Insights into chaperonin function from studies on ... - Semantic Scholar

94

Biochemical Society Transactions (2011) Volume 39, part 1

Insights into chaperonin function from studies on archaeal thermosomes Peter Lund1 School of Biosciences, University of Birmingham, Birmingham B15 2TT, U.K.

Abstract 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.

The roles of molecular chaperones A classic experiment, published in 1988, was one of the earliest demonstrations of the cellular role of molecular chaperones [1]. In this experiment, a strain of Escherichia coli was constructed lacking a gene called rpoH, which encodes an RNA polymerase subunit that, when present, leads to transcription of the E. coli heat-shock genes. Lack of rpoH leads unsurprisingly to temperature-sensitivity, but it was perhaps unexpected to discover that such strains cannot grow above 20◦ C, well below the optimal growth temperature for E. coli, thus showing that the genes of the heat-shock response are expressed and required for growth at normal temperatures. This study posed the question what mutations are found if strains that lacked rpoH are selected for growth at temperatures above 20◦ C? The answer was that such mutants were found to overexpress an operon which encodes two of the heat-shock genes, groES and groEL; the higher the temperature that was used for selection, the greater the degree of overexpression that was found. A subsequent study showed that many cellular proteins fail to fold in strains lacking rpoH, and that the overexpression of the groES and groEL genes largely suppresses this defect [2]. Before this paper was published, a separate study had shown that the bacterial groEL gene was homologous with a chloroplast protein [Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase)-binding protein] which was required for the assembly of oligomeric ribulose bisphosphate Key words: archaeon, chaperonin, heat shock, molecular chaperone, protein folding, thermosome. Abbreviations used: Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase. 1 email [email protected]

C The

C 2011 Biochemical Society Authors Journal compilation

carboxylase, an important and significant finding in the days before the onset of complete genomes and BLAST servers [3]. Subsequently, it was shown that the GroEL and GroES proteins could help Rubisco subunits to fold in an ATPdependent fashion [4,5]. The name ‘molecular chaperone’ was proposed for proteins which could help other proteins to fold, and, in many subsequent experiments, large numbers of such chaperones have been identified and studied. For a newly discovered protein to be admitted to the rather exclusive molecular chaperone club, it should be shown that it does indeed assist in protein folding both in vivo and in vitro. In vivo experiments usually involve the study of mutated strains or organisms to see whether specific proteins fail to fold (and are hence either degraded or found in insoluble aggregates) when the gene for a particular molecular chaperone is deleted. In vitro experiments involve reconstitution of the folding reaction for suitable client proteins of the molecular chaperone with purified components, showing that the client, when starting from its denatured state, becomes aggregated, or at least fails to fold, in the absence of the molecular chaperone, and that aggregation is blocked or reversed, and ideally folding is enhanced, in the chaperone’s presence. Such experiments are often called neo-Anfinsen experiments, as they are extensions of the Nobel prize-winning work of Peter Anfinsen, which showed that denatured proteins would refold spontaneously to their native state when the denaturant was removed [6]. The existence of molecular chaperones does not violate Anfinsen’s original conclusion that the native state of proteins represents the lowest free-energy state of the protein. The problem with protein folding inside the cell is kinetic, not thermodynamic: Biochem. Soc. Trans. (2011) 39, 94–98; doi:10.1042/BST0390094

Molecular Biology of Archaea II

proteins which fold in the highly crowded intracellular milieu may finish up in aggregates with other proteins, largely because hydrophobic residues on a folding polypeptide chain make inappropriate contacts with other similar residues on other polypeptides which are folding in close proximity, rather than with hydrophobic residues on the same chain [7,8]. The main roles of molecular chaperones is to block the formation of these aggregates or other misfolded structures, or to reverse their formation if they are formed.

The chaperonins Among the many classes of molecular chaperone which have been defined by sequence homology, the GroEL protein belongs to a group referred to as the chaperonins. These proteins are found in nearly all organisms, a few mycoplasmas being the only known exceptions. All are approx. 60 kDa in size, and they break down phylogenetically into two related but clearly distinct groups: the group I chaperonins, found in bacteria and organelles of endosymbiotic descent, and the group II chaperonins, found in archaea and the eukaryotic cytosol. Chaperonin proteins assemble into large oligomeric complexes consisting of two stacked rings of subunits, each enclosing a central cavity which is the site where their client proteins fold. The group I chaperonins all have rings with 7-fold symmetry, whereas the group II chaperonins are 8- or 9-fold symmetrical [9,10]. The structures of several of these complexes have been solved with atomic resolution and they have been subject to significant genetic, biochemical and biophysical dissection. The way in which the group I chaperonins function is quite well understood, although the precise mechanisms of their action is not without controversy; the group II chaperonins are less well studied, but certain features of both their mechanism of action and their in vivo roles are beginning to emerge. Chaperonins act by sequestering early protein-folding intermediates within the central cavity, where they are unlikely to aggregate, given the absence of any other folding proteins. Whether this process is entirely passive or whether the chaperonin has a more active role to play is still under study [10,11]. For the group I chaperonins, the reaction involves the binding of a client protein to hydrophobic patches at the end of one of the rings (the cis ring), followed by the binding of ATP and the co-chaperonin GroES to the same ring, causing a large conformational change in the complex and the displacement of the client protein into the cavity. Folding can take place here for as long as GroES remains bound, capping the cavity, and this is timed by the hydrolysis of ATP, since negative co-operativity between the rings prevents anything binding to the opposite (trans) ring. Once ATP has been hydrolysed, which takes some seconds, client protein, ATP and GroES bind to the trans ring, and the cis ring-bound GroES dissociates, leaving the client free to leave the complex [10]. The basic mechanism of the group II chaperonins is thought to be broadly similar, but as archaea lack a co-chaperonin protein, an important question to address is how the folding cavity is closed during

the reaction cycle and whether the alternating action of the two rings is also part of the group II chaperonin mechanism. Structural studies, combined with neo-Anfinsen experiments on the archaeal chaperonins, provide the answers.

Structural and functional studies on thermosomes The first structure of a thermosome oligomer, from Thermoplasma acidophilum, showed an 8-fold symmetry with two rings of eight subunits each, arranged with alternating α and β subunits in the rings [12]. The fold of the protomers is very reminiscent of GroEL protomers, despite the relatively low sequence similarity between the two types, but a striking difference between the structures of the group I and group II chaperonins is the presence of a ‘helical protrusion’ of 33 residues which acts as lid to the cavities in the two rings, and hence is an excellent candidate for providing the function which GroES fulfils with the group I chaperonins. The first crystal structure was obtained in the presence of various different nucleotides or in the nucleotide-free state, but this lid was always closed. Other studies with different methods including cryoelectron microscopy and SANS (small-angle neutron scattering) [13– 16] showed the existence of an open form where at either one or both of the ends of the complex, the helical protusions were raised, opening the cavity (and also presumably exposing potential client protein binding sites). These studies did not distinguish the nature of the event that brings about the transition between the two forms, and this was unclear for some time, but this question appears to have been settled by the demonstration that a thermosome mutant that can bind, but not hydrolyse, ATP remains in the open conformation, strongly suggesting that ATP hydrolysis is required for lid closure [17]. Structural studies have revealed the existence of a partially closed state [18], which, it has been proposed, may be needed for folding clients that are too large to fit in the fully closed complex. A combination of single-particle cryoelectron microscopy, computational modelling and X-ray crystallogaphy has revealed structures for the thermosome from Methanococcus maripaludis both in the closed and the open state, which should facilitate the identification of key residues needed for binding of client proteins and progression through the reaction cycle [19,20] (Figure 1). The open structure was obtained for a form of the chaperonin where the regions which make up the lid were deleted, and replaced by a short amino acid linker. The properties of this mutated protein are revealing: it can still bind client protein and hydrolyse ATP, but it can no longer mediate the refolding of the protein in neo-Anfinsen-type experiments [17]. The reasons for this are surprisingly complex. The simplest way to consider the action of a chaperonin is as a single ring, which exists in two conformations: an open state which is competent to bind nucleotide and client proteins, and a closed state which is competent to fold them. Binding and hydrolysis of nucleotide by one ring is highly co-operative, which may be important in enabling C The


95

96


Figure 1 Top and side views of closed and open conformations of a lidless version of the M. maripaludis thermosome, based on single-particle cryo-electron microscopy and computational modelling as described in [19] The images were derived from PDB codes 3LOS and 3IYF respectively, visualized using PyMOL (DeLano Scientific; http://www.pymol.org). (A) Top view, closed; (B) side view, closed; (C) top view, open; (D) side view, open.

the co-ordinated movements in the chaperonin complex that occur during the reaction cycle, as well as ensuring nearly simultaneous release of bound client into the folding cavity (since it may initially be bound to more than one subunit of the ring). A second level of complexity is then introduced by considering the second ring and the allosteric connection between the two. In both group I and group II chaperonins, it has been shown that negative co-operativity exists between the two rings, such that the affinity of a ring for ATP is drastically lowered if the other ring has ATP bound [21,22]. This property forces the chaperonins to work as a twostroke motor where the folding of client proteins alternates C The


between the two rings. Remarkably, loss of the thermosome lid leads to loss of both intra- and inter-ring co-operativity, showing that the lid functions not only to cap the cavity where protein folding occurs, but also to regulate the progression of the folding complex through the reaction cycle, a feature which is unique to group II chaperonins [17].

Thermosomes: genetic and in vivo studies Genes encoding one or more thermosome subunits are found in all complete archaeal genomes which are currently available. A few also contain genes encoding group I

Molecular Biology of Archaea II

chaperonins, and a few group II chaperonin genes are found in bacterial genomes [23–25]. The number of genes varies from one to five, with two being the commonest number [24] Structural studies show that many thermosomes are heterooligomeric, but in the one case studied genetically (Haloferax volcanii, which has three chaperonin genes), cells remain viable even after two of these genes have been deleted [26]. If the remaining copy is placed under the control of a tightly repressible promoter which is then turned off by growth in the appropriate medium, cells grow for a few generations, but then stop and eventually lyse, proving that, for archaea (and consistent with results from bacteria and eukaryotes), chaperonins are essential for viability [27]. Phylogenetic evidence suggests that chaperonin gene duplication and loss is common in archaeal lineages, unlike in the eukaryotes, where eight distinct chaperonin genes arose very early in the eukaryotic lineage [28]. Moreover, the part of the gene encoding the region of the protein which is predicted to be involved in client protein recognition is subject to significant gene conversion, the implication being that, in archaea, the duplicated genes do not encode proteins with significant client specificity [29], in contrast with the eukaryotic proteins, where all eight genes have to be present for viability. However, it has been shown in the only genetic study to date that, of two chaperonin genes from Haloarcula morismortui that can be successfully expressed in the closely related H. volcanii, only one can replace the essential function of the H. volcanii chaperonins, implying that there may be some specialization of function in some archaeal chaperonins, which is also probably the case with the duplicated group I chaperonins found in many bacterial genomes [27,30]. Chaperonin genes are generally members of the heat-shock regulon in archaea, being transcribed under all conditions, but strongly up-regulated upon heat shock. Indeed, in some cases, they are virtually the only protein synthesized when the cells are subjected to heat shock [31–33]. The heat-shock regulon of archaea is surprisingly restricted when compared with that of most bacteria, and some common chaperones (such as homologues of the Hsp70, Hsp90 and Hsp100 genes found in bacteria and eukaryotes) are rare in, or absent from, many archaeal genomes [34]. The functional significance of this, and in particular the possibility that there are other chaperones unique to archaea, has not been explored. Chaperonins in bacteria are essential because several proteins which are essential for cell growth are among their client proteins [35,36]. Archaeal chaperonins are also essential, so this is presumably true also for archaea, but to date only one study has addressed this directly, and this was done in an organism (Methanosarcina mazei) which also expresses group I chaperonins, making this a rather special case [37]. In this study, co-immunoprecipitation experiments coupled with proteomics methods did detect approx. 250 potential client proteins for the thermosome in this organism, of which 14 were predicted to be essential. The presence of both group I and group II chaperonins in this organism enabled the authors to address an interesting question which was whether there was evidence for some

selectivity between the two chaperonin types, or whether they bound the same proteins. The answer was that, although many proteins were identified that were apparently bound by both chaperonins, a significant number were bound by one, but not the other, clearly suggesting some selectivity by the two different chaperonin families. In total, of the 252 thermosome clients identified, 150 (60 %) showed evidence of binding to the thermosome, but not to the endogenous group I chaperonins in M. mazei. This points to a degree of difference in the mode of binding of the two chaperonins, and indeed a bioinformatics analysis of the properties of the client sets showed the group II chaperonins to be capable of binding a wider range of fold types of clients than the group I chaperonins.

Summary and conclusions Good progress has been made in studies on the group II chaperonins in recent years, in terms of understanding their mechanism of action and their in vivo role, and the thermosomes have proven especially valuable as a model system both for the group II chaperonins and for chaperonins in general. However, the detailed reaction cycle is still better understood for the group I than for the group II chaperonins, and the fine details of the structural changes that occur during the reaction cycle, including the ways in which allosteric information is transmitted between the two rings, remain to be elucidated for both types. The improvement of genetic tools which are available for use in archaea means that a better understanding of the in vivo roles of the thermosomes is emerging, and it is now possible to probe their structure– function relationships with the combined power of detailed structural models and good in vivo methods.

Funding Work in my laboratory on archaeal thermosomes has been supported by the Biotechnology and Biological Sciences Research Council [grant numbers C15311 and BBF0024831] and by research studentships from the Darwin Trust of Edinburgh.

References 1 Kusukawa, N. and Yura, T. (1988) Heat shock protein GroE of Escherichia coli: key protective roles against thermal stress. Genes Dev. 2, 874–884 2 Gragerov, A., Nudler, E., Komissarova, N., Gaitanaris, G.A., Gottesman, M.E. and Nikiforov, V. (1992) Cooperation of GroEL/GroES and DnaK/DnaJ heat shock proteins in preventing protein misfolding in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 89, 10341–10344 3 Hemmingsen, S.M., Woolford, C., Van Der Vies, S.M., Tilly, K., Dennis, D.T., Georgopoulos, C.P., Hendrix, R.W. and Ellis, R.J. (1988) Homologous plant and bacterial proteins chaperone oligomeric protein assembly. Nature 333, 330–334 4 Goloubinoff, P., Gatenby, A.A. and Lorimer, G.H. (1989) GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escherichia coli. Nature 337, 44–47 5 Goloubinoff, P., Christeller, J.T., Gatenby, A.A. and Lorimer, G.H. (1989) Reconstitution of active dimeric ribulose bisphosphate carboxylase from an unfoleded state depends on two chaperonin proteins and Mg-ATP. Nature 342, 884–889 6 Anfinsen, C.B. (1973) Principles that govern the folding of protein chains. Science 181, 223–230 C The


97

98


7 Ellis, R.J. (1997) Molecular chaperones: avoiding the crowd. Curr. Biol. 7, 531–533 8 Ellis, R.J. (2001) Macromolecular crowding: obvious but underappreciated. 26, 597–604 9 Sigler, P.B., Xu, Z.H., Rye, H.S., Burston, S.G., Fenton, W.A. and Horwich, A.L. (1998) Structure and function in GroEL-mediated protein folding. Annu. Rev. Biochem. 67, 581–608 10 Horwich, A.L., Fenton, W.A., Chapman, E. and Farr, G.W. (2007) Two families of chaperonin: physiology and mechanism. Annu. Rev. Cell Dev. Biol. 23, 115–145 11 Horwich, A.L. and Fenton, W.A. (2009) Chaperonin-mediated protein folding: using a central cavity to kinetically assist polypeptide chain folding. Q. Rev. Biophys. 42, 83–116 12 Ditzel, L., Lowe, J., Stock, D., Stetter, K.O., Huber, H., Huber, R. and Steinbacher, S. (1998) Crystal structure of the thermosome, the archaeal chaperonin and homolog of CCT. Cell 93, 125–138 13 Quaite-Randall, E., Trent, J.D., Josephs, R. and Joachimiak, A. (1995) Conformational cycle of the archaeosome, a TCP1-like chaperonin from Sulfolobus shibatae. J. Biol. Chem. 270, 28818–28823 14 Nitsch, M., Walz, J., Typke, D., Klumpp, M., Essen, L.O. and Baumeister, W. (1998) Group II chaperonin in an open conformation examined by electron tomography. Nat. Struct. Biol. 5, 855–857 15 Schoehn, G., Hayes, M., Cliff, M., Clarke, A.R. and Saibil, H.R. (2000) Domain rotations between open, closed and bullet-shaped forms of the thermosome, an archaeal chaperonin. J. Mol. Biol. 301, 323–332 16 Schoehn, G., Quaite-Randall, E., Jimenez, ´ J.L., Joachimiak, A. and Saibil, H.R. (2000) Three conformations of an archaeal chaperonin, TF55 from Sulfolobus shibatae. J. Mol. Biol. 296, 813–819 17 Reissmann, S., Parnot, C., Booth, C.R., Chiu, W. and Frydman, J. (2007) Essential function of the built-in lid in the allosteric regulation of eukaryotic and archaeal chaperonins. Nat. Struct. Mol. Biol. 14, 432–440 18 Clare, D.K., Stagg, S., Quispe, J., Farr, G.W., Horwich, A.L. and Saibil, H.R. (2008) Multiple states of a nucleotide-bound group 2 chaperonin. Structure 16, 528–534 19 Zhang, J., Baker, M.L., Schroder, ¨ G.F., Douglas, N.R., Reissmann, S., Jakana, J., Dougherty, M., Fu, C.J., Levitt, M., Ludtke, S.J. et al. (2010) Mechanism of folding chamber closure in a group II chaperonin. Nature 463, 379–383 20 Pereira, J.H., Ralston, C.Y., Douglas, N.R., Meyer, D., Knee, K.M., Goulet, D.R., King, J.A., Frydman, J. and Adams, P.D. (2010) Crystal structures of a group II chaperonin reveal the open and closed states associated with the protein folding cycle. J. Biol. Chem. 285, 27958–27966 21 Yifrach, O. and Horovitz, A. (1995) Nested cooperativity in the ATPase activity of the oligomeric chaperonin GroEL. Biochemistry 34, 5303–5308 22 Bigotti, M.G. and Clarke, A.R. (2005) Cooperativity in the thermosome. J. Mol. Biol. 348, 13–26 23 Klunker, D., Haas, B., Hirtreiter, A., Figueiredo, L., Naylor, D.J., Pfeifer, G., Muller, ¨ V., Deppenmeier, U., Gottschalk, G., Hartl, F.U. and Hayer-Hartl, M. (2003) Coexistence of group I and group II chaperonins in the archaeon Methanosarcina mazei. J. Biol. Chem. 278, 33256–33267

C The


24 Large, A.T. and Lund, P.A. (2009) Archaeal chaperonins. Front. Biosci. 14, 1304–1324 25 Williams, T.A., Codoner, ˜ F.M., Toft, C. and Fares, M.A. (2010) Two chaperonin systems in bacterial genomes with distinct ecological roles. Trends Genet. 26, 47–51 26 Kapatai, G., Large, A., Benesch, J.L., Robinson, C.V., Carrascosa, J.L., Valpuesta, J.M., Gowrinathan, P. and Lund, P.A. (2006) All three chaperonin genes in the archaeon Haloferax volcanii are individually dispensable. Mol. Microbiol. 61, 1583–1597 27 Large, A., Stamme, C., Lange, C., Duan, Z., Allers, T., Soppa, J. and Lund, P.A. (2007) Characterization of a tightly controlled promoter of the halophilic archaeon Haloferax volcanii and its use in the analysis of the essential cct1 gene. Mol. Microbiol. 66, 1092–1106 28 Archibald, J.M., Logsdon, J.M. and Doolittle, W.F. (1999) Recurrent paralogy in the evolution of archaeal chaperonins. Curr. Biol. 9, 1053–1056 29 Archibald, J.M. and Roger, A.J. (2002) Gene duplication and gene conversion shape the evolution of archaeal chaperonins. J. Mol. Biol. 316, 1041–1050 30 Lund, P.A. (2009) Multiple chaperonins in bacteria: why so many? FEMS Microbiol. Rev. 33, 784–790 31 Trent, J.D., Osipiuk, J. and Pinkau, T. (1990) Acquired thermotolerance and heat shock in the extremely thermophilic archaebacterium Sulfolobus sp. strain B12. J. Bacteriol. 172, 1478–1484 32 Trent, J.D., Nimmesgern, E., Wall, J.S., Hartl, F.U. and Horwich, A.L. (1991) A molecular chaperone from a thermophilic archaebacterium is related to the eukaryotic protein t-complex polypeptide-1. Nature 354, 490–493 33 Phipps, B.M., Typke, D., Hegerl, R., Volker, S., Hoffmann, A., Stetter, K.O. and Baumeister, W. (1993) Structure of a molecular chaperone from a thermophilic archaebacterium. Nature 361, 475–477 34 Laksanalamai, P., Whitehead, T.A. and Robb, F.T. (2004) Minimal protein-folding systems in hyperthermophilic archaea. Nat. Rev. Microbiol. 2, 315–324 35 Houry, W.A., Frishman, D., Eckerskorn, C., Lottspeich, F. and Hartl, F.U. (1999) Identification of in vivo substrates of the chaperonin GroEL. Nature 402, 147–154 36 Kerner, M.J., Naylor, D.J., Ishihama, Y., Maier, T., Chang, H.C., Stines, A.P., Georgopoulos, C., Frishman, D., Hayer-Hartl, M., Mann, M. and Hartl, F.U. (2005) Proteome-wide analysis of chaperonin-dependent protein folding in Escherichia coli. Cell 122, 209–220 37 Hirtreiter, A.M., Calloni, G., Forner, F., Scheibe, B., Puype, M., Vandekerckhove, J., Mann, M., Hartl, F.U. and Hayer-Hartl, M. (2009) Differential substrate specificity of group I and group II chaperonins in the archaeon Methanosarcina mazei. Mol. Microbiol. 74, 152–168

Received 14 September 2010 doi:10.1042/BST0390094