ATPase cycle of an archaeal chaperonin

Recent structural data imply differences in allosteric behavior of the group I chaperonins, typified by GroEL from Escherichia coli, and the group II chaperonins, which comprise archaeal thermosome and eukaryotic TRiC/CCT. Therefore, this study addresses the mechanism of interaction of adenine nucleotides with recombinant alpha-only and native alphabeta-thermosomes from Thermoplasma acidophilum, which also enables us to analyze the role of the heterooligomeric composition of the natural thermosome. Although all subunits of the alpha-only thermosome seem to bind nucleotides tightly and independently, the native chaperonin has two different classes of ATP-binding sites. Furthermore, for the alpha-only thermosome, the steady-state ATPase rate is determined by the cleavage reaction itself, whereas, for the alphabeta-thermosome, the rate-limiting step is associated with a post-hydrolysis isomerisation into a non-covalent ADP*P(i) species prior to the release of the gamma-phosphate group. After half-saturation with ATP, a negative cooperativity in hydrolysis is observed for both thermosomes. The effect of Mg(2+) and K(+) nucleotide cycling is documented. We conclude that archaeal chaperonins have unique allosteric properties and discuss them in the light of the mechanism established for the group I chaperonins.     

Coexistence of group I and group II chaperonins in the archaeon Methanosarcina mazei

Two distantly related classes of cylindrical chaperonin complexes assist in the folding of newly synthesized and stress-denatured proteins in an ATP-dependent manner. Group I chaperonins are thought to be restricted to the cytosol of bacteria and to mitochondria and chloroplasts, whereas the group II chaperonins are found in the archaeal and eukaryotic cytosol. Here we show that members of the archaeal genus Methanosarcina co-express both the complete group I (GroEL/GroES) and group II (thermosome/prefoldin) chaperonin systems in their cytosol. These mesophilic archaea have acquired between 20 and 35% of their genes by lateral gene transfer from bacteria. In Methanosarcina mazei G?1, both chaperonins are similarly abundant and are moderately induced under heat stress. The M. mazei GroEL/GroES proteins have the structural features of their bacterial counterparts. The thermosome contains three paralogous subunits, alpha, beta, and gamma, which assemble preferentially at a molar ratio of 2:1:1. As shown in vitro, the assembly reaction is dependent on ATP/Mg2+ or ADP/Mg2+ and the regulatory role of the beta subunit. The co-existence of both chaperonin systems in the same cellular compartment suggests the Methanosarcina species as useful model systems in studying the differential substrate specificity of the group I and II chaperonins and in elucidating how newly synthesized proteins are sorted from the ribosome to the proper chaperonin for folding.     

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.     

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.     

Gene duplication and the evolution of group II chaperonins: implications for structure and function

Chaperonins are multisubunit protein-folding assemblies. They are composed of two distinct structural classes, which also have a characteristic phylogenetic distribution. Group I chaperonins (called GroEL/cpn60/hsp60) are present in Bacteria and eukaryotic organelles while group II chaperonins are found in Archaea (called the thermosome or TF55) and the cytoplasm of eukaryotes (called CCT or TriC). Gene duplication has been an important force in the evolution of group II chaperonins: Archaea possess one, two, or three homologous chaperonin subunit-encoding genes, and eight distinct CCT gene families (paralogs) have been described in eukaryotes. Phylogenetic analyses indicate that while the duplications in archaeal chaperonin genes have occurred numerous times independently in a lineage-specific fashion, the eight different CCT subunits found in eukaryotes are the products of duplications that occurred early and very likely only once in the evolution of the eukaryotic nuclear genome. Analyses of CCT sequences from diverse eukaryotic species reveal that each of the CCT subunits possesses a suite of invariant subunit-specific amino acid residues ("signatures"). When mapped onto the crystal structure of the archaeal chaperonin from Thermoplasma acidophilum, these signatures are located in the apical, intermediate, and equatorial domains. Regions that were found to be variable in length and/or amino acid sequence were localized primarily to the exterior of the molecule and, significantly, to the extreme tip of the apical domain (the "helical protrusion"). In light of recent biochemical and electron microscopic data describing specific CCT-substrate interactions, our results have implications for the evolution of subunit-specific functions in CCT.     

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.     

Conformational rearrangements of an archaeal chaperonin upon ATPase cycling

Chaperonins are double-ring protein assemblies with a central cavity that provides a sequestered environment for in vivo protein folding. Their reaction cycle is thought to consist of a nucleotide-regulated alternation between an open substrate-acceptor state and a closed folding-active state. The cavity of ATP-charged group I chaperonins, typified by Escherichia coli GroEL [1], is sealed off by a co-chaperonin, whereas group II chaperonins--the archaeal thermosome and eukaryotic TRiC/CCT [2]--possess a built-in lid [3-5]. The mechanism of the lid's rearrangements requires clarification, as even in the absence of nucleotides, thermosomes of Thermoplama acidophilum appear open in vitrified ice [6] and closed in crystals [4]. Here we analyze the conformation of the thermosome at each step of the ATPase cycle by small-angle neutron scattering. The apo-chaperonin is open in solution, and ATP binding induces its further expansion. Closure seems to occur during ATP hydrolysis and before phosphate release, and represents the rate-limiting step of the cycle. The same closure can be triggered by the crystallization buffer. Thus, the allosteric regulation of group II chaperonins appears different from that of their group I counterparts.     

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.     

Domain rotations between open, closed and bullet-shaped forms of the thermosome, an archaeal chaperonin

Three conformations of the thermosome, an archaeal group II chaperonin, have been determined by cryo-electron microscopy (EM). We describe an open form of the double-ring oligomer, a closed form and a bullet-shaped form with one ring open and the other closed. Domain movements have been deduced by docking atomic coordinates into the EM maps. The subunit apical domains, bearing the putative substrate binding sites, rotate about 30 degrees upwards and twist in the plane of the ring from the closed to the open conformation. The closed rings have their nucleotide binding pockets closed by the intermediate domains, but in the open rings, the pocket is accessible.     

Cryo-EM structure of a group II chaperonin in the prehydrolysis ATP-bound state leading to lid closure

Chaperonins are large ATP-driven molecular machines that mediate cellular protein folding. Group II chaperonins use their "built-in lid" to close their central folding chamber. Here we report the structure of an archaeal group II chaperonin in its prehydrolysis ATP-bound state at subnanometer resolution using single particle cryo-electron microscopy (cryo-EM). Structural comparison of Mm-cpn in ATP-free, ATP-bound, and ATP-hydrolysis states reveals that ATP binding alone causes the chaperonin to close slightly with a ～45° counterclockwise rotation of the apical domain. The subsequent ATP hydrolysis drives each subunit to rock toward the folding chamber and to close the lid completely. These motions are attributable to the local interactions of specific active site residues with the nucleotide, the tight couplings between the apical and intermediate domains within the subunit, and the aligned interactions between two subunits across the rings. This mechanism of structural changes in response to ATP is entirely different from those found in group I chaperonins.     

Different mechanistic requirements for prokaryotic and eukaryotic chaperonins: a lattice study

MOTIVATION: The folding of many proteins in vivo and in vitro is assisted by molecular chaperones. A well-characterized molecular chaperone system is the chaperonin GroEL/GroES from Escherichia coli which has a homolog found in the eukaryotic cytosol called CCT. All chaperonins have a ring structure with a cavity in which the substrate protein folds. An interesting difference between prokaryotic and eukaryotic chaperonins is in the nature of the ATP-mediated conformational changes that their ring structures undergo during their reaction cycle. Prokaryotic chaperonins are known to exhibit a highly cooperative concerted change of their cavity surface while in eukaryotic chaperonins the change is sequential. Approximately 70% of proteins in eukaryotic cells are multi-domain whereas in prokaryotes single-domain proteins are more common. Thus, it was suggested that the different modes of action of prokaryotic and eukaryotic chaperonins can be explained by the need of eukaryotic chaperonins to facilitate folding of multi-domain proteins. RESULTS: Using a 2D square lattice model, we generated two large populations of single-domain and double-domain substrate proteins. Chaperonins were modeled as static structures with a cavity wall with which the substrate protein interacts. We simulated both concerted and sequential changes of the cavity surfaces and demonstrated that folding of single-domain proteins benefits from concerted but not sequential changes whereas double-domain proteins benefit also from sequential changes. Thus, our results support the suggestion that the different modes of allosteric switching of prokaryotic and eukaryotic chaperonin rings have functional implications as it enables eukaryotic chaperonins to better assist multi-domain protein folding.     

Functional characterization of an archaeal GroEL/GroES chaperonin system: significance of substrate encapsulation

In all three kingdoms of life chaperonins assist the folding of a range of newly synthesized proteins. As shown recently, Archaea of the genus Methanosarcina contain both group I (GroEL/GroES) and group II (thermosome) chaperonins in the cytosol. Here we report on a detailed functional analysis of the archaeal GroEL/GroES system of Methanosarcina mazei (Mm) in comparison to its bacterial counterpart from Escherichia coli (Ec). We find that the groESgroEL operon of M. mazei is unable to functionally replace groESgroEL in E. coli. However, the MmGroES protein can largely complement a mutant EcGroES protein in vivo. The ATPase rate of MmGroEL is very low and the dissociation of MmGroES from MmGroEL is 15 times slower than for the EcGroEL/GroES system. This slow ATPase cycle results in a prolonged enclosure time for model substrate proteins, such as rhodanese, in the MmGroEL:GroES folding cage before their release into the medium. Interestingly, optimal functionality of MmGroEL/GroES and its ability to encapsulate larger proteins, such as malate dehydrogenase, requires the presence of ammonium sulfate in vitro. In the absence of ammonium sulfate, malate dehydrogenase fails to be encapsulated by GroES and rather cycles on and off the GroEL trans ring in a non-productive reaction. These results indicate that the archaeal GroEL/GroES system has preserved the basic encapsulation mechanism of bacterial GroEL and suggest that it has adjusted the length of its reaction cycle to the slower growth rates of Archaea. Additionally, the release of only the folded protein from the GroEL/GroES cage may prevent adverse interactions of the GroEL substrates with the thermosome, which is not normally located within the same compartment.     

Salt bridges at the inter-ring interface regulate the thermostat of GroEL

The chaperonin GroEL consists of a double-ring structure made of identical subunits and displays unusual allosteric properties caused by the interaction between its constituent subunits. Cooperative binding of ATP to a protein ring allows binding of GroES to that ring, and at the same time negative inter-ring cooperativity discharges the ligands from the opposite ring, thus driving the protein-folding cycle. Biochemical and electron microscopy analysis of wild type GroEL, a single-ring mutant (SR1), and two mutants with one inter-ring salt bridge of the chaperonin disrupted (E461K and E434K) indicate that these ion pairs form part of the interactions that allow the inter-ring allosteric signal to be transmitted. The wild type-like activities of the ion pair mutants at 25 degrees C are in contrast with their lack of inter-ring communication and folding activity at physiological temperatures. These salt bridges stabilize the inter-ring interface and maintain the inter-ring spacing so that functional communication between protein heptamers takes place. The characterization of GroEL hybrids containing different amounts of wild type and mutant subunits also indicates that as the number of inter-ring salt bridges increases the functional properties of the hybrids recover. Taken together, these results strongly suggest that inter-ring salt bridges form a stabilizing ring-shaped, ionic zipper that ensures inter-ring communication at the contact sites and therefore a functional protein-folding cycle. Furthermore, they regulate the chaperonin thermostat, allowing GroEL to distinguish physiological (37 degrees C) from stress temperatures (42 degrees C).     

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

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

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.     

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.     

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.     

Crystal Structures of a Group II Chaperonin Reveal the Open and Closed States Associated with the Protein Folding Cycle

Chaperonins are large protein complexes consisting of two stacked multisubunit rings, which open and close in an ATP-dependent manner to create a protected environment for protein folding. Here, we describe the first crystal structure of a group II chaperonin in an open conformation. We have obtained structures of the archaeal chaperonin from Methanococcus maripaludis in both a peptide acceptor (open) state and a protein folding (closed) state. In contrast with group I chaperonins, in which the equatorial domains share a similar conformation between the open and closed states and the largest motions occurs at the intermediate and apical domains, the three domains of the archaeal chaperonin subunit reorient as a single rigid body. The large rotation observed from the open state to the closed state results in a 65% decrease of the folding chamber volume and creates a highly hydrophilic surface inside the cage. These results suggest a completely distinct closing mechanism in the group II chaperonins as compared with the group I chaperonins.