The chaperonin of the archaeon Sulfolobus solfataricus is an RNA-binding protein that participates in ribosomal RNA processing.

The 60 kDa molecular chaperones (chaperonins) are high molecular weight protein complexes having a characteristic double-ring toroidal shape; they are thought to aid the folding of denatured or newly synthesized polypeptides. These proteins exist as two functionally similar, but distantly related families, one comprising the bacterial and organellar chaperonins and another (the so-called CCT-TRiC family) including the chaperonins of the archaea and the eukaryotes. Although some evidence exists that the archaeal chaperonins are implicated in protein folding, much remains to be learned about their precise cellular function. In this work, we report that the chaperonin of the thermophilic archaeon Sulfolobus solfataricus is an RNA-binding protein that interacts specifically in vivo with the 16S rRNA and participates in the maturation of its 5' extremity in vitro. We further show that the chaperonin binds RNA as the native heterooligomeric complex and that RNA binding and processing are inhibited by ATP. These results agree with previous reports indicating a role for the bacterial/organellar chaperonins in RNA protection or processing and suggest that all known chaperonin families share specific and evolutionarily ancient functions in RNA metabolism.     

Recurrent paralogy in the evolution of archaeal chaperonins.

Chaperonins are multisubunit double-ring complexes that mediate the folding of nascent proteins [1] [2]. In bacteria, chaperonins are homo-oligomeric and are composed of seven-membered rings. Eukaryotic and most archaeal chaperonin rings are eight-membered and exhibit varying degrees of hetero-oligomerism [3] [4]. We have cloned and sequenced seven new genes encoding chaperonin subunits from the crenarchaeotes Sulfolobus solfataricus, S. acidocaldarius, S. shibatae and Desulfurococcus mobilis. Although some archaeal genomes possess a single chaperonin gene, most have two. We describe a third chaperonin-encoding gene (TF55-gamma) from two Sulfolobus species; phylogenetic analyses indicate that the gene duplication producing TF55-gamma occurred within crenarchaeal evolution. The presence of TF55-gamma in Sulfolobus correlates with their unique nine-membered chaperonin rings. Duplicate genes (paralogs) for chaperonins within archaeal genomes very often resemble each other more than they resemble chaperonin genes from other archaea. Our phylogenetic analyses suggest multiple independent gene duplications - at least seven among the archaea examined. The persistence of paralogous genes for chaperonin subunits in multiple archaeal lineages may involve a process of co-evolution, where chaperonin subunit heterogeneity changes independently of selection on function.     

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.     

All three chaperonin genes in the archaeon Haloferax volcanii are individually dispensable

The Hsp60 or chaperonin class of molecular chaperones is divided into two phylogenetic groups: group I, found in bacteria, mitochondria and chloroplasts, and group II, found in eukaryotic cytosol and archaea. Group I chaperonins are generally essential in bacteria, although when multiple copies are found one or more of these are dispensable. Eukaryotes contain eight genes for group II chaperonins, all of which are essential, and it has been shown that these proteins assemble into double-ring complexes with eightfold symmetry where all proteins occupy specific positions in the ring. In archaea, there are one, two or three genes for the group II chaperonins, but whether they are essential for growth is unknown. Here we describe a detailed genetic, structural and biochemical analysis of these proteins in the halophilic archaeon, Haloferax volcanii. This organism contains three genes for group II chaperonins, and we show that all are individually dispensable but at least one must be present for growth. Two of the three possible double mutants can be constructed, but only one of the three genes is capable of fully complementing the stress-dependent phenotypes that these double mutants show. The chaperonin complexes are made up of hetero-oligomers with eightfold symmetry, and the properties of the different combinations of subunits derived from the mutants are distinct. We conclude that, although they are more homologous to eukaryotic than prokaryotic chaperonins, archaeal chaperonins have some redundancy of function.     

An engineered chaperonin caging a guest protein: Structural insights and potential as a protein expression tool

The structure of a chaperonin caging a substrate protein is not quite clear. We made engineered group II chaperonins fused with a guest protein and analyzed their structural and functional features. Thermococcus sp. KS-1 chaperonin alpha-subunit (TCP) which forms an eightfold symmetric double-ring structure was used. Expression plasmids were constructed which carried two or four TCP genes ligated head to tail in phase and a target protein gene at the 3' end of the linked TCP genes. Electron microscopy showed that the expressed gene products with the molecular sizes of ~120 kDa (di-TCP) and ~230 kDa (tetra-TCP) formed double-ring complexes similar to those of wild-type TCP. The tetra-TCP retained ATPase activity and its thermostability was significantly higher than that of the wild type. A 260-kDa fusion protein of tetra-TCP and green fluorescent protein (GFP, 27 kDa) was able to form the double-ring complexes with green fluorescence. Image analyses indicated that the GFP moiety of tetra-TCP/GFP fusion protein was accommodated in the central cavity, and tetra-TCP/GFP formed the closed-form similar to that crystallographically resolved in group II chaperonins. Furthermore, it was suggested that caging GFP expanded the cavity around the bottom. Using this tetra-TCP fusion strategy, two virus structural proteins (21-25 kDa) toxic to host cells or two antibody fragments (25-36 kDa) prone to aggregate were well expressed in the soluble fraction of Escherichia coli. These fusion products also assembled to double-ring complexes, suggesting encapsulation of the guest proteins. The antibody fragments liberated by site-specific protease digestion exhibited ligand-binding activities.     

Inter-Ring Communication Is Dispensable in the Reaction Cycle of Group II Chaperonins

Chaperonins are ubiquitous molecular chaperones with the subunit molecular mass of 60 kDa. 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.     

The plastid chaperonin

The discovery and properties of the plastid chaperonin are described. This chaperonin is implicated in the folding and assembly of the enzyme ribulose bisphosphate carboxylase-oxygenase and in the folding of proteins imported into plastids from the cytosol. The plastid chaperonin appears to be unique in that it contains two distinct types of 60 kDa polypeptide, whereas only a single subunit type has been reported for the bacterial and mitochondrial chaperonins. The plastid chaperonin polypeptides are encoded by nuclear genes which are subject to complex regulation.     

The human mitochondrial Hsp60 in the APO conformation forms a stable tetradecameric complex

The human mitochondrial chaperonin is a macromolecular machine that catalyzes the proper folding of mitochondrial proteins and is of vital importance to all cells. This chaperonin is composed of 2 distinct proteins, Hsp60 and Hsp10, that assemble into large oligomeric complexes that mediate the folding of non-native polypeptides in an ATP dependent manner. Here, we report the bacterial expression and purification of fully assembled human Hsp60 and Hsp10 recombinant proteins and that Hsp60 forms a stable tetradecameric double-ring conformation in the absence of co-chaperonin and nucleotide. Evidence of the stable double-ring conformation is illustrated by the 15 Å resolution electron microscopy reconstruction presented here. Furthermore, our biochemical analyses reveal that the presence of a non-native substrate initiates ATP-hydrolysis within the Hsp60/10 chaperonin to commence protein folding. Collectively, these data provide insight into the architecture of the intermediates used by the human mitochondrial chaperonin along its protein folding pathway and lay a foundation for subsequent high resolution structural investigations into the conformational changes of the mitochondrial chaperonin.     

Chaperonins are cell-signalling proteins: the unfolding biology of molecular chaperones

The chaperonins are a subgroup of oligomeric molecular chaperones; the best-studied examples are chaperonin 60 (GroEL) and chaperonin 10 (GroES), both from the bacterium Escherichia coli. At the end of the 20th century, the paradigm of chaperonins as protein folders had emerged, but it is likely that during the 21st century these proteins will come to be viewed as intercellular signals. Indeed, it is possible that the chaperonins were among the first intercellular signalling proteins to evolve. During the past few years, it has emerged that chaperonin 10 and chaperonin 60 can be found on the surface of various prokaryotic and eukaryotic cells, and can even be released from cells. Secreted chaperonins can interact with a variety of cell types, including leukocytes, vascular endothelial cells and epithelial cells, and activate key cellular activities such as the synthesis of cytokines and adhesion proteins. Much has been made of the high degree of sequence conservation among the chaperonins, particularly in terms of the immunogenicity of these proteins. However, different chaperonin 60 proteins can bind to different cell-surface receptors, including the Toll-like receptors, suggesting that this family of proteins cannot be treated as one biological entity and that several subfamilies may exist. Chaperonins have been implicated in human diseases on the basis of their immunogenicity. The finding that chaperonins can also induce tissue pathology suggests that they may play roles in infections and in idiopathic diseases such as atherosclerosis and arthritis.     

Weak Intra-Ring Allosteric Communications of the Archaeal Chaperonin Thermosome Revealed by Normal Mode Analysis

Chaperonins are molecular machines that use ATP-driven cycles to assist misfolded substrate proteins to reach the native state. During the functional cycle, these machines adopt distinct nucleotide-dependent conformational states, which reflect large-scale allosteric changes in individual subunits. Distinct allosteric kinetics has been described for the two chaperonin classes. Bacterial (group I) chaperonins, such as GroEL, undergo concerted subunit motions within each ring, whereas archaeal and eukaryotic chaperonins (group II) undergo sequential subunit motions. We study these distinct mechanisms through a comparative normal mode analysis of monomer and double-ring structures of the archaeal chaperonin thermosome and GroEL. We find that thermosome monomers of each type exhibit common low-frequency behavior of normal modes. The observed distinct higher-frequency modes are attributed to functional specialization of these subunit types. The thermosome double-ring structure has larger contribution from higher-frequency modes, as it is found in the GroEL case. We find that long-range intersubunit correlation of amino-acid pairs is weaker in the thermosome ring than in GroEL. Overall, our results indicate that distinct allosteric behavior of the two chaperonin classes originates from different wiring of individual subunits as well as of the intersubunit communications.     

Mammalian mitochondrial chaperonin 60 functions as a single toroidal ring.

Chaperonins are thought to participate in the process of protein folding in bacteria and in eukaryotic mitochondria and chloroplasts. While some chaperonins are relatively well characterized, the structures of the mammalian chaperonins are unknown. We have expressed a mammalian mitochondrial chaperonin 60 in Escherichia coli and purified the recombinant protein to homogeneity. Structural and biochemical analyses of this protein establish a single toroidal structure of seven subunits, in contrast to the homologous bacterial, fungal, and plant chaperonin 60s, which have double toroidal structures comprising two layers of seven identical subjects each. The recombinant mammalian chaperonin 60, together with the mammalian chaperonin 10 (but not with bacterial chaperonin 10), facilitates the formation of catalytically active ribulose-bisphosphate carboxylase from an unfolded state in the presence of K+ and MgATP. Analysis of the partial reactions involved in this in vitro reconstitution reveals that the single toroid of chaperonin 60 can form stable complexes with both unfolded or partially folded [35S]ribulose-bisphosphate carboxylase and mitochondrial (but not bacterial) chaperonin 10 in the presence of MgATP. We conclude that the minimal functional unit of chaperonin 60 is a single hepatmeric toroid.     

Differential substrate specificity of group I and group II chaperonins in the archaeon Methanosarcina mazei

Chaperonins are macromolecular machines that assist in protein folding. The archaeon Methanosarcina mazei has acquired numerous bacterial genes by horizontal gene transfer. As a result, both the bacterial group I chaperonin, GroEL, and the archaeal group II chaperonin, thermosome, coexist. A proteome‐wide analysis of chaperonin interactors was performed to determine the differential substrate specificity of GroEL and thermosome. At least 13% of soluble M. mazei proteins interact with chaperonins, with the two systems having partially overlapping substrate sets. Remarkably, chaperonin selectivity is independent of phylogenetic origin and is determined by distinct structural and biochemical features of proteins. GroEL prefers well‐conserved proteins with complex α/β domains. In contrast, thermosome substrates comprise a group of faster‐evolving proteins and contain a much wider range of different domain folds, including small all‐α and all‐β modules, and a greater number of large multidomain proteins. Thus, the group II chaperonins may have facilitated the evolution of the highly complex proteomes characteristic of eukaryotic cells.     

On the oligomeric state of chloroplast chaperonin 10 and chaperonin 20

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

The Escherichia coli groE chaperonins

The E.coli groES and groEL genes have been shown to form an operon, to be essential for E. coli viability, and to belong to the so-called heat-shock class of genes whose expression is regulated by the intracellular levels of sigma factor sigma 32. Both groE chaperonin proteins possess a seven-fold axis of symmetry, groES being composed of seven identical subunits of 97 amino acids each, and groEL of fourteen identical subunits of 548 amino acids each. The two groE chaperonins interact intimately as judged by both genetic and biochemical criteria. This interaction has been shown to be required for both bacteriophage morphogenesis and bacterial growth. The groEL chaperonin has been shown to bind to a number of incomplete or unfolded polypeptides in vitro. Such binding may prevent misfolding and promote rapid intra- or intermolecular folding of polypeptides in vivo. The proposed role of the groES chaperonin is to displace the polypeptides bound to groEL, thus effectively promoting the recycling of groEL.     

Chloroplast chaperonins: evidence for heterogeneous assembly of alpha and beta Cpn60 polypeptides into a chaperonin oligomer

Higher plant chloroplasts contain two chaperonin 60 family proteins, Cpn60alpha and Cpn60beta, which are known to have divergent amino acid sequences. Although Cpn60alpha and Cpn60beta are present in roughly equal amounts and copurify in their native states, a heterogeneous ensemble of the chaperonin oligomer has not yet been demonstrated. We separately purified Cpn60alpha and Cpn60beta proteins from spinach leaves as the monomeric form. Either antibody raised against one chaperonin 60 protein could coimmunoprecipitate the other chaperonin 60 protein in their oligomeric state but not in its monomeric state, suggesting that the chloroplast Cpn60alpha and Cpn60beta polypeptides actually reside in the same chaperonin oligomer in the stroma. Moreover, the chaperonin oligomers migrated as at least two distinct bands on the native gel electrophoresis, each of which contained both chaperonin 60 proteins. These results suggest that chloroplast chaperonin oligomers might be composed of at least two distinct sets of two chaperonin proteins.     

GroEL-mediated protein folding: making the impossible, possible

Protein folding is a spontaneous process that is essential for life, yet the concentrated and complex interior of a cell is an inherently hostile environment for the efficient folding of many proteins. Some proteins-constrained by sequence, topology, size, and function-simply cannot fold by themselves and are instead prone to misfolding and aggregation. This problem is so deeply entrenched that a specialized family of proteins, known as molecular chaperones, evolved to assist in protein folding. Here we examine one essential class of molecular chaperones, the large, oligomeric, and energy utilizing chaperonins or Hsp60s. The bacterial chaperonin GroEL, along with its co-chaperonin GroES, is probably the best-studied example of this family of protein-folding machine. In this review, we examine some of the general properties of proteins that do not fold well in the absence of GroEL and then consider how folding of these proteins is enhanced by GroEL and GroES. Recent experimental and theoretical studies suggest that chaperonins like GroEL and GroES employ a combination of protein isolation, unfolding, and conformational restriction to drive protein folding under conditions where it is otherwise not possible.     

Identification and characterization of a testis-specific isoform of a chaperonin in a moth, Heliothis virescens

Two relatively abundant proteins having subunit molecular weights of 60,000 and 63,000 (p60 and p63, respectively) have been purified as a 16 to 18S complex from sperm mitochondria of a moth. Heliothis virescens. Although the function of these proteins had heretofore not been established, interest in the p63 polypeptide stemmed from its sperm-specific expression and its striking occurrence as a net charge variant among several insect species surveyed, using two-dimensional gel electrophoresis. Genomic and cDNA clones corresponding to the p63 protein have now been isolated and their sequencing has revealed extensive amino acid sequence identity with both the Escherichia coli GroEL protein and its eukaryotic homologues, the chaperonins. Immunoblot studies with a Tetrahymena chaperonin antiserum demonstrated that the p60 protein, which is expressed in all cell types, is structurally related to p63 and is itself a chaperonin subunit. While the chaperonin complex from Heliothis sperm shares certain properties with GroEL, including the ability to hydrolyze ATP and organization of its subunits into a seven-member ring, electron microscopic analysis revealed that its higher-order structure differed from GroEL (and other lower eukaryotic chaperonins) in that the native particle comprises one such ring rather than a doublet. It is not yet known whether the two chaperonin isoforms coexpressed in moth sperm assemble separately or give rise to hybrid particles. In either case, the existence of multiple chaperonin subunits in sperm leaves open the possibility that some aspect of mitochondrial biogenesis that is dependent upon the activity of these proteins is qualitatively or quantitatively different in this cell type.     

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.     

GroEL-Mediated Protein Folding: Making the Impossible,Possible

ABSTRACT

Protein folding is a spontaneous process that is essential for life, yet the concentrated and complex interior of a cell is an inherently hostile environment for the efficient folding of many proteins. Some proteins—constrained by sequence, topology, size, and function—simply cannot fold by themselves and are instead prone to misfolding and aggregation. This problem is so deeply entrenched that a specialized family of proteins, known as molecular chaperones, evolved to assist in protein folding. Here we examine one essential class of molecular chaperones, the large, oligomeric, and energy utilizing chaperonins or Hsp60s. The bacterial chaperonin GroEL, along with its co-chaperonin GroES, is probably the best-studied example of this family of protein-folding machine. In this review, we examine some of the general properties of proteins that do not fold well in the absence of GroEL and then consider how folding of these proteins is enhanced by GroEL and GroES. Recent experimental and theoretical studies suggest that chaperonins like GroEL and GroES employ a combination of protein isolation, unfolding, and conformational restriction to drive protein folding under conditions where it is otherwise not possible.