Structural analysis of thermosome from hyperthermophilic archaeon Thermococcus

The purification and characterization of thermostable chaperonin of the thermosome family from hyperthermophilic archaeon Thermococcus profunds are described. The purified thermosome is a homooligomeric complex and an ATPase with maximal activity at 80 degrees C. The electron micrographs obtained from negatively stained as well as frozen-hydrated specimen showed an eight-fold symmetry of chaperonin. They were about 15 nm height and 16 nm in diameter with a central cavity of 5 nm. In order to understand the ATPase cycling of thermosome, we analyzed the oligomeric structure of thermosome treated with several nucleotides.     

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.     

Group II chaperonins: new TRiC(k)s and turns of a protein folding machine.

In the past decade, the eubacterial group I chaperonin GroEL became the paradigm of a protein folding machine. More recently, electron microscopy and X-ray crystallography offered insights into the structure of the thermosome, the archetype of the group II chaperonins which also comprise the chaperonin from the eukaryotic cytosol TRiC. Some structural differences from GroEL were revealed, namely the existence of a built-in lid provided by the helical protrusions of the apical domains instead of a GroES-like co-chaperonin. These structural studies provide a framework for understanding the differences in the mode of action between the group II and the group I chaperonins. In vitro analyses of the folding of non-native substrates coupled to ATP binding and hydrolysis are progressing towards establishing a functional cycle for group II chaperonins. A protein complex called GimC/prefoldin has recently been found to cooperate with TRiC in vivo, and its characterization is under way.     

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

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

Budding yeast silencing complexes and regulation of Sir2 activity by protein-protein interactions

Gene silencing in the budding yeast Saccharomyces cerevisiae requires the enzymatic activity of the Sir2 protein, a highly conserved NAD-dependent deacetylase. In order to study the activity of native Sir2, we purified and characterized two budding yeast Sir2 complexes: the Sir2/Sir4 complex, which mediates silencing at mating-type loci and at telomeres, and the RENT complex, which mediates silencing at the ribosomal DNA repeats. Analyses of the protein compositions of these complexes confirmed previously described interactions. We show that the assembly of Sir2 into native silencing complexes does not alter its selectivity for acetylated substrates, nor does it allow the deacetylation of nucleosomal histones. The inability of Sir2 complexes to deacetylate nucleosomes suggests that additional factors influence Sir2 activity in vivo. In contrast, Sir2 complexes show significant enhancement in their affinities for acetylated substrates and their sensitivities to the physiological inhibitor nicotinamide relative to recombinant Sir2. Reconstitution experiments showed that, for the Sir2/Sir4 complex, these differences stem from the physical interaction of Sir2 with Sir4. Finally, we provide evidence that the different nicotinamide sensitivities of Sir2/Sir4 and RENT in vitro could contribute to locus-specific differences in how Sir2 activity is regulated in vivo.     

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.     

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.     

Identification of a critical chaperoning region on an archaeal recombinant thermosome

Chaperone function in water-miscible organic co-solvents is useful for biocatalytic applications requiring enzyme stability in semi-aqueous media and for understanding chaperone behavior in hydrophobic environments. Previously, we have shown that a recombinant single subunit thermosome (rTHS) from Methanocaldococcus jannaschii functions in multiple co-solvents to hydrolyze ATP, prevent protein aggregation, and refold enzymes following solvent denaturation. For the present study, a truncated analog to the thermosome in which 70 N-terminal amino acids are removed is used to identify important regions within the thermosome for its chaperoning functions in organic co-solvents. Data presented herein indicate that the N-terminal region of rTHS is essential for the chaperone to restore the native state of the enzyme citrate synthase, but it is not a critical region for either binding of unfolded proteins or ATP hydrolysis. This is the first demonstration that direct refolding by a Group II chaperonin requires the N-terminal region of the protein.     

Three conformations of an archaeal chaperonin, TF55 from Sulfolobus shibatae

Chaperonins are cylindrical, oligomeric complexes, essential for viability and required for the folding of other proteins. The GroE (group I) subfamily, found in eubacteria, mitochondria and chloroplasts, have 7-fold symmetry and provide an enclosed chamber for protein subunit folding. The central cavity is transiently closed by interaction with the co-protein, GroES. The most prominent feature specific to the group II subfamily, found in archaea and in the eukaryotic cytosol, is a long insertion in the substrate-binding region. In the archaeal complex, this forms an extended structure acting as a built-in lid, obviating the need for a GroES-like co-factor. This extension occludes a site known to bind non-native polypeptides in GroEL. The site and nature of substrate interaction are not known for the group II subfamily. The atomic structure of the thermosome, an archaeal group II chaperonin, has been determined in a fully closed form, but the entry and exit of protein substrates requires transient opening. Although an open form has been investigated by electron microscopy, conformational changes in group II chaperonins are not well characterized. Using electron cryo-microscopy and three-dimensional reconstruction, we describe three conformations of a group II chaperonin, including an asymmetric, bullet-shaped form, revealing the range of domain movements in this subfamily.     

The intercellular signaling activity of the Mycobacterium tuberculosis chaperonin 60.1 protein resides in the equatorial domain

The major heat shock protein, chaperonin 60, has been established to have intercellular signaling activity in addition to its established protein-folding function. Mycobacterium tuberculosis is one of a small proportion of bacteria to encode two chaperonin 60 proteins. We have demonstrated that chaperonin 60.1 from this bacterium is a very active stimulator of human monocytes. To determine structure/function relationships of chaperonin 60.1 we have cloned and expressed the apical, equatorial, and intermediate domains of this protein. We have found that the signaling activity of M. tuberculosis chaperonin 60.1 resides in the equatorial domain. This activity of the recombinant equatorial domain was completely blocked by treating the protein with proteinase K, ruling out lipopolysaccharide contamination as the cause of the cell activation. Blockade of the activity of the equatorial domain by anti-CD14 monoclonal antibodies reveals that this domain activates monocytes by binding to CD14. Looking at the oligomeric state of the active proteins, using native gel electrophoresis and protein cross-linking we found that recombinant M. tuberculosis chaperonin 60.1 fails to form the prototypic tetradecameric structure of chaperonin 60 proteins under the conditions tested and only forms dimers. It is therefore concluded that the monocyte-stimulating activity of M. tuberculosis Cpn60.1 resides in the monomeric subunit and within this subunit the biological activity is due to the equatorial domain.     

Purification and characterization of chaperonins 60 and 10 from Methylobacillus glycogenes

Two proteins belonging to the group I chaperonin family were isolated from an obligate methanotroph, Methylobacillus glycogenes. The two proteins, one a GroEL homologue (cpn60: M. glycogenes 60 kDa chaperonin) and the other a GroES homologue (cpn10: M. glycogenes 10 kDa chaperonin), composed a heteropolymeric complex in the presence of ATP. Both proteins were purified from crude extracts of M. glycogenes by anion-exchange (DEAE-Toyopearl) and gel-filtration (Sephacryl S-400) chromatography. The native molecular weights of each chaperonin protein as determined by high-performance liquid chromatography (HPLC) gel-filtration were 820 000 for cpn60 and 65 000 for cpnl0. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the subunit molecular weights of cpn60 and cpnl0 were 58 000 and 10 000, respectively. Both cpn60 and cpnl0 possessed amino acid sequences which were highly homologous to other group I chaperonins. M. glycogenes cpn60 displayed an ATPase activity which was inhibited in the presence of cpn10. The chaperonins also displayed an ability to interact with and facilitate the refolding of Thermus malate dehydrogenase and yeast enolase in a manner similar to that of GroEL/ES. The similarities between the Escherichia coli GroE proteins are discussed.     

Chaperonin TRiC assists the refolding of sperm-specific glyceraldehyde-3-phosphate dehydrogenase

The cytosolic chaperonin TRiC was isolated from ovine testes using ultracentrifugation and heparin-Sepharose chromatography. The molecular mass of the obtained preparation was shown to exceed 900 kDa (by Blue Native PAGE). SDS–PAGE yielded a set of bands in the range of 50–60 kDa. Electron microscopy examination revealed ring-shaped complexes with the outer diameter of 15 nm and the inner diameter of approximately 6 nm. The results suggest that the purified chaperonin is an oligomeric complex composed of two 8-membered rings.The chaperonin TRiC was shown to assist an ATP-dependent refolding of recombinant forms of sperm-specific glyceraldehyde-3-phosphate dehydrogenase, an enzyme that is expressed only in precursor cells of the sperms in the seminiferous tubules of the testes. In contrast, TRiC did not influence the refolding of muscle isoform of glyceraldehyde-3-phosphate dehydrogenase and assisted the refolding of muscle lactate dehydrogenase by an ATP-independent mechanism. The obtained results suggest that TRiC is likely to be involved in the refolding of sperm-specific proteins.     

Identification of in vivo substrates of the yeast mitochondrial chaperonins reveals overlapping but non-identical requirement for hsp60 and hsp10.

The mechanism of chaperonin-assisted protein folding has been mostly analyzed in vitro using non-homologous substrate proteins. In order to understand the relative importance of hsp60 and hsp10 in the living cell, homologous substrate proteins need to be identified and analyzed. We have devised a novel screen to test the folding of a large variety of homologous substrates in the mitochondrial matrix in the absence or presence of functional hsp60 or hsp10. The identified substrates have an Mr of 15-90 kDa and fall into three groups: (i) proteins that require both hsp60 and hsp10 for correct folding; (ii) proteins that completely fail to fold after inactivation of hsp60 but are unaffected by the inactivation of hsp10; and (iii) newly imported hsp60 itself, which is more severely affected by inactivation of hsp10 than by inactivation of pre-existing hsp60. The majority of the identified substrates are group I proteins. For these, the lack of hsp60 function has a more pronounced effect than inactivation of hsp10. We suggest that homologous substrate proteins have differential chaperonin requirements, indicating that hsp60 and hsp10 do not always act as a single functional unit in vivo.     

Functional characterization of recombinant prefoldin complexes from a hyperthermophilic archaeon, Thermococcus sp. strain KS-1

Prefoldin is a heterohexameric molecular chaperone complex that is found in the eukaryotic cytosol and also in archaea. It captures a nonnative protein and subsequently delivers it to a group II chaperonin for proper folding. Archaeal prefoldin is a heterocomplex containing two α subunits and four β subunits with the structure of a double β-barrel assembly, with six long coiled coils protruding from it like a jellyfish with six tentacles. We have studied the protein folding mechanism of group II chaperonin using those of Thermococcus sp. strain KS-1 (T. KS-1) because they exhibit high protein folding activity in vitro. We have also demonstrated functional cooperation between T. KS-1 chaperonins and prefoldin from Pyrococcus horikoshii OT3. Recent genome analysis has shown that Thermococcus kodakaraensis KOD1 contains two pairs of prefoldin subunit genes, correlating with the existence of two different chaperonin subunits. In this study, we characterized four different recombinant prefoldin complexes composed of two pairs of prefoldin subunits (α1, α2, β1, and β2) from T. KS-1. All of them (α1-β1, α2-β1, α1-β2, and α2-β2) exist as α₂β₄ heterohexamers and can protect several proteins from forming aggregates with different activities. We have also compared the collaborative activity between the prefoldin complexes and the cognate chaperonins. Prefoldin complexes containing the β1 subunit interacted with the chaperonins more strongly than those with the β2 subunit. The results suggest that Thermococcus spp. express different prefoldins for different substrates or conditions as chaperonins.     

Molecular chaperones: containers and surfaces for folding, stabilising or unfolding proteins

Newly solved chaperone structures include the thermosome, a group II chaperonin, and a small heat-shock protein. Novel ideas on chaperone mechanism are presented in the forced unfolding hypothesis of GroEL action. Structures of chaperone-pilin complexes reveal the mechanism of chaperone interaction in bacterial pilus assembly and there have been major advances in understanding the structure and function of Hsp100 unfoldases.     

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.     

Properties of the alpha subunit of a Chaperonin from the hyperthermophilic Crenarchaeon Aeropyrum pernix K1

The gene encoding for a putative thermosome from the hyperthermophilic crenarchaeon Aeropyrum pernix K1 (ApcpnA) was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (accession no. APE0907) from A. pernix K1 showed some homology with other group II chaperonins from archaea. The recombinant ApcpnA protein has a molecular mass of 60 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and exhibited ATPase activity with an optimum temperature and pH of 90 degrees C and 5.0, respectively. The ATPase activity was found to be dependent on manganese and potassium ions, but not magnesium ion. The K(m) for ATP at pH 5.0 and 90 degrees C was 10.04 (+/- 1.31) microM, and k(cat) was determined to be 2.21 (+/- 0.11) min(-1) for the ApcpnA monomer. The recombinant ApcpnA prevents thermal aggregation of bovine rhodanese and enhances the thermal stability of alcohol dehydrogenase in vitro, indicating that the protein is suitable as a molecular chaperonin in the high-temperature environment.     

Cooperativity in the thermosome

The thermosome from Thermoplasma acidophilum is a type II chaperonin composed of eight alpha and eight beta subunits. The genes encoding the two types of subunit were co-expressed in Escherichia coli and the alpha8/beta8 complex purified from the cell extract. The isolated complex showed steady-state ATPase properties characteristic of the thermosome purified from the native organism and was capable of enhancing the folding yield of a thermostable enzyme at elevated temperature (55 degrees C). To compare the nucleotide response of this double-ring structure with the type I and more compositionally heterogeneous type II chaperonins, the tryptophan residue within the alpha subunit was used as a fluorescence reporter of the conformational changes within the thermosome induced by the binding of nucleotides. Stopped-flow measurements of indole fluorescence at 55 degrees C showed that there is a fast (approximately 350 s(-1)) and a slow (approximately 0.6 s(-1)) structural rearrangement when ATP binds to the thermosome. Further examination of the fast rearrangement showed that the associated rate constant followed a two-phase saturation profile, as it does for GroEL and for the type II chaperonin from the eukaryotic cytoplasm. This result, in keeping with these precedents, reveals that the thermosome is also a negatively cooperative system with respect to inter-ring communications, i.e. the first ring loads with higher affinity than the second. As in the case of GroEL, the loading of the second ring is weakened by ADP, implying that asymmetric ATP/ADP complexes are favoured over symmetric ones. Despite the difference in co-protein involvement in the type I and II chaperonins, these observations show that negative cooperativity is a common feature of all chaperonins thus far examined. This property results in a strong preference for asymmetry in nucleotide occupancy and implies at least some commonality with the reciprocating encapsulation mechanism shown for the GroE chaperonins.     

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.     

Identification of a 66 KDa protein associated with yeast mitochondrial ATP synthase as heat shock protein hsp60

A 66 kDa protein, denoted P66, not hitherto classified as an integral component of yeast mitochondrial ATPase, is often observed in preparations of this enzyme complex. A physical association exists between P66 and the assembled ATPase complex since both components are coimmunoprecipitated by anti-F1 beta monoclonal antibody. Two recombinant clones expressing proteins immunologically similar to P66 were isolated from a yeast genomic library in lambda gt11 by screening with a polyclonal anti-holo-ATPase antibody. Based on restriction site mapping and partial nucleotide sequence analysis, both clones encompass the gene encoding the yeast heat shock protein hsp60. The identification of P66 with hsp60, taken together with its demonstrated association with the mitochondrial ATPase complex, is consistent with recent suggestions that hsp60 is involved in assembly of the ATP synthase complex.