blindness was observed. Early diagnosis would play a role in providing counseling, and in some cases, therapeutic interventions for BBS patients.

Intraring allostery controls the function and assembly of a hetero-oligomeric class II chaperonin.

Class II chaperonins are essential multisubunit complexes that aid the folding of nonnative proteins in the cytosol of archaea and eukarya. They use energy derived from ATP to drive a series of structural rearrangements that enable polypeptides to fold within their central cavity. These events are regulated by an elaborate allosteric mechanism in need of elucidation. We employed mutagenesis and experimental analysis in concert with in silico molecular dynamics simulations and interface-binding energy calculations to investigate the class II chaperonin from Thermoplasma acidophilum. Here we describe the effects on the asymmetric allosteric mechanism and on hetero-oligomeric complex formation in a panel of mutants in the ATP-binding pocket of the α and ß subunits. Our observations reveal a potential model for a nonconcerted folding mechanism optimized for protecting and refolding a range of nonnative substrates under different environmental conditions, starting to unravel the role of subunit heterogeneity in this folding machine and establishing important links with the behavior of the most complex eukaryotic chaperonins.-Shoemark, D. K., Sessions, R. B., Brancaccio, A., Bigotti, M. G. Intraring allostery controls the function and assembly of a hetero-oligomeric class II chaperonin.

An information theoretic framework reveals a tunable allosteric network in group II chaperonins.

ATP-dependent allosteric regulation of the ring-shaped group II chaperonins remains ill defined, in part because their complex oligomeric topology has limited the success of structural techniques in suggesting allosteric determinants. Further, their high sequence conservation has hindered the prediction of allosteric networks using mathematical covariation approaches. Here, we develop an information theoretic strategy that is robust to residue conservation and apply it to group II chaperonins. We identify a contiguous network of covarying residues that connects all nucleotide-binding pockets within each chaperonin ring. An interfacial residue between the networks of neighboring subunits controls positive cooperativity by communicating nucleotide occupancy within each ring. Strikingly, chaperonin allostery is tunable through single mutations at this position. Naturally occurring variants at this position that double the extent of positive cooperativity are less prevalent in nature. We propose that being less cooperative than attainable allows chaperonins to support robust folding over a wider range of metabolic conditions.

Replacement of GroEL in Escherichia coli by the Group II Chaperonin from the Archaeon Methanococcus maripaludis.

UNLABELLED: Chaperonins are required for correct folding of many proteins. They exist in two phylogenetic groups: group I, found in bacteria and eukaryotic organelles, and group II, found in archaea and eukaryotic cytoplasm. The two groups, while homologous, differ significantly in structure and mechanism. The evolution of group II chaperonins has been proposed to have been crucial in enabling the expansion of the proteome required for eukaryotic evolution. In an archaeal species that expresses both groups of chaperonins, client selection is determined by structural and biochemical properties rather than phylogenetic origin. It is thus predicted that group II chaperonins will be poor at replacing group I chaperonins. We have tested this hypothesis and report here that the group II chaperonin from Methanococcus maripaludis (Mm-cpn) can partially functionally replace GroEL, the group I chaperonin of Escherichia coli Furthermore, we identify and characterize two single point mutations in Mm-cpn that have an enhanced ability to replace GroEL function, including one that allows E. coli growth after deletion of the groEL gene. The biochemical properties of the wild-type and mutant Mm-cpn proteins are reported. These data show that the two groups are not as functionally diverse as has been thought and provide a novel platform for genetic dissection of group II chaperonins. IMPORTANCE: The two phylogenetic groups of the essential and ubiquitous chaperonins diverged approximately 3.7 billion years ago. They have similar structures, with two rings of multiple subunits, and their major role is to assist protein folding. However, they differ with regard to the details of their structure, their cofactor requirements, and their reaction cycles. Despite this, we show here that a group II chaperonin from a methanogenic archaeon can partially substitute for the essential group I chaperonin GroEL in E. coli and that we can easily isolate mutant forms of this chaperonin with further improved functionality. This is the first demonstration that these two groups, despite the long time since they diverged, still overlap significantly in their functional properties.

Contribution of the C-Terminal Region of a Group II Chaperonin to its Interaction with Prefoldin and Substrate Transfer.

Prefoldin is a molecular chaperone that captures an unfolded protein substrate and transfers it to a group II chaperonin. Previous studies have shown that the interaction sites for prefoldin are located in the helical protrusions of group II chaperonins. However, it does not exclude the possibility of the existence of other interaction sites. In this study, we constructed C-terminal truncation mutants of a group II chaperonin and examined the effects of these mutations on the chaperone's function and interaction with prefoldin. Whereas the mutants with up to 6 aa truncation from the C-terminus retained more than 90% chaperone activities for protecting citrate synthase from thermal aggregation and refolding of green fluorescent protein and isopropylmalate dehydrogenase, the truncation mutants showed decreased affinities for prefoldin. Consequently, the truncation mutants showed reduced transfer efficiency of the denatured substrate protein from prefoldin and subsequent chaperonin-dependent refolding. The results clearly show that the C-terminal region of group II chaperonins contributes to their interactions with prefoldin, the transfer of the substrate protein from prefoldin and its refolding.

A Model for the Molecular Mechanism of an Engineered Light-Driven Protein Machine.

Controllable protein-based machines and materials are of considerable interest for diverse biotechnological applications. We previously re-engineered an ATP-driven protein machine, a group II chaperonin, to function as a light-gated nanocage. Here we develop and test a model for the molecular mechanism of the re-engineered chaperonin, which undergoes a large-scale closed to open conformational change triggered by reversible photo-isomerization of a site-specifically attached azobenzene crosslinker. In silico experiments using all-atom simulations suggest that rigid body motions of protein subdomains couple the length changes of the crosslinker to rearrangements of the nucleotide-binding pocket, leading to cage opening. We tested this model by designing a mutant for which the orientation of the two protein subdomains forming the nucleotide-binding pocket is directly controlled by the crosslinker, and confirmed successful reversible photoswitching in vitro. The model probes the conformational cycle of group II chaperonins and offers a design principle for engineering other light-driven protein-based molecular machines.

Structural and Functional Insights into the Evolution and Stress Adaptation of Type II Chaperonins.

Chaperonins are essential biological complexes assisting protein folding in all kingdoms of life. Whereas homooligomeric bacterial GroEL binds hydrophobic substrates non-specifically, the heterooligomeric eukaryotic CCT binds specifically to distinct classes of substrates. Sulfolobales, which survive in a wide range of temperatures, have evolved three different chaperonin subunits (α, ß, γ) that form three distinct complexes tailored for different substrate classes at cold, normal, and elevated temperatures. The larger octadecameric ß complexes cater for substrates under heat stress, whereas smaller hexadecameric αß complexes prevail under normal conditions. The cold-shock complex contains all three subunits, consistent with greater substrate specificity. Structural analysis using crystallography and electron microscopy reveals the geometry of these complexes and shows a novel arrangement of the α and ß subunits in the hexadecamer enabling incorporation of the γ subunit.

The Mechanism and Function of Group II Chaperonins.

Protein folding in the cell requires the assistance of enzymes collectively called chaperones. Among these, the chaperonins are 1-MDa ring-shaped oligomeric complexes that bind unfolded polypeptides and promote their folding within an isolated chamber in an ATP-dependent manner. Group II chaperonins, found in archaea and eukaryotes, contain a built-in lid that opens and closes over the central chamber. In eukaryotes, the chaperonin TRiC/CCT is hetero-oligomeric, consisting of two stacked rings of eight paralogous subunits each. TRiC facilitates folding of approximately 10% of the eukaryotic proteome, including many cytoskeletal components and cell cycle regulators. Folding of many cellular substrates of TRiC cannot be assisted by any other chaperone. A complete structural and mechanistic understanding of this highly conserved and essential chaperonin remains elusive. However, recent work is beginning to shed light on key aspects of chaperonin function and how their unique properties underlie their contribution to maintaining cellular proteostasis.

New Export Pathway in Plasmodium falciparum-Infected Erythrocytes: Role of the Parasite Group II Chaperonin, PfTRiC.

The export of numerous proteins to the plasma membrane of its host erythrocyte is essential for the virulence and survival of the malaria parasite Plasmodium falciparum. The Maurer's clefts, membrane structures transposed by the parasite in the cytoplasm of its host erythrocyte, play the role of a marshal platform for such exported parasite proteins. We identify here the export pathway of three resident proteins of the Maurer's clefts membrane: the proteins are exported as soluble forms in the red cell cytoplasm to the Maurer's clefts membrane in association with the parasite group II chaperonin (PfTRIC), a chaperone complex known to bind and address a large spectrum of unfolded proteins to their final location. We have also located the domain of interaction with PfTRiC within the amino-terminal domain of one of these Maurer's cleft proteins, PfSBP1. Because several Maurer's cleft membrane proteins with different export motifs seem to follow the same route, we propose a general role for PfTRiC in the trafficking of malarial parasite proteins to the host erythrocyte.

Inter-ring communication is dispensable in the reaction cycle of group II chaperonins.

Chaperonins are ubiquitous molecular chaperones with the subunit molecular mass of 60kDa. They exist as double-ring oligomers with central cavities. An ATP-dependent conformational change of the cavity induces the folding of an unfolded protein that is captured in the cavity. In the group I chaperonins, which are present in eubacteria and eukaryotic organelles, inter-ring communication takes important role for the reaction cycle. However, there has been limited study on the inter-ring communication in the group II chaperonins that exist in archaea and the eukaryotic cytosol. In this study, we have constructed the asymmetric ring complex of a group II chaperonin using circular permutated covalent mutants. Although one ring of the asymmetric ring complex lacks ATPase or ATP binding activity, the other wild-type ring undergoes an ATP-dependent conformational change and maintains protein-folding activity. The results clearly demonstrate that inter-ring communication is dispensable in the reaction cycle of group II chaperonins.

Group II archaeal chaperonin recognition of partially folded human γD-crystallin mutants.

The features in partially folded intermediates that allow the group II chaperonins to distinguish partially folded from native states remain unclear. The archaeal group II chaperonin from Methanococcus Mauripaludis (Mm-Cpn) assists the in vitro refolding of the well-characterized ß-sheet lens protein human γD-crystallin (HγD-Crys). The domain interface and buried cores of this Greek key conformation include side chains, which might be exposed in partially folded intermediates. We sought to assess whether particular features buried in the native state, but absent from the native protein surface, might serve as recognition signals. The features tested were (a) paired aromatic side chains, (b) side chains in the interface between the duplicated domains of HγD-Crys, and (c) side chains in the buried core which result in congenital cataract when substituted. We tested the Mm-Cpn suppression of aggregation of these HγD-Crys mutants upon dilution out of denaturant. Mm-Cpn was capable of suppressing the off-pathway aggregation of the three classes of mutants indicating that the buried residues were not recognition signals. In fact, Mm-Cpn recognized the HγD-Crys mutants better than (wild-type) WT and refolded most mutant HγD-Crys to levels twice that of WT HγD-Crys. This presumably represents the increased population or longer lifetimes of the partially folded intermediates of the mutant proteins. The results suggest that Mm-Cpn does not recognize the features of HγD-Crys tested-paired aromatics, exposed domain interface, or destabilized core-but rather recognizes other features of the partially folded ß-sheet conformation that are absent or inaccessible in the native state of HγD-Crys.