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.     

Nested cooperativity and salt dependence of the ATPase activity of the archaeal chaperonin Mm-cpn

The properties of the ATPase activity of the type II chaperonin from Methanococcus maripaludis (Mm-cpn) were examined. Mm-cpn can hydrolyze not only ATP, but also CTP, UTP, and GTP, albeit with different effectiveness. The ATPase activity is dependent on magnesium and potassium ions, and is effectively inhibited by sodium ions. Maximal rates of ATP hydrolysis are achieved at 600 mM potassium. Initial rates of ATP hydrolysis by Mm-cpn were determined at various ATP concentrations, revealing for the first time the presence of both positive intra-ring and negative inter-ring cooperativity in the archaeal chaperonin.     

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.     

Nucleotide specificity of an archaeal group II chaperonin from Thermococcus strain KS-1 with reference to the ATP-dependent protein folding cycle

The archaeal chaperonin-mediated folding of green fluorescent protein (GFP) was examined in the presence of various nucleotides. The recombinant alpha- and beta-subunit homo-oligomers and natural chaperonin oligomer from Thermococcus strain KS-1 exhibited folding activity with not only ATP but also with CTP, GTP, or UTP. The ADP-bound form of both recombinant and natural chaperonin had the ability to capture non-native GFP, but could not refold it in the presence of CTP, GTP or UTP until ATP was supplied. The archaeal chaperonin thus utilized ATP, but could not use other nucleoside triphosphates in the cytoplasm where ADP was present.     

Oligomerization of an archaeal group II chaperonin is mediated by N-terminal salt bridges

Group II chaperonins (Cpns) are essential mediators of cellular protein folding in eukaryotes and archaea. They consist of two back-to-back rings forming symmetrical cavities in which non-native substrates undergo appropriate folding, but the primary structural basis for the double ring formation remains unclear. To address this, we carried out systematic mutagenesis on the Cpn from the hyperthermophilic archaeon Pyrococcus furiosus, which is assembled from identical subunits. In our study, ²¹GRDAQRMNIL³⁰ was found to be a critical domain for double ring formation. Deletion of this section stepwise beyond residue 20 resulted in failure to assemble double-ring oligomers and the progressive loss of chaperone function. A key domain spanning the residues 21–50 that is essential for the formation of tetramers that appear to be the intermediates for double ring assembly. Mutation of either Arg22 to Ala22 or Glu37 to Ala37 resulted in similar defects in double-ring assembly and functional deficits. A mutant with Arg22 and Glu37 switched assembled double rings efficiently and exhibited chaperone functions similar to the wild-type. Therefore, Arg22 and Glu37 could form inter-ring salt bridges critical for double ring formation. In addition, Asn28 and Ile29 were found to contribute significantly to ring formation. Sequence alignment revealed that these four residues are highly conserved among group II Cpns. This is the first report of a comprehensive N-terminal mutational analysis for elucidating the oligomerization of group II Cpns.     

Rapid degradation kinetics of amyloid fibrils under mild conditions by an archaeal chaperonin

Amyloid depositions containing exceptionally stable β-sheet rich protein aggregates, called fibrils are associated with prevalent and incurable neurodegenerative diseases. Chaperones are proteins that facilitate protein folding in both eukaryotes and prokaryotes. We found that a cold-adapted mutant ATP-dependant chaperonins (Hsp60) from a hyperthermophilic archaeon binds to and fragments insulin fibrils very rapidly with local targeted entry points. Individual fragments swell and the fibrillar β-sheet is quickly transformed into a mix of α-helical and unordered protein structures. After further incubation, the fragments coalesced, forming large amorphous aggregates with poly-disperse topologies. This finding represents a new approach to the disassembly of refractory protein aggregates under physiological conditions.     

ATPase site architecture and helicase mechanism of an archaeal MCM

The subunits of the presumptive replicative helicase of archaea and eukaryotes, the MCM complex, are members of the AAA+ (ATPase-associated with various cellular activities) family of ATPases. Proteins within this family harness the chemical energy of ATP hydrolysis to perform a broad range of cellular processes. Here, we investigate the function of the AAA+ site in the mini-chromosome maintenance (MCM) complex of the archaeon Sulfolobus solfataricus (SsoMCM). We find that SsoMCM has an unusual active-site architecture, with a unique blend of features previously found only in distinct families of AAA+ proteins. We additionally describe a series of mutant doping experiments to investigate the mechanistic basis of intersubunit coordination in the generation of helicase activity. Our results indicate that MCM can tolerate catalytically inactive subunits and still function as a helicase, leading us to propose a semisequential model for helicase activity of this complex.     

Insights into chaperonin function from studies on archaeal thermosomes

It is now well understood that, although proteins fold spontaneously (in a thermodynamic sense), many nevertheless require the assistance of helpers called molecular chaperones to reach their correct and active folded state in living cells. This is because the pathways of protein folding are full of traps for the unwary: the forces that drive proteins into their folded states can also drive them into insoluble aggregates, and, particularly when cells are stressed, this can lead, without prevention or correction, to cell death. The chaperonins are a family of molecular chaperones, practically ubiquitous in all living organisms, which possess a remarkable structure and mechanism of action. They act as nanoboxes in which proteins can fold, isolated from their environment and from other partners with which they might, with potentially deleterious consequences, interact. The opening and closing of these boxes is timed by the binding and hydrolysis of ATP. The chaperonins which are found in bacteria are extremely well characterized, and, although those found in archaea (also known as thermosomes) and eukaryotes have received less attention, our understanding of these proteins is constantly improving. This short review will summarize what we know about chaperonin function in the cell from studies on the archaeal chaperonins, and show how recent work is improving our understanding of this essential class of molecular chaperones.     

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.     

Testing the neutral fixation of hetero-oligomerism in the archaeal chaperonin CCT

The evolutionary transition from homo-oligomerism to hetero-oligomerism in multimeric proteins and its contribution to function innovation and organism complexity remain to be investigated. Here, we undertake the challenge of contributing to this theoretical ground by investigating the hetero-oligomerism in the molecular chaperonin cytosolic chaperonin containing tailless complex polypeptide 1 (CCT) from archaea. CCT is amenable to this study because, in contrast to eukaryotic CCTs where sub-functionalization after gene duplication has been taken to completion, archaeal CCTs present no evidence for subunit functional specialization. Our analyses yield additional information to previous reports on archaeal CCT paralogy by identifying new duplication events. Analyses of selective constraints show that amino acid sites from 1 subunit have fixed slightly deleterious mutations at inter-subunit interfaces after gene duplication. These mutations have been followed by compensatory mutations in nearby regions of the same subunit and in the interface contact regions of its paralogous subunit. The strong selective constraints in these regions after speciation support the evolutionary entrapment of CCTs as hetero-oligomers. In addition, our results unveil different evolutionary dynamics depending on the degree of CCT hetero-oligomerism. Archaeal CCT protein complexes comprising 3 distinct classes of subunits present 2 evolutionary processes. First, slightly deleterious and compensatory mutations were fixed neutrally at inter-subunit regions. Second, sub-functionalization may have occurred at substrate-binding and adenosine triphosphate-binding regions after the 2nd gene duplication event took place. CCTs with 2 distinct types of subunits did not present evidence of sub-functionalization. Our results provide the 1st in silico evidence for the neutral fixation of hetero-oligomerism in archaeal CCTs and provide information on the evolution of hetero-oligomerism toward sub-functionalization in archaeal CCTs.     

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.     

The chaperonin cycle and protein folding

The process of protein folding in the cell is now known to depend on the action of other proteins. These proteins include molecular chaperones, Which interact non-covalently with proteins as they fold and improve the final yields of active protein in the cell. The precise mechanism by which molecular chaperones act is obscure. Experiments reported recently⁽¹⁾ show that for one molecular chaperone (Cpn60, typified by the E. coli protein GroEL), the folding reaction is driven by cycles of binding and release of the co-chaperone Cpn10 (known as GroES in E. coli). These alternate with binding and release of the unfolded protein substrate. These cycles come about because of the opposite effects of Cpn10 and unfolded protein on the Cpn60 complex: the former stabilises the ADP-bound state of Cpn60, whereas the latter stimulates ADP-ATP exchange. This model proposes that the substrate protein goes through multiple cycles of binding and release, and is released into the cavity of the Cpn60 complex where it can undergo folding without interacting with other nearby folding intermediates. This is consistent with the ability of Cpn60 proteins to enhance folding by blocking pathways to aggregation.     

Two kinds of archaeal chaperonin with different temperature dependency from a hyperthermophile

Thermococcus kodakaraensis KOD1 produces two kinds of chaperonin subunits, CpkA and CpkB. To monitor the expression levels of CpkA and CpkB, anti-CpkA and anti-CpkB antisera were obtained by using synthesized peptides as the haptens. These haptens were prepared based on the carboxyl terminus regions of CpkA and CpkB, which show clear differences in amino acid sequence. Immunoblotting analysis using obtained antisera revealed that the expression levels of CpkA and CpkB changed depending on the cultivation temperature. When cells were grown at 95 degrees C, intracellular amount of CpkA was low, while CpkB was expressed at extremely high level in KOD1. In the case of 70 degrees C cultivation, CpkA existed as the major chaperonin in the cell, whereas CpkB existed as the minor one. Temperature-shift experiments showed that the expression of CpkB was induced by the up-shift and reduced by the down-shift of temperature. In contrast, the expression of CpkA was reduced by the up-shift and induced by the down-shift of temperature. Furthermore, native PAGE and immunoprecipitation experiments revealed that the stoichiometrical ratio of CpkA and CpkB in chaperonin complex changed according to growth temperature.     

GroEL-substrate-GroES ternary complexes are an important transient intermediate of the chaperonin cycle

GroEL C138W is a mutant form of Escherichia coli GroEL, which forms an arrested ternary complex composed of GroEL, the co-chaperonin GroES and the refolding protein molecule rhodanese at 25 degrees C. This state of arrest could be reversed with a simple increase in temperature. In this study, we found that GroEL C138W formed both stable trans- and cis-ternary complexes with a number of refolding proteins in addition to bovine rhodanese. These complexes could be reactivated by a temperature shift to obtain active refolded protein. The simultaneous binding of GroES and substrate to the cis ring suggested that an efficient transfer of substrate protein into the GroEL central cavity was assured by the binding of GroES prior to complete substrate release from the apical domain. Stopped-flow fluorescence spectroscopy of the mutant chaperonin revealed a temperature-dependent conformational change in GroEL C138W that acts as a trigger for complete protein release. The behavior of GroEL C138W was reflected closely in its in vivo characteristics, demonstrating the importance of this conformational change to the overall activity of GroEL.     

Single-molecule fluorescence polarization study of conformational change in archaeal group II chaperonin

Group II chaperonins found in archaea and in eukaryotic cytosol mediate protein folding without a GroES-like cofactor. The function of the cofactor is substituted by the helical protrusion at the tip of the apical domain, which forms a built-in lid on the central cavity. Although many studies on the change in lid conformation coupled to the binding and hydrolysis of nucleotides have been conducted, the molecular mechanism of lid closure remains poorly understood. Here, we performed a single-molecule polarization modulation to probe the rotation of the helical protrusion of a chaperonin from a hyperthermophilic archaeum, Thermococcus sp. strain KS-1. We detected approximately 35° rotation of the helical protrusion immediately after photorelease of ATP. The result suggests that the conformational change from the open lid to the closed lid state is responsible for the approximately 35° rotation of the helical protrusion.     

A novel cochaperonin that modulates the ATPase activity of cytoplasmic chaperonin

The folding of alpha- and beta-tubulin requires three proteins: the heteromeric TCP-1-containing cytoplasmic chaperonin and two additional protein cofactors (A and B). We show that these cofactors participate in the folding process and do not merely trigger release, since in the presence of Mg-ATP alone, alpha- and beta-tubulin target proteins are discharged from cytoplasmic chaperonin in a nonnative form. Like the prokaryotic cochaperonin GroES, which interacts with the prototypical Escherichia coli chaperonin GroEL and regulates its ATPase activity, cofactor A modulates the ATPase activity of its cognate chaperonin. However, the sequence of cofactor A derived from a cloned cDNA defines a 13-kD polypeptide with no significant homology to other known proteins. Moreover, while GroES functions as a heptameric ring, cofactor A behaves as a dimer. Thus, cofactor A is a novel cochaperonin that is structurally unrelated to GroES.     

ATP binding is critical for the conformational change from an open to closed state in archaeal group II chaperonin

Group II chaperonins, found in archaea and in eukaryotic cytosol, do not have a co-chaperonin corresponding to GroES. Instead, it is suggested that the helical protrusion extending from the apical domain acts as a built-in lid for the central cavity and that the opening and closing of the lid is regulated by ATP binding and hydrolysis. However, details of this conformational change remain unclear. To investigate the conformational change associated with the ATP-driven cycle, we conducted protease sensitivity analyses and tryptophan fluorescence spectroscopy of alpha-chaperonin from a hyperthermophilic archaeum, Thermococcus strain KS-1. In the nucleotide-free or ADP-bound state, the chaperonin, especially in the helical protrusion region, was highly sensitive to proteases. Addition of ATP and ammonium sulfate induced the transition to the relatively protease-resistant form. The fluorescence intensity of the tryptophan residue introduced at the tip of the helical protrusion was enhanced by the presence of ATP or ammonium sulfate. We conclude that ATP binding induces the conformational change from the lid-open to lid-closed form in archaeal group II chaperonin.     

Analysis of the interaction mode between hyperthermophilic archaeal group II chaperonin and prefoldin using a platform of chaperonin oligomers of various subunit arrangements

Prefoldin is a co-chaperone that captures an unfolded protein substrate and transfers it to the group II chaperonin for completion of protein folding. Group II chaperonin of a hyperthermophilic archaeon, Thermococcus strain KS-1, interacts and cooperates with archaeal prefoldins. Although the interaction sites within chaperonin and prefoldin have been analyzed, the binding mode between jellyfish-like hexameric prefoldin and the double octameric ring group II chaperonin remains unclear. As prefoldin binds the chaperonin β subunit more strongly than the α subunit, we analyzed the binding mode between prefoldin and chaperonin in the context of Thermococcus group II chaperonin complexes of various subunit compositions and arrangements. The oligomers exhibited various affinities for prefoldins according to the number and order of subunits. Binding affinity increased with the number of Cpnβ subunits. Interestingly, chaperonin complexes containing two β subunits adjacently exhibited stronger affinities than other chaperonin complexes containing the same number of β subunits. The result suggests that all four β tentacles of prefoldin interact with the helical protrusions of CPN in the PFD–CPN complex as the previously proposed model that two adjacent PFD β subunits seem to interact with two CPN adjacent subunits.     

The archaeal cell cycle: current issues

The recently discovered structural similarities between the archaeal Orc1/Cdc6 and bacterial DnaA initiator proteins for chromosome replication have exciting implications for cell cycle regulation. Together with current attempts to identify archaeal chromosome replication origins, the information is likely to yield fundamental insights into replication control in both archaea and eukaryotes within the near future. Several proteins that affect, or are likely to affect, chromatin structure and genome segregation in archaea have been described recently, including Sph1 and 2, ScpA and B, Sir2, Alba and Rio1p. Important insights into the properties of the MinD and FtsZ cell division proteins, and of putative cytoskeletal elements, have recently been gained in bacteria. As these proteins also are present among archaea, it is likely that the new information will also be essential for understanding archaeal genome segregation and cell division. A series of interesting cell cycle issues has been brought to light through the discovery of the novel Nanoarchaeota phylum, and these are outlined briefly. Exciting areas for extended cell cycle investigations of archaea are identified, including termination of chromosome replication, application of in situ cytological techniques for localization of cell cycle proteins and the regulatory roles of GTP-binding proteins and small RNAs.