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Chaperonin-mediated Protein Folding

2013; Elsevier BV; Volume: 288; Issue: 33 Linguagem: Inglês

10.1074/jbc.x113.497321

1083-351X

Arthur L. Horwich,

Enzyme Structure and Function

We have been studying chaperonins these past twenty years through an initial discovery of an action in protein folding, analysis of structure, and elucidation of mechanism. Some of the highlights of these studies were presented recently upon sharing the honor of the 2013 Herbert Tabor Award with my early collaborator, Ulrich Hartl, at the annual meeting of the American Society for Biochemistry and Molecular Biology in Boston. Here, some of the major findings are recounted, particularly recognizing my collaborators, describing how I met them and how our great times together propelled our thinking and experiments. We have been studying chaperonins these past twenty years through an initial discovery of an action in protein folding, analysis of structure, and elucidation of mechanism. Some of the highlights of these studies were presented recently upon sharing the honor of the 2013 Herbert Tabor Award with my early collaborator, Ulrich Hartl, at the annual meeting of the American Society for Biochemistry and Molecular Biology in Boston. Here, some of the major findings are recounted, particularly recognizing my collaborators, describing how I met them and how our great times together propelled our thinking and experiments. My pathway to studying a protein folding machine could not have been predicted from the trajectory of my training. I finished a six-year medical program at Brown University in 1975 and then trained in pediatrics at Yale University. During residency, I became fascinated with cell transformation and, in 1978, went off to the Salk Institute to train in tumor virology with Walter Eckhart and Tony Hunter. I participated in early recombinant DNA-mediated expression of tumor virus-transforming proteins and watched Tony discover tyrosine phosphorylation (1.Eckhart W. Hutchinson M.A. Hunter T. An activity phosphorylating tyrosine in polyoma T antigen immunoprecipitates.Cell. 1979; 18: 925-933Abstract Full Text PDF PubMed Scopus (293) Google Scholar). In 1981, with the cloning of human coding sequences becoming increasingly possible, I returned to Yale to pursue genetics training with Leon Rosenberg. In particular, I cloned the cDNA for a nuclear-coded urea cycle enzyme, ornithine transcarbamylase (OTC), which is involved in an X-linked deficiency that results in lethal ammonia intoxication in newborn male infants. It is a devastating clinical situation I indelibly observed during pediatric training. With the cloned cDNA in hand (2.Horwich A.L. Fenton W.A. Williams K.R. Kalousek F. Kraus J.P. Doolittle R.F. Konigsberg W. Rosenberg L.E. Structure and expression of a cDNA for the nuclear coded precursor of human mitochondrial ornithine transcarbamylase.Science. 1984; 224: 1068-1074Crossref PubMed Scopus (204) Google Scholar), we were able to develop DNA diagnostic approaches to help affected families by providing prenatal diagnosis of the condition, but the cloned cDNA also allowed us to see the cleavable N-terminal mitochondrial targeting sequence in OTC. We soon showed that the targeting sequence contained sufficient information to direct mitochondrial localization because, when we fused it to the cytosolic protein dihydrofolate reductase (DHFR), it now directed DHFR into mitochondria (3.Horwich A.L. Kalousek F. Mellman I. Rosenberg L.E. A leader peptide is sufficient to direct mitochondrial import of a chimeric protein.EMBO J. 1985; 4: 1129-1135Crossref PubMed Scopus (137) Google Scholar). I found these studies of mitochondrial trafficking to be of fundamental interest. When I moved across the hall as an independent investigator in 1984, I wanted to isolate the “machinery” of the mitochondria themselves that was involved with protein import. To develop a screen for import machinery, we first tested whether expression of the human OTC cDNA in yeast would lead to its proper targeting, signal cleavage, and maturation into active enzyme in the mitochondrial matrix. Happily, we observed this to be the case (4.Cheng M.Y. Pollock R.A. Hendrick J.P. Horwich A.L. Import and processing of human ornithine transcarbamoylase precursor by mitochondria from Saccharomyces cerevisiae.Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 4063-4067Crossref PubMed Scopus (25) Google Scholar), allowing us to use OTC as a reporter protein. We produced a bank of temperature-sensitive lethal yeast mutants containing OTC as an inducible reporter protein. The idea was that at the nonpermissive temperature (37 °C), if proteins failed to be imported into mitochondria and no new mitochondria could be produced, cell growth would halt. For the human OTC reporter protein, we could turn on its expression after a shift from 23 °C to 37 °C using a Gal operon promoter (yeast cells lack a mitochondrial OTC enzyme, and we deleted the gene for their cytosolic OTC). The idea was that if OTC protein was made but no enzyme activity was detected in one or more of the temperature-sensitive lethal mutants after temperature shift, this would indicate that mitochondrial import was blocked. This could occur at any number of possible points, e.g. recognition of the newly translated precursor protein at the mitochondrial outer membrane, translocation through the membranes, or maturational cleavage of the N-terminal signal peptide. We began screening our temperature-sensitive lethal mutants and soon identified ones with mutations that affected the cleavage of the signal peptide by a matrix-localized peptidase. Then one night, after a long day of screening, it dawned on Ming Cheng (one of my first graduate students) and me that a very interesting type of mutant might be present in our bank, one that would affect the folding of the newly imported OTC to its native form. After all, there were studies from Gottfried Schatz's group in Basel showing that imported proteins had to be completely unfolded to pass through the mitochondrial membranes (5.Eilers M. Schatz G. Binding of a specific ligand inhibits import of a purified precursor protein into mitochondria.Nature. 1986; 322: 228-232Crossref PubMed Scopus (466) Google Scholar). Also, there was increasing evidence, much of it coming from Hugh Pelham's group at the MRC Laboratory of Molecular Biology, that there was a class of specialized proteins known as heat shock proteins (induced by thermal exposure of cells) that could adjust the conformation of other proteins, preventing them from aggregating with each other under stress conditions (6.Pelham H.R.B. Speculations on the functions of the major heat-shock and glucose-regulated proteins.Cell. 1986; 46: 959-961Abstract Full Text PDF PubMed Scopus (1145) Google Scholar). Could there be such a thing as a machine that supported de novo protein folding of imported proteins? Here, in our mitochondrial system, we were in a position to address this question. Within a week, Ming found such a mutant in our library; in particular, OTC was imported and its signal peptide was cleaved, but there was no detectable OTC enzyme activity (7.Cheng M.Y. Hartl F.-U. Martin J. Pollock R.A. Kalousek F. Neupert W. Hallberg E.M. Hallberg R.L. Horwich A.L. Mitochondrial heat shock protein HSP60 is essential for assembly of proteins imported into yeast mitochondria.Nature. 1989; 337: 620-625Crossref PubMed Scopus (670) Google Scholar). We were stunned. People had always believed that mitochondrial proteins spontaneously refolded into their native form after import through the membranes. After all, the work of Christian Anfinsen at the National Institutes of Health had shown that the primary structure of a polypeptide chain contains all of the information required to reach the native state (8.Anfinsen C.B. Principles that govern the folding of protein chains.Science. 1973; 181: 223-230Crossref PubMed Scopus (5135) Google Scholar), but even Anfinsen had pondered the observation that not all proteins could refold in vitro after dilution from denaturant (9.Epstein C.J. Goldberger R.F. Anfinsen C.B. The genetic control of tertiary protein structure: studies with model systems.Cold Spring Harb. Symp. Quant. Biol. 1963; 28: 439-449Crossref Google Scholar), and many proteins expressed in bacteria failed to properly fold, lodging in inclusion bodies. It had seemed as if something more was needed in some situations providing, for example, kinetic assistance. We next tested an endogenous yeast mitochondrial matrix protein, the β-subunit of the F1-ATPase. We found that at the nonpermissive temperature, it also failed to reach its destination in the stalk structure that faces the mitochondrial matrix (Fig. 1). The protein was present, but it appeared to be lodged in an insoluble fraction, suggesting that it had aggregated (7.Cheng M.Y. Hartl F.-U. Martin J. Pollock R.A. Kalousek F. Neupert W. Hallberg E.M. Hallberg R.L. Horwich A.L. Mitochondrial heat shock protein HSP60 is essential for assembly of proteins imported into yeast mitochondria.Nature. 1989; 337: 620-625Crossref PubMed Scopus (670) Google Scholar). Now, we were really excited but also uncertain how to proceed to further prove that this yeast mutant failed to fold newly imported proteins. Amazingly, at this point, the phone rang, and it was Walter Neupert and Ulrich Hartl calling from the University of Munich to ask if we could use a little biochemical help to study our yeast mitochondrial protein import mutants. The answer was unequivocally yes, as soon as possible. I went immediately to Munich and presented a seminar about our mutants. At the end of my talk, I mentioned the mutant that we thought might have a defect in protein folding. Ulrich and Walter were quite surprised to hear about this phenotype. They were a little worried that we might be studying a translocation defect, i.e. the imported protein could be jammed in the translocation channel. This would allow the N terminus to be cleaved in the matrix, but the mature portion of the protein would be lodged in the import site and unable to fold properly (see Fig. 1). This possibility seemed to be easily addressed by isolating mitochondria from the mutant yeast and applying an exogenous protease to an import reaction. I returned home and mailed off the yeast mutant. Two weeks later, Ulrich called excitedly to report that imported proteins were indeed localized within the matrix, fully protected from the exogenously added protease, apparently failing to refold in the matrix. This was both tremendously exciting and comforting. Now, working together, our labs could employ the wealth of anti-mitochondrial protein antibody reagents in the Neupert lab, as well as their biochemical expertise with isolated organelles, to study other imported proteins, including monomeric ones. We soon observed that the monomeric Rieske iron-sulfur protein failed to undergo normal biogenesis (Fig. 1) (7.Cheng M.Y. Hartl F.-U. Martin J. Pollock R.A. Kalousek F. Neupert W. Hallberg E.M. Hallberg R.L. Horwich A.L. Mitochondrial heat shock protein HSP60 is essential for assembly of proteins imported into yeast mitochondria.Nature. 1989; 337: 620-625Crossref PubMed Scopus (670) Google Scholar). This lent enormous strength to the contention that we were studying the nascent folding of newly imported proteins in the mitochondrial matrix, as opposed to oligomeric assembly of already folded proteins. In additional studies with Joachim Ostermann, Ulrich showed that newly imported monomeric DHFR, sent into the mitochondria via an attached targeting peptide, became associated with a large complex, occupying a protease-susceptible, apparently non-native state in this complex in the absence of ATP. However, upon addition of ATP, DHFR was released and became native-like in its protease resistance (Fig. 1) (10.Ostermann J. Horwich A.L. Neupert W. Hartl F.-U. Protein folding in mitochondria requires complex formation with HSP60 and ATP hydrolysis.Nature. 1989; 341: 125-130Crossref PubMed Scopus (476) Google Scholar). These latter observations further indicated that the yeast mutant was affecting folding of imported proteins as opposed to oligomeric assembly. We rescued the mutant cells with a yeast library and found that the gene that was able to rescue the folding-defective phenotype encoded an abundant mitochondrial matrix protein. This protein, first identified in Tetrahymena thermophila by Richard Hallberg's group at Iowa State University, was induced by ∼2-fold by heat shock and found to be present in a double-ring assembly (11.McMullin T.W. Hallberg R.L. A highly evolutionarily conserved mitochondrial protein is structurally related to the protein encoded by the Escherichia coli groEL gene.Mol. Cell. Biol. 1988; 8: 371-380Crossref PubMed Scopus (189) Google Scholar). Hallberg had been studying the yeast homolog, so we called him up and found that our rescuing sequence was a dead match to his. Thus, much to our delight, a double-ring assembly in the mitochondrial matrix, which we collectively dubbed Hsp60 (but actually is essential for cell growth at all temperatures), appeared to mediate protein folding to the native state in that compartment (Fig. 1). There also was the indication from this conclusion that other similar double-ring assemblies were likely to mediate folding of proteins in their cellular compartments as well, because their subunits bore substantial sequence identity to Hsp60. One of these assemblies was GroEL in the bacterial cytoplasm, which had been previously implicated in phage assembly (e.g. Ref. 12.Georgopoulos C.P. Hendrix R.W. Kaiser A.D. Wood W.B. Role of the host cell in bacteriophage morphogenesis: effects of a bacterial mutation on T4 head assembly.Nat. New Biol. 1972; 239: 38-41Crossref PubMed Scopus (132) Google Scholar), and another was a complex inside chloroplasts, which had been previously implicated in ribulose-bisphosphate carboxylase/oxygenase (Rubisco) assembly (13.Barraclough R. Ellis R.J. Protein synthesis in chloroplasts. IX. Assembly of newly-synthesized large subunit into ribulose bisphosphate carboxylase in isolated intact pea chloroplasts.Biochim. Biophys. Acta. 1980; 608: 19-31Crossref PubMed Scopus (189) Google Scholar). With such an understanding in hand, Ulrich (Fig. 2) and I had enormous fun thinking further about how the double-ring chaperonin machines could assist folding. Our conversations took place everywhere: the Munich airport while we waited for me to catch a plane and tried to deduce whether the two major domains of the subunits in the rings were arranged “ABAB” versus “ABBA”; my kitchen in Connecticut, where we mused about how the central cavity lining the rings might operate; and on the Merritt Parkway in Connecticut, where we conjectured that maybe Hsp70 class proteins could “hand off” nascent unfolded chains to the chaperonin cavity. It was a time of intense ferment. Perhaps the most fun of all was had by Ming Cheng (Fig. 2), who had miraculously pulled out the Hsp60 mutant from our library. Ming was a young physician from Taiwan who wanted to gain basic molecular biology training and so joined the Yale Genetics Graduate Program. She was, in two words, experimentally fearless. Her bench reflected this attitude, with the most dense collection of yeast reagents I have ever seen (Fig. 2). There seemed to be simply no room in which to carry out an experimental manipulation, but she was deft. During a recent visit to Taiwan, I observed that her kitchen is no different from the laboratory, with every counter space fully occupied (Fig. 2). Regardless, there were many stories concerning Ming's various outside activities. Driver education was one feature. One morning, we learned that she and her car had done battle with a bush next to her garage and defeated it, but Ming ultimately became a very good driver and also a good skier, regularly participating in what were termed “von Horwich” ski trips to Vermont. She has subsequently pursued both research and teaching at the National Yang-Ming Medical College in Taiwan. Shortly after our mitochondrial experiments, George Lorimer and his colleagues at DuPont reconstituted a chaperonin reaction in vitro using the bacterial homolog GroEL and a “lid”-like co-chaperonin, a seven-member ring assembly called GroES, whose coding sequence is in an operon with GroEL (14.Goloubinoff P. Christeller J.T. Gatenby A.A. Lorimer G.H. Reconstitution of active dimeric ribulose bisphosphate carboxylase from an unfolded state depends on two chaperonin proteins and MgATP.Nature. 1989; 342: 884-889Crossref PubMed Scopus (546) Google Scholar). In a first step, a dimeric form of Rubisco, unfolded in denaturant, was diluted into a mixture with GroEL. One Rubisco monomer formed a stoichiometric complex with one GroEL tetradecamer, in which Rubisco was inactive. In contrast, if Rubisco was diluted from denaturant into buffer without any GroEL present, it underwent wholesale aggregation. In a second step, upon addition of the GroES lid and ATP to the Rubisco-GroEL complex, Rubisco became properly folded over a period of a few minutes and subsequently dimerized to its native active form. Ulrich and our collective soon carried out similar reconstitution studies using GroEL-GroES-ATP to refold monomeric rhodanese and DHFR (15.Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.-U. Chaperonin-mediated protein folding occurs at the surface of GroEL via a molten globule-like intermediate.Nature. 1991; 352: 36-42Crossref PubMed Scopus (725) Google Scholar). By 1992, there were almost as many models for how GroEL-GroES might be working as there were investigators in the field. We were busy localizing bound protein to the central cavity of the GroEL ring (16.Braig K. Simon M. Furuya F. Hainfeld J.F. Horwich A.L. A polypeptide bound to the chaperonin GroEL is localized within a central cavity.Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 3978-3982Crossref PubMed Scopus (154) Google Scholar, 17.Langer T. Pfeifer G. Martin J. Baumeister W. Hartl F.-U. Chaperonin-mediated protein folding: GroES binds to one end of the GroEL cylinder, which accommodates the protein substrate within its central cavity.EMBO J. 1992; 11: 4757-4765Crossref PubMed Scopus (356) Google Scholar), but we were unsure how to proceed to a more general understanding of the reaction mechanism. This was changed by an administrative mission that took me across the Yale campus to chat with Paul Sigler, a newly arrived crystallographer from the University of Chicago (Fig. 3). Paul immediately brushed the administrative matter aside and began to discuss the chaperonins, marveling at their symmetries (two back-to-back 7-fold radially symmetric rings) and enjoining us that we would never figure out how the chaperonins work without an x-ray structure. The collaboration was born at that minute. He brought Zbyszek Otwinowski into the room (Fig. 3), who just shook his head at the computational problem of dealing with an 800-kDa assembly. Zbyszek reminded us that it would take us a few years to crystallize the molecular machine and that the computing capability would be ready by then to deal with it. He was right! Once we produced two data sets, one of the native complex and another with an ethyl mercury derivative, Zbyszek astonishingly solved the structure in a single day in the fall of 1993 using a search program he had devised to sort through the large number of heavy atoms (18.Braig K. Otwinowski Z. Hegde R. Boisvert D.C. Joachimiak A. Horwich A.L. Sigler P.B. Crystal structure of GroEL at 2.8 Å.Nature. 1994; 371: 578-586Crossref PubMed Scopus (1191) Google Scholar). As added testimony to Zbyszek's brilliance and daring, when we initially went to collect native data at the Cornell High Energy Synchrotron Source (CHESS) beamline for the first time in the summer of 1993, he wondered if we could collect a few reflections at very low resolution from which he might be able to phase the structure. We tried moving the detector way back and collecting a reflection or two at 150 Å. Obviously, this did not succeed, but it was surely fun to try. Andrzej Joachimiak from Paul's lab (Fig. 3) was a wonderful companion and collaborator during the period of generating GroEL protein and testing crystallizations. The two of us would converse about expression and purification of GroEL every day, our collaboration accelerated to white heat by the proximity of our group and the Sigler group next to each other in the newly built Boyer Center for Molecular Medicine at the Yale School of Medicine in late 1991. Ultimately, we produced GroEL to the level of 95% of total bacterial protein. I remember the look on Andrzej's face when I showed him a gel with a massive Coomassie blotch at the position of GroEL. I said, “I have a gram of this stuff, Andrzej, from 2 liters.” “This is amazing,” was his response. It was enormous fun some months later to drive up to CHESS with Andrzej for the first synchrotron data collection. I remember walking down the hill to grab breakfast after an overnight fire drill with putting up image plates, shooting x-rays, and then fetching the plates and putting them into the Fuji scanner at the F1 beamline (this was before charge-coupled device cameras), thinking, “This is really going to change our understanding of this machine.” Equally good times have subsequently been had visiting Andrzej at the ID19 beamline at the Argonne National Laboratory, trying any and all means to see GroEL-bound polypeptide by crystallography. However, we did not have much success. These were trips made with George Farr, who came as a postdoctoral fellow to my laboratory in the mid-1990s but stayed for fifteen years, enjoying the biochemistry and structural aspects of the chaperonin system. I can only say that as we have migrated toward cell biology and physiology of neurodegeneration, I have missed going to the synchrotron, but I continue to enjoy Andrzej's Christmas cards, loaded with images of newly solved structures. As for Paul, he was like a father to me for a period of ten years. We had the Chicago scene in common (I grew up there), the Bulls, the Cubs, the Ohio Street pizza places, but we also shared a love of jazz and an early training in medicine. Like me, Paul had been medically trained and had been an intern with my postdoctoral genetics mentor, Leon Rosenberg. There was a memorable account of his taking care of Marilyn Monroe as a patient: no further comment needed. Anyway, he osmotically taught me crystallography, showing all aspects of the process when we were next-door neighbors in the Boyer Center. It was wonderful to hear the stories about the MRC Laboratory of Molecular Biology in the 1960s, where Paul had trained with David Blow, working on chymotrypsin, in the circle of Max Perutz, Sydney Brenner, and many others. Many of these stories were shared in our lunchroom, where Paul, an avid fan of Slim-Fast as the solution to good health, would rant at the messy state of the kitchen as he prepared the next dose of the reagent. I can attest that it remains, even ten years after Paul's passing, a constant challenge to keep the lab kitchen workable. It was a magical time, where ideas flowed freely between our two labs on the mechanism of GroEL-GroES, and we watched the Sigler group reveal the beauties of heterotrimeric G proteins, NF-κB, phospholipases, and other structures. It was inspiring. Kerstin Braig was the true artist through all of this (Fig. 3). She produced the first well diffracting crystals of GroEL. She was a visiting student from Berlin, supposedly for one year, but she stayed for five years. Her first study visualized a gold particle-labeled substrate polypeptide in the GroEL cavity by scanning transmission electron microscopy at the Brookhaven National Laboratory (16.Braig K. Simon M. Furuya F. Hainfeld J.F. Horwich A.L. A polypeptide bound to the chaperonin GroEL is localized within a central cavity.Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 3978-3982Crossref PubMed Scopus (154) Google Scholar), in collaboration with Joe Wall and Jim Hainfeld. She then took on the crystallization and structure determination of GroEL. In her outside life, Kerstin required a strong artistic involvement, such as sculpture classes, and many other outside activities, probably to maintain her sanity during the difficult task of crystallization trials, which were a manual undertaking at that point of history. Perhaps her boldest exploit was a weekend jaunt to Mongolia. She spent one day flying and two days on the ground enjoying the local scene and then flew back to have a look at her new crystals. Indeed, when she pulled the initial well diffracting ammonium sulfate crystal, it emerged from a literal wall of trays stacked to massive heights side-by-side at room temperature on shelves above her bench. Kerstin was helped considerably by another student, David Boisvert, who also brought a lot of personality to the lab, as well as experience in crystallization. David had assisted in crystallization studies of reverse transcriptase in Tom Steitz's laboratory at Yale before matriculating in the graduate school (Fig. 3). David brought with him a wealth of expertise and ultimately crystallized GroEL in complex with adenosine 5′-O-(3-thiotriphosphate) (ATPγS) (19.Boisvert D.C. Wang J. Otwinowski Z. Horwich A.L. Sigler P.B. The 2.4 Å crystal structure of the bacterial chaperonin GroEL complexes with ATPγS.Nat. Struct. Biol. 1996; 3: 170-177Crossref PubMed Scopus (244) Google Scholar). These wonderful people and many others, including Axel Brunger, contributed as a team to the crystallization and refinement efforts. With a structure of GroEL, we saw a central cavity that is 45 Å in diameter (Fig. 4, left). This cavity is blocked at the waistline of the cylinder by disordered C-terminal tails of each subunit (25 residues each, amounting to ∼20 kDa per ring), visible as collective masses by EM but not visible by crystallography. Thus, GroEL contains a central cavity at either end of the cylinder, large enough to house a polypeptide of 30–40 kDa. Of course, in the absence of a bound GroES, larger proteins can be both bound in the cavity and partly present in the bulk solution, bound as if a champagne cork (20.Thiyagarajan P. Henderson S.J. Joachimiak A. Solution structures of GroEL and its complex with rhodanese from small-angle neutron scattering.Structure. 1996; 4: 79-88Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Mutational studies soon revealed that the polypeptide-binding site is present on the cavity surface of the terminal apical domains of the subunits, presenting hydrophobic residues to the solvent (Fig. 4, right, bottom ring) (21.Fenton W.A. Kashi Y. Furtak K. Horwich A.L. Residues in chaperonin GroEL required for polypeptide binding and release.Nature. 1994; 371: 614-619Crossref PubMed Scopus (574) Google Scholar). Non-native proteins, presenting their own hydrophobic surfaces, which would be buried in the interior in the native state, are bound on this surface. This binding prevents them from forming associations that would lead to aggregation. Later studies, both genetic and EM, have also shown that such binding is multivalent in character, with a polypeptide bound to three or four consecutive apical domains (22.Farr G.W. Furtak K. Rowland M.B. Ranson N.A. Saibil H.R. Kirchhausen T. Horwich A.L. Multivalent binding of non-native substrate proteins by the chaperonin GroEL.Cell. 2000; 100: 561-573Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 23.Elad N. Farr G.W. Clare D.K. Orlova E.V. Horwich A.L. Saibil H.R. Topologies of a substrate protein bound to the chaperonin GroEL.Mol. Cell. 2007; 26: 415-426Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The polypeptide occupies an unstructured state while bound (24.Horst R. Bertelsen E.B. Fiaux J. Wider G. Horwich A.L. Wüthrich K. Direct NMR observation of a substrate protein bound to the chaperonin GroEL.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 12748-12753Crossref PubMed Scopus (101) Google Scholar); indeed, fluorescence studies have indicated that a loosely folded but kinetically trapped state can be “pulled apart” upon binding to the apical domains (25.Lin Z. Rye H.S. Expansion and compression of a protein folding intermediate by GroEL.Mol. Cell. 2004; 16: 23-34Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The apical domains of GroEL are connected to a “base” composed of α-helical equatorial domains that make tight contacts both going around a ring and across the ring-ring interface (Fig. 4). Each of these domains exhibits a pocket that could house an ATP molecule (Fig. 4, middle, top ring). David Boisvert soon co-crystallized GroEL-ATPγS, observing the stereochemistry of nucleotide binding in the pocket (19.Boisvert D.C. Wang J. Otwinowski Z. Horwich A.L. Sigler P.B. The 2.4 Å crystal structure of the bacterial chaperonin GroEL complexes with ATPγS.Nat. Struct. Biol. 1996; 3: 170-177Crossref PubMed Scopus (244) Google Scholar). How does folding ensue in the GroEL system? Early EM studies had shown that the lid-shaped GroES co-chaperonin can bind to GroEL in the presence of ATP to form asymmetric complexes (Fig. 4, middle and right) (26.Saibil H. Dong Z. Wood S. auf der Mauer A. Binding of chaperonins.Nature. 1991; 353: 25-26Crossref PubMed Scopus (82) Google Scholar), but does the polypeptide fold inside these complexes or only outside in solution? When we looked at the native structure of one of our favorite substrate proteins, rhodanese, even in its native state, it clashed with the GroEL apical domains when imposed on the unliganded GroEL x-ray model. It seemed that folding might have to occur in solution, but our EM collaborator, Helen Saibil at Birkbeck College in London, changed our thinking about that about a month later with beautiful new images in both negative stain and cryo-EM (27.Saibil H.R. Zheng D. Roseman A.M. Hunter A.S. Watson G.M. Chen S. auf der Mauer A. O'Hara B.P. Wood S.P. Mann