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Role of the GroEL Chaperonin Intermediate Domain in Coupling ATP Hydrolysis to Polypeptide Release

1998; Elsevier BV; Volume: 273; Issue: 13 Linguagem: Inglês

10.1074/jbc.273.13.7351

1083-351X

Jörg Martin,

Protein Structure and Dynamics

Modification of the Escherichia colichaperonin GroEL with N-ethylmaleimide at residue Cys138 affects the structural and functional integrity of the complex. Nucleotide affinity and ATPase activity of the modified chaperonin are increased, whereas cooperativity of ATP hydrolysis and affinity for GroES are reduced. As a consequence, release and folding of substrate proteins are strongly impaired and uncoupled from ATP hydrolysis in a temperature-dependent manner. Folding of dihydrofolate reductase at 25 °C becomes dependent on GroES, whereas folding of typically GroES-dependent proteins is blocked completely. At 37 °C, GroES binding is restored to normal levels, and the modified GroEL regains its chaperone activity to some extent. These results assign a central role to the intermediate GroEL domain for transmitting conformational changes between apical and central domains, and for coupling ATP hydrolysis to productive protein release. Modification of the Escherichia colichaperonin GroEL with N-ethylmaleimide at residue Cys138 affects the structural and functional integrity of the complex. Nucleotide affinity and ATPase activity of the modified chaperonin are increased, whereas cooperativity of ATP hydrolysis and affinity for GroES are reduced. As a consequence, release and folding of substrate proteins are strongly impaired and uncoupled from ATP hydrolysis in a temperature-dependent manner. Folding of dihydrofolate reductase at 25 °C becomes dependent on GroES, whereas folding of typically GroES-dependent proteins is blocked completely. At 37 °C, GroES binding is restored to normal levels, and the modified GroEL regains its chaperone activity to some extent. These results assign a central role to the intermediate GroEL domain for transmitting conformational changes between apical and central domains, and for coupling ATP hydrolysis to productive protein release. The Escherichia coli chaperonin GroEL plays a pivotal role in the folding of proteins in the cell. Assisted by the cofactor GroES, it allows newly synthesized polypeptide chains to gain their native state under the crowded conditions of the cytosol (for review, see Refs. 1Martin J. Hartl F.U. Curr. Opin. Struct. Biol. 1997; 7: 41-52Crossref PubMed Scopus (163) Google Scholar and 2Fenton W.A. Horwich A.L. Protein Sci. 1997; 6: 743-760Crossref PubMed Scopus (325) Google Scholar). GroEL forms a cylinder composed of two symmetrically stacked rings, each with seven identicalMr 57,000 subunits (3Hemmingsen S.M. Woolford C. van der Vies S.M. Tilly K. Dennis D.T. Georgopoulos C.P. Hendrix R.W. Ellis R.J. Nature. 1988; 333: 330-334Crossref PubMed Scopus (918) Google Scholar, 4Langer T. Pfeifer G. Martin J. Baumeister W. Hartl F.U. EMBO J. 1992; 11: 4757-4765Crossref PubMed Scopus (352) Google Scholar). Substrate proteins bind to the interior surface of this cylinder at the level of the apical domains (5Braig K. Simon M. Furuya F. Hainfeld J.F. Horwich A.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3978-3982Crossref PubMed Scopus (152) Google Scholar, 6Fenton W.A. Kashi Y. Furtak K. Horwich A.L. Nature. 1994; 371: 614-619Crossref PubMed Scopus (569) Google Scholar). Typically, only one substrate protein binds to one GroEL tetradecamer (5Braig K. Simon M. Furuya F. Hainfeld J.F. Horwich A.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3978-3982Crossref PubMed Scopus (152) Google Scholar, 7Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.-U. Nature. 1991; 352: 36-42Crossref PubMed Scopus (718) Google Scholar). The conformation of substrate protein in its GroEL-associated state has been analyzed by tryptophan fluorescence, binding of the hydrophobic fluorescent dye anilino naphthalene sulfonate, and hydrogen-exchange experiments coupled with mass-spectrometry and NMR (7Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.-U. Nature. 1991; 352: 36-42Crossref PubMed Scopus (718) Google Scholar, 8Hayer-Hartl M.K. Ewbank J.J. Creigthon T.E. Hartl F.U. EMBO J. 1994; 13: 3192-3202Crossref PubMed Scopus (156) Google Scholar, 9Robinson C.V. Groβ M. Eyles S.J. Ewbank J.J. Mayhew M. Hartl F.U. Dobson C.M. Radford S.E. Nature. 1995; 372: 646-651Crossref Scopus (194) Google Scholar, 10Goldberg M.S. Zhang J. Sondek S. Matthews C.R. Fox R.O. Horwich A.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1080-1085Crossref PubMed Scopus (89) Google Scholar, 11Groβ M. Robinson C.V. Mayhew M. Hartl F.U. Radford S.E. Protein Sci. 1996; 5: 2506-2513Crossref PubMed Scopus (66) Google Scholar, 12Zahn R. Perrett S. Stenberg G. Fersht A.R. Science. 1996; 271: 642-645Crossref PubMed Scopus (134) Google Scholar). According to these results, the bound proteins are in a “molten globule”-like folding state, marked by the presence of secondary structure, lack of persistent tertiary interactions, and the exposure of hydrophobic structure elements at the protein surface. GroEL has a relatively weak ATPase activity which is regulated by GroES (7Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.-U. Nature. 1991; 352: 36-42Crossref PubMed Scopus (718) Google Scholar, 13Chandrasekhar G.N. Tilly K. Woolford C. Hendrix R. Georgopoulos C. J. Biol. Chem. 1986; 261: 12414-12419Abstract Full Text PDF PubMed Google Scholar). This smaller cofactor is comprised of sevenMr 10,000 subunits that are arranged in a single ring. GroES can bind on top of the GroEL cylinder in the presence of nucleotides. A result of this interaction is an increased nucleotide affinity of GroEL and a tight coordination of the cycles of ATP binding, ATP hydrolysis, and ADP release.The mechanism of GroEL/GroES-mediated protein folding has been subject of extensive investigation. The current data can be summarized in a model (1Martin J. Hartl F.U. Curr. Opin. Struct. Biol. 1997; 7: 41-52Crossref PubMed Scopus (163) Google Scholar, 2Fenton W.A. Horwich A.L. Protein Sci. 1997; 6: 743-760Crossref PubMed Scopus (325) Google Scholar), according to which ATP hydrolysis in the GroEL ring that is opposite GroES leads to release of ADP in the GroES-containing ring, accompanied by transient GroES release and binding of ATP. Once ATP is bound, GroES caps the end of the GroEL cylinder that also contains the substrate protein, thereby discharging the unfolded polypeptide into the shielded cavity for folding. Subsequent ATP hydrolysis in the opposite GroEL ring leads to the release of GroES and allows a folded substrate protein eventually to exit the cylinder. However, polypeptides that fold incompletely and still expose binding sites for the chaperonin after one such reaction cycle, will rebind. Typically, a polypeptide has to undergo several reaction cycles in association with GroEL before it leaves the chaperonin as a native or native-like protein (14Martin J. Mayhew M. Langer T. Hartl F.U. Nature. 1993; 366: 228-233Crossref PubMed Scopus (235) Google Scholar, 15Mayhew M. da Silva A.C.R. Martin J. Erdjument-Bromage H. Tempst P. Hartl F.-U. Nature. 1996; 379: 420-426Crossref PubMed Scopus (342) Google Scholar, 16Weissman J.S. Hohl C.M. Kovalenko O. Kashi Y. Chen S. Braig K Saibil H.R. Fenton W.A. Horwich A.L. Cell. 1995; 83: 577-587Abstract Full Text PDF PubMed Scopus (387) Google Scholar).The crystal structures of GroEL, GroES, and complexes between these two proteins have been solved (17Braig K. Otwinowski Z. Hegde R. Boisvert D.C. Joachimiak A. Horwich A.L. Sigler P.B. Nature. 1994; 371: 578-586Crossref PubMed Scopus (1182) Google Scholar, 18Boisvert D.C. Wang J.M. Otwinowski Z. Horwich A.L. Sigler P.B. Nat. Struct. Biol. 1996; 3: 170-177Crossref PubMed Scopus (243) Google Scholar, 19Xu Z. Horwich A.L. Sigler P.B. Nature. 1997; 388: 741-749Crossref PubMed Scopus (1031) Google Scholar), indicating the positions where nucleotides and the unfolded substrate proteins bind. Binding elements for GroES and polypeptide reside, and partially overlap, within the outer domains, whereas nucleotides bind to the central amino-terminal domains (7Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.-U. Nature. 1991; 352: 36-42Crossref PubMed Scopus (718) Google Scholar, 18Boisvert D.C. Wang J.M. Otwinowski Z. Horwich A.L. Sigler P.B. Nat. Struct. Biol. 1996; 3: 170-177Crossref PubMed Scopus (243) Google Scholar). Electron microscopy studies, and the recently solved GroEL/GroES crystal structure have revealed the significant conformational changes that occur after nucleotide-dependent binding of GroES to GroEL (4Langer T. Pfeifer G. Martin J. Baumeister W. Hartl F.U. EMBO J. 1992; 11: 4757-4765Crossref PubMed Scopus (352) Google Scholar, 19Xu Z. Horwich A.L. Sigler P.B. Nature. 1997; 388: 741-749Crossref PubMed Scopus (1031) Google Scholar, 20Roseman A.M. Chen S. White H. Braig K. Saibil H.R. Cell. 1996; 87: 241-251Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Most prominently, the apical GroEL domains move outward about 60°, leading to a significant widening and enlargement of the central cavity. These domain movements seem to be mediated by an intermediary hinge region (residues 134–190 and 377–408) which links the central and apical domains. In addition to its critical role in effecting these GroES-induced conformational changes, the hinge region is also thought to be involved in mediating the changes in the central ATP-binding region which are induced by the binding of substrate protein in the apical domain. Conversely, nucleotide binding and ATP hydrolysis in the central domain affect binding and release of substrate protein from a distance, and the intermediary domain is expected to be involved in this process as well.We have previously observed that modification of GroEL with NEM 1The abbreviations used are: NEM,N-ethylmaleimide; [α-32P]8-N3ADP and [α-32P]8-N3ATP, azido-labeled, radioactive adenosine diphosphate and adenosine triphosphate, respectively; DHFR, dihydrofolate reductase; MOPS, 3-(N-morpholino)propanesulfonic acid; GdmCl, guanidinium chloride; PAGE, polyacrylamide gel electrophoresis; ATPγS, adenosine 5′-O-(3-thiotriphosphate). 1The abbreviations used are: NEM,N-ethylmaleimide; [α-32P]8-N3ADP and [α-32P]8-N3ATP, azido-labeled, radioactive adenosine diphosphate and adenosine triphosphate, respectively; DHFR, dihydrofolate reductase; MOPS, 3-(N-morpholino)propanesulfonic acid; GdmCl, guanidinium chloride; PAGE, polyacrylamide gel electrophoresis; ATPγS, adenosine 5′-O-(3-thiotriphosphate). leads to structural changes in the intermediary hinge region and, unlike wild-type GroEL, makes it accessible to attack by proteinase K (21Martin J. Goldie K.N. Engel A. Hartl F.-U. Biol. Chem. Hoppe-Seyler. 1994; 375: 635-639PubMed Google Scholar). In this study, Cys138, a residue located in the intermediate domain, is identified as the target for NEM modification. As it seemed likely that NEM modification not only affects the structure, but has even more profound effects on the function of the GroEL chaperonin, this study analyzes the consequences of NEM modification for the ability of GroEL to successfully mediate the folding of substrate proteins.DISCUSSIONModification of GroEL with NEM changes the properties of the chaperonin profoundly, both structurally and functionally. Only one of the three cysteines in GroEL, Cys138, is modified by NEM. The small intermediate domain (residues 134–190 and 377–408), in which this residue resides, joins the apical domain of a GroEL subunit with its large equatorial domain. It has been proposed that this intermediate segment allows a hinge-like opening and twisting of the apical domain about the common domain junction (Gly192 and Gly375), which is caused by nucleotide binding in the equatorial domains and GroES binding to the apical domains of one GroEL ring (19Xu Z. Horwich A.L. Sigler P.B. Nature. 1997; 388: 741-749Crossref PubMed Scopus (1031) Google Scholar). As a result, the volume of the central GroEL chamber in the GroES-associated ring increases significantly, forming the cage in which protein folding proceeds. The now dome-shaped cavity is spacious enough to allow the enclosed protein to explore its folding pathway without direct interference from the chaperonin itself. In addition to moving upward the apical domain becomes twisted. The affinity of GroEL for unfolded polypeptide is strongly reduced when GroES is bound in the presence of nucleotides because rotation of the apical domains about the hinge between the apical and intermediate domains then occludes most of the hydrophobic-binding regions in the intersubunit interface. The opening movement of the apical domain is made possible by a downward movement of the intermediate segment toward the central channel which occurs at the domain junction between intermediate and equatorial domain around residues Pro137 and Gly410 (19Xu Z. Horwich A.L. Sigler P.B. Nature. 1997; 388: 741-749Crossref PubMed Scopus (1031) Google Scholar). Further evidence for the importance of that second hinge region comes from the present study showing that modification of Cys138 adjacent to Pro137 with a bulky NEM moiety interferes with this movement. Structural changes in the vicinity of Pro137 can explain not only the observations described here but also our findings from an earlier study (21Martin J. Goldie K.N. Engel A. Hartl F.-U. Biol. Chem. Hoppe-Seyler. 1994; 375: 635-639PubMed Google Scholar) where we analyzed fragments of NEM-GroEL that were obtained after proteolysis in the presence of nucleotides. Cleavage by proteinase K occurred between residues 143 and 153. Whereas Pro137 is highly conserved in members of the chaperonin family, one encounters more variability in the adjacent residue. In addition to Cys, one frequently finds Val at this position, and mutations to other small uncharged residues like Ala or Ser result in functional proteins (32Bochkareva E.S. Horovitz A. Girshovich A.S. J. Biol. Chem. 1994; 269: 44-46Abstract Full Text PDF PubMed Google Scholar, 33Luo G.-X. Horowitz P.M. J. Biol. Chem. 1994; 269: 32151-32154Abstract Full Text PDF PubMed Google Scholar, 34Yifrach O. Horovitz A. Biochemistry. 1995; 34: 5303-5308Crossref PubMed Scopus (263) Google Scholar). However, when we attempted to mimic the NEM effect by exchanging Cys138 to the larger hydrophobic residues Trp or Phe, we found defects similar to that observed with the chemically modified chaperonin. 3J. L. Mark and J. Martin, unpublished results. It appears that only modification with bulky hydrophobic groups perturbs the structural integrity in the complex in such a way that proteinase K gains access to an otherwise inaccessible region.The primary consequences of modification of the critical residue Cys138 in NEM-GroEL are changes in nucleotide binding, rate and cooperativity of ATP hydrolysis, and GroES binding. NEM-GroEL has an increased ATPase activity and binds nucleotides with higher affinity than wild-type GroEL. This phenotype is unique among the various GroEL mutants that have been generated so far in that none of these analyzed mutants is characterized by an accelerated rate of ATP hydrolysis (6Fenton W.A. Kashi Y. Furtak K. Horwich A.L. Nature. 1994; 371: 614-619Crossref PubMed Scopus (569) Google Scholar); typically, the rate is reduced. In a GroEL·GroES complex the downward movement around Pro137 impedes dissociation of ADP, locking the chaperonin complex in the nucleotide-bound state. It is conceivable that NEM·GroEL adopts a conformation that mimics part of this movement and thereby leads to an increase in nucleotide affinity. This conformational switch could bring Cys138 closer to the nucleotide-binding site, explaining earlier data from cross-linking experiments with ATPγS that had placed Cys138 in the vicinity of the ATP phosphate groups (32Bochkareva E.S. Horovitz A. Girshovich A.S. J. Biol. Chem. 1994; 269: 44-46Abstract Full Text PDF PubMed Google Scholar). Remarkably, despite its higer affinity for nucleotides, the affinity of NEM-GroEL for GroES is reduced compared with wild-type GroEL. This result demonstrates that a tighter binding of ATP or ADP to the chaperonin does not automatically correlate with better binding of the chaperonin cofactor as expected. As a consequence of the weak interaction of NEM-GroEL with GroES, the modified chaperonin is compromised in its ability to promote protein folding at 25 °C. Substrate proteins are likely to make contact with GroEL at several of the seven subunits in the cylindrical ring. A coordinated release of substrate from all these sites during ATP hydrolysis would give the protein time to partially fold within the cavity until the high affinity ADP state is regenerated. Without GroES, nucleotide binding and ATP hydrolysis by the individual GroEL subunits result in nonproductive release of folding intermediates into the bulk solution. For smaller proteins that can fold relatively easily, such as DHFR, release per se may be sufficient. In contrast, for those substrates that are more likely to engage in non-productive intermolecular interactions and are prone to misfolding, such as rhodanese, full coordination of the conformational changes in the individual subunits and the presence of GroES is required for a productive release into the chaperonin cavity. Unsynchronized release of substrate protein, as in NEM-GroEL, would shorten the time window for folding, because at any given time a binding site in NEM-GroEL would be competent for (re)binding the substrate. GroES binding to NEM-GroEL may facilitate coordinated release at least to such a degree that is sufficient for the release and folding of the weakly binding substrate DHFR, whose reactivation consequently becomes GroES-dependent. However, for tight binding substrate proteins like rhodanese, the GroES interaction with NEM-GroEL is still too weak to promote their efficient displacement into the cavity. At 37 °C, several effects may contribute to the partially restored chaperonin activity of NEM-GroEL. First, folding of substrate proteins is expected to occur with faster kinetics, which might enable the folding proteins to escape an untimely rebinding to NEM-GroEL. Second, the conformational changes induced by NEM modification may be less pronounced at 37 °C, or may affect the chaperonin conformation less than at 25 °C due to an increased flexibility of the chaperonin structure. Third, and most importantly, there is a strong improvement in GroES binding to NEM-GroEL at higher temperatures. A firmly anchored GroES is required to displace the substrate protein from its binding site at GroEL which partially overlaps with that of the smaller cofactor. In fact, it appears that this aspect of GroES function is the most critical one, more important than a precise regulation of the GroEL ATPase activity (35Hayer-Hartl M.K. Weber F. Hartl F.U. EMBO J. 1996; 15: 6111-6121Crossref PubMed Scopus (132) Google Scholar). High affinity binding of GroES to the apical domain after nucleotide binding to the central domain would be made possible by the intermediate domain, coupling not only these two events, but also ATP hydrolysis to productive protein release. The Escherichia coli chaperonin GroEL plays a pivotal role in the folding of proteins in the cell. Assisted by the cofactor GroES, it allows newly synthesized polypeptide chains to gain their native state under the crowded conditions of the cytosol (for review, see Refs. 1Martin J. Hartl F.U. Curr. Opin. Struct. Biol. 1997; 7: 41-52Crossref PubMed Scopus (163) Google Scholar and 2Fenton W.A. Horwich A.L. Protein Sci. 1997; 6: 743-760Crossref PubMed Scopus (325) Google Scholar). GroEL forms a cylinder composed of two symmetrically stacked rings, each with seven identicalMr 57,000 subunits (3Hemmingsen S.M. Woolford C. van der Vies S.M. Tilly K. Dennis D.T. Georgopoulos C.P. Hendrix R.W. Ellis R.J. Nature. 1988; 333: 330-334Crossref PubMed Scopus (918) Google Scholar, 4Langer T. Pfeifer G. Martin J. Baumeister W. Hartl F.U. EMBO J. 1992; 11: 4757-4765Crossref PubMed Scopus (352) Google Scholar). Substrate proteins bind to the interior surface of this cylinder at the level of the apical domains (5Braig K. Simon M. Furuya F. Hainfeld J.F. Horwich A.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3978-3982Crossref PubMed Scopus (152) Google Scholar, 6Fenton W.A. Kashi Y. Furtak K. Horwich A.L. Nature. 1994; 371: 614-619Crossref PubMed Scopus (569) Google Scholar). Typically, only one substrate protein binds to one GroEL tetradecamer (5Braig K. Simon M. Furuya F. Hainfeld J.F. Horwich A.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3978-3982Crossref PubMed Scopus (152) Google Scholar, 7Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.-U. Nature. 1991; 352: 36-42Crossref PubMed Scopus (718) Google Scholar). The conformation of substrate protein in its GroEL-associated state has been analyzed by tryptophan fluorescence, binding of the hydrophobic fluorescent dye anilino naphthalene sulfonate, and hydrogen-exchange experiments coupled with mass-spectrometry and NMR (7Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.-U. Nature. 1991; 352: 36-42Crossref PubMed Scopus (718) Google Scholar, 8Hayer-Hartl M.K. Ewbank J.J. Creigthon T.E. Hartl F.U. EMBO J. 1994; 13: 3192-3202Crossref PubMed Scopus (156) Google Scholar, 9Robinson C.V. Groβ M. Eyles S.J. Ewbank J.J. Mayhew M. Hartl F.U. Dobson C.M. Radford S.E. Nature. 1995; 372: 646-651Crossref Scopus (194) Google Scholar, 10Goldberg M.S. Zhang J. Sondek S. Matthews C.R. Fox R.O. Horwich A.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1080-1085Crossref PubMed Scopus (89) Google Scholar, 11Groβ M. Robinson C.V. Mayhew M. Hartl F.U. Radford S.E. Protein Sci. 1996; 5: 2506-2513Crossref PubMed Scopus (66) Google Scholar, 12Zahn R. Perrett S. Stenberg G. Fersht A.R. Science. 1996; 271: 642-645Crossref PubMed Scopus (134) Google Scholar). According to these results, the bound proteins are in a “molten globule”-like folding state, marked by the presence of secondary structure, lack of persistent tertiary interactions, and the exposure of hydrophobic structure elements at the protein surface. GroEL has a relatively weak ATPase activity which is regulated by GroES (7Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.-U. Nature. 1991; 352: 36-42Crossref PubMed Scopus (718) Google Scholar, 13Chandrasekhar G.N. Tilly K. Woolford C. Hendrix R. Georgopoulos C. J. Biol. Chem. 1986; 261: 12414-12419Abstract Full Text PDF PubMed Google Scholar). This smaller cofactor is comprised of sevenMr 10,000 subunits that are arranged in a single ring. GroES can bind on top of the GroEL cylinder in the presence of nucleotides. A result of this interaction is an increased nucleotide affinity of GroEL and a tight coordination of the cycles of ATP binding, ATP hydrolysis, and ADP release. The mechanism of GroEL/GroES-mediated protein folding has been subject of extensive investigation. The current data can be summarized in a model (1Martin J. Hartl F.U. Curr. Opin. Struct. Biol. 1997; 7: 41-52Crossref PubMed Scopus (163) Google Scholar, 2Fenton W.A. Horwich A.L. Protein Sci. 1997; 6: 743-760Crossref PubMed Scopus (325) Google Scholar), according to which ATP hydrolysis in the GroEL ring that is opposite GroES leads to release of ADP in the GroES-containing ring, accompanied by transient GroES release and binding of ATP. Once ATP is bound, GroES caps the end of the GroEL cylinder that also contains the substrate protein, thereby discharging the unfolded polypeptide into the shielded cavity for folding. Subsequent ATP hydrolysis in the opposite GroEL ring leads to the release of GroES and allows a folded substrate protein eventually to exit the cylinder. However, polypeptides that fold incompletely and still expose binding sites for the chaperonin after one such reaction cycle, will rebind. Typically, a polypeptide has to undergo several reaction cycles in association with GroEL before it leaves the chaperonin as a native or native-like protein (14Martin J. Mayhew M. Langer T. Hartl F.U. Nature. 1993; 366: 228-233Crossref PubMed Scopus (235) Google Scholar, 15Mayhew M. da Silva A.C.R. Martin J. Erdjument-Bromage H. Tempst P. Hartl F.-U. Nature. 1996; 379: 420-426Crossref PubMed Scopus (342) Google Scholar, 16Weissman J.S. Hohl C.M. Kovalenko O. Kashi Y. Chen S. Braig K Saibil H.R. Fenton W.A. Horwich A.L. Cell. 1995; 83: 577-587Abstract Full Text PDF PubMed Scopus (387) Google Scholar). The crystal structures of GroEL, GroES, and complexes between these two proteins have been solved (17Braig K. Otwinowski Z. Hegde R. Boisvert D.C. Joachimiak A. Horwich A.L. Sigler P.B. Nature. 1994; 371: 578-586Crossref PubMed Scopus (1182) Google Scholar, 18Boisvert D.C. Wang J.M. Otwinowski Z. Horwich A.L. Sigler P.B. Nat. Struct. Biol. 1996; 3: 170-177Crossref PubMed Scopus (243) Google Scholar, 19Xu Z. Horwich A.L. Sigler P.B. Nature. 1997; 388: 741-749Crossref PubMed Scopus (1031) Google Scholar), indicating the positions where nucleotides and the unfolded substrate proteins bind. Binding elements for GroES and polypeptide reside, and partially overlap, within the outer domains, whereas nucleotides bind to the central amino-terminal domains (7Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.-U. Nature. 1991; 352: 36-42Crossref PubMed Scopus (718) Google Scholar, 18Boisvert D.C. Wang J.M. Otwinowski Z. Horwich A.L. Sigler P.B. Nat. Struct. Biol. 1996; 3: 170-177Crossref PubMed Scopus (243) Google Scholar). Electron microscopy studies, and the recently solved GroEL/GroES crystal structure have revealed the significant conformational changes that occur after nucleotide-dependent binding of GroES to GroEL (4Langer T. Pfeifer G. Martin J. Baumeister W. Hartl F.U. EMBO J. 1992; 11: 4757-4765Crossref PubMed Scopus (352) Google Scholar, 19Xu Z. Horwich A.L. Sigler P.B. Nature. 1997; 388: 741-749Crossref PubMed Scopus (1031) Google Scholar, 20Roseman A.M. Chen S. White H. Braig K. Saibil H.R. Cell. 1996; 87: 241-251Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Most prominently, the apical GroEL domains move outward about 60°, leading to a significant widening and enlargement of the central cavity. These domain movements seem to be mediated by an intermediary hinge region (residues 134–190 and 377–408) which links the central and apical domains. In addition to its critical role in effecting these GroES-induced conformational changes, the hinge region is also thought to be involved in mediating the changes in the central ATP-binding region which are induced by the binding of substrate protein in the apical domain. Conversely, nucleotide binding and ATP hydrolysis in the central domain affect binding and release of substrate protein from a distance, and the intermediary domain is expected to be involved in this process as well. We have previously observed that modification of GroEL with NEM 1The abbreviations used are: NEM,N-ethylmaleimide; [α-32P]8-N3ADP and [α-32P]8-N3ATP, azido-labeled, radioactive adenosine diphosphate and adenosine triphosphate, respectively; DHFR, dihydrofolate reductase; MOPS, 3-(N-morpholino)propanesulfonic acid; GdmCl, guanidinium chloride; PAGE, polyacrylamide gel electrophoresis; ATPγS, adenosine 5′-O-(3-thiotriphosphate). 1The abbreviations used are: NEM,N-ethylmaleimide; [α-32P]8-N3ADP and [α-32P]8-N3ATP, azido-labeled, radioactive adenosine diphosphate and adenosine triphosphate, respectively; DHFR, dihydrofolate reductase; MOPS, 3-(N-morpholino)propanesulfonic acid; GdmCl, guanidinium chloride; PAGE, polyacrylamide gel electrophoresis; ATPγS, adenosine 5′-O-(3-thiotriphosphate). leads to structural changes in the intermediary hinge region and, unlike wild-type GroEL, makes it accessible to attack by proteinase K (21Martin J. Goldie K.N. Engel A. Hartl F.-U. Biol. Chem. Hoppe-Seyler. 1994; 375: 635-639PubMed Google Scholar). In this study, Cys138, a residue located in the intermediate domain, is identified as the target for NEM modification. As it seemed likely that NEM modification not only affects the structure, but has even more profound effects on the function of the GroEL chaperonin, this study analyzes the consequences of NEM modification for the ability of GroEL to successfully mediate the folding of substrate proteins. DISCUSSIONModification of GroEL with NEM changes the properties of the chaperonin profoundly, both structurally and functionally. Only one of the three cysteines in GroEL, Cys138, is modified by NEM. The small intermediate domain (residues 134–190 and 377–408), in which this residue resides, joins the apical domain of a GroEL subunit with its large equatorial domain. It has been proposed that this intermediate segment allows a hinge-like opening and twisting of the apical domain about the common domain junction (Gly192 and Gly375), which is caused by nucleotide binding in the equatorial domains and GroES binding to the apical domains of one GroEL ring (19Xu Z. Horwich A.L. Sigler P.B. Nature. 1997; 388: 741-749Crossref PubMed Scopus (1031) Google Scholar). As a result, the volume of the central GroEL chamber in the GroES-associated ring increases significantly, forming the cage in which protein folding proceeds. The now dome-shaped cavity is spacious enough to allow the enclosed protein to explore its folding pathway without direct interference from the chaperonin itself. In addition to moving upward the apical domain becomes twisted. The affinity of GroEL for unfolded polypeptide is strongly reduced when GroES is bound in the presence of nucleotides because rotation of the apical domains about the hinge between the apical and intermediate domains then occludes most of the hydrophobic-binding regions in the intersubunit interface. The opening movement of the apical domain is made possible by a downward movement of the intermediate segment toward the central channel which occurs at the domain junction between intermediate and equatorial domain around residues Pro137 and Gly410 (19Xu Z. Horwich A.L. Sigler P.B. Nature. 1997; 388: 741-749Crossref PubMed Scopus (1031) Google Scholar). Further evidence for the importance of that second hinge region comes from the present study showing that modification of Cys138 adjacent to Pro137 with a bulky NEM moiety interferes with this movement. Structural changes in the vicinity of Pro137 can explain not only the observations described here but also our findings from an earlier study (21Martin J. Goldie K.N. Engel A. Hartl F.-U. Biol. Chem. Hoppe-Seyler. 1994; 375: 635-639PubMed Google Scholar) where we analyzed fragments of NEM-GroEL that were obtained after proteolysis in the presence of nucleotides. Cleavage by proteinase K occurred between residues 143 and 153. Whereas Pro137 is highly conserved in members of the chaperonin family, one encounters more variability in the adjacent residue. In addition to Cys, one frequently finds Val at this position, and mutations to other small uncharged residues like Ala or Ser result in functional proteins (32Bochkareva E.S. Horovitz A. Girshovich A.S. J. Biol. Chem. 1994; 269: 44-46Abstract Full Text PDF PubMed Google Scholar, 33Luo G.-X. Horowitz P.M. J. Biol. Chem. 1994; 269: 32151-32154Abstract Full Text PDF PubMed Google Scholar, 34Yifrach O. Horovitz A. Biochemistry. 1995; 34: 5303-5308Crossref PubMed Scopus (263) Google Scholar). However, when we attempted to mimic the NEM effect by exchanging Cys138 to the larger hydrophobic residues Trp or Phe, we found defects similar to that observed with the chemically modified chaperonin. 3J. L. Mark and J. Martin, unpublished results. It appears that only modification with bulky hydrophobic groups perturbs the structural integrity in the complex in such a way that proteinase K gains access to an otherwise inaccessible region.The primary consequences of modification of the critical residue Cys138 in NEM-GroEL are changes in nucleotide binding, rate and cooperativity of ATP hydrolysis, and GroES binding. NEM-GroEL has an increased ATPase activity and binds nucleotides with higher affinity than wild-type GroEL. This phenotype is unique among the various GroEL mutants that have been generated so far in that none of these analyzed mutants is characterized by an accelerated rate of ATP hydrolysis (6Fenton W.A. Kashi Y. Furtak K. Horwich A.L. Nature. 1994; 371: 614-619Crossref PubMed Scopus (569) Google Scholar); typically, the rate is reduced. In a GroEL·GroES complex the downward movement around Pro137 impedes dissociation of ADP, locking the chaperonin complex in the nucleotide-bound state. It is conceivable that NEM·GroEL adopts a conformation that mimics part of this movement and thereby leads to an increase in nucleotide affinity. This conformational switch could bring Cys138 closer to the nucleotide-binding site, explaining earlier data from cross-linking experiments with ATPγS that had placed Cys138 in the vicinity of the ATP phosphate groups (32Bochkareva E.S. Horovitz A. Girshovich A.S. J. Biol. Chem. 1994; 269: 44-46Abstract Full Text PDF PubMed Google Scholar). Remarkably, despite its higer affinity for nucleotides, the affinity of NEM-GroEL for GroES is reduced compared with wild-type GroEL. This result demonstrates that a tighter binding of ATP or ADP to the chaperonin does not automatically correlate with better binding of the chaperonin cofactor as expected. As a consequence of the weak interaction of NEM-GroEL with GroES, the modified chaperonin is compromised in its ability to promote protein folding at 25 °C. Substrate proteins are likely to make contact with GroEL at several of the seven subunits in the cylindrical ring. A coordinated release of substrate from all these sites during ATP hydrolysis would give the protein time to partially fold within the cavity until the high affinity ADP state is regenerated. Without GroES, nucleotide binding and ATP hydrolysis by the individual GroEL subunits result in nonproductive release of folding intermediates into the bulk solution. For smaller proteins that can fold relatively easily, such as DHFR, release per se may be sufficient. In contrast, for those substrates that are more likely to engage in non-productive intermolecular interactions and are prone to misfolding, such as rhodanese, full coordination of the conformational changes in the individual subunits and the presence of GroES is required for a productive release into the chaperonin cavity. Unsynchronized release of substrate protein, as in NEM-GroEL, would shorten the time window for folding, because at any given time a binding site in NEM-GroEL would be competent for (re)binding the substrate. GroES binding to NEM-GroEL may facilitate coordinated release at least to such a degree that is sufficient for the release and folding of the weakly binding substrate DHFR, whose reactivation consequently becomes GroES-dependent. However, for tight binding substrate proteins like rhodanese, the GroES interaction with NEM-GroEL is still too weak to promote their efficient displacement into the cavity. At 37 °C, several effects may contribute to the partially restored chaperonin activity of NEM-GroEL. First, folding of substrate proteins is expected to occur with faster kinetics, which might enable the folding proteins to escape an untimely rebinding to NEM-GroEL. Second, the conformational changes induced by NEM modification may be less pronounced at 37 °C, or may affect the chaperonin conformation less than at 25 °C due to an increased flexibility of the chaperonin structure. Third, and most importantly, there is a strong improvement in GroES binding to NEM-GroEL at higher temperatures. A firmly anchored GroES is required to displace the substrate protein from its binding site at GroEL which partially overlaps with that of the smaller cofactor. In fact, it appears that this aspect of GroES function is the most critical one, more important than a precise regulation of the GroEL ATPase activity (35Hayer-Hartl M.K. Weber F. Hartl F.U. EMBO J. 1996; 15: 6111-6121Crossref PubMed Scopus (132) Google Scholar). High affinity binding of GroES to the apical domain after nucleotide binding to the central domain would be made possible by the intermediate domain, coupling not only these two events, but also ATP hydrolysis to productive protein release. Modification of GroEL with NEM changes the properties of the chaperonin profoundly, both structurally and functionally. Only one of the three cysteines in GroEL, Cys138, is modified by NEM. The small intermediate domain (residues 134–190 and 377–408), in which this residue resides, joins the apical domain of a GroEL subunit with its large equatorial domain. It has been proposed that this intermediate segment allows a hinge-like opening and twisting of the apical domain about the common domain junction (Gly192 and Gly375), which is caused by nucleotide binding in the equatorial domains and GroES binding to the apical domains of one GroEL ring (19Xu Z. Horwich A.L. Sigler P.B. Nature. 1997; 388: 741-749Crossref PubMed Scopus (1031) Google Scholar). As a result, the volume of the central GroEL chamber in the GroES-associated ring increases significantly, forming the cage in which protein folding proceeds. The now dome-shaped cavity is spacious enough to allow the enclosed protein to explore its folding pathway without direct interference from the chaperonin itself. In addition to moving upward the apical domain becomes twisted. The affinity of GroEL for unfolded polypeptide is strongly reduced when GroES is bound in the presence of nucleotides because rotation of the apical domains about the hinge between the apical and intermediate domains then occludes most of the hydrophobic-binding regions in the intersubunit interface. The opening movement of the apical domain is made possible by a downward movement of the intermediate segment toward the central channel which occurs at the domain junction between intermediate and equatorial domain around residues Pro137 and Gly410 (19Xu Z. Horwich A.L. Sigler P.B. Nature. 1997; 388: 741-749Crossref PubMed Scopus (1031) Google Scholar). Further evidence for the importance of that second hinge region comes from the present study showing that modification of Cys138 adjacent to Pro137 with a bulky NEM moiety interferes with this movement. Structural changes in the vicinity of Pro137 can explain not only the observations described here but also our findings from an earlier study (21Martin J. Goldie K.N. Engel A. Hartl F.-U. Biol. Chem. Hoppe-Seyler. 1994; 375: 635-639PubMed Google Scholar) where we analyzed fragments of NEM-GroEL that were obtained after proteolysis in the presence of nucleotides. Cleavage by proteinase K occurred between residues 143 and 153. Whereas Pro137 is highly conserved in members of the chaperonin family, one encounters more variability in the adjacent residue. In addition to Cys, one frequently finds Val at this position, and mutations to other small uncharged residues like Ala or Ser result in functional proteins (32Bochkareva E.S. Horovitz A. Girshovich A.S. J. Biol. Chem. 1994; 269: 44-46Abstract Full Text PDF PubMed Google Scholar, 33Luo G.-X. Horowitz P.M. J. Biol. Chem. 1994; 269: 32151-32154Abstract Full Text PDF PubMed Google Scholar, 34Yifrach O. Horovitz A. Biochemistry. 1995; 34: 5303-5308Crossref PubMed Scopus (263) Google Scholar). However, when we attempted to mimic the NEM effect by exchanging Cys138 to the larger hydrophobic residues Trp or Phe, we found defects similar to that observed with the chemically modified chaperonin. 3J. L. Mark and J. Martin, unpublished results. It appears that only modification with bulky hydrophobic groups perturbs the structural integrity in the complex in such a way that proteinase K gains access to an otherwise inaccessible region. The primary consequences of modification of the critical residue Cys138 in NEM-GroEL are changes in nucleotide binding, rate and cooperativity of ATP hydrolysis, and GroES binding. NEM-GroEL has an increased ATPase activity and binds nucleotides with higher affinity than wild-type GroEL. This phenotype is unique among the various GroEL mutants that have been generated so far in that none of these analyzed mutants is characterized by an accelerated rate of ATP hydrolysis (6Fenton W.A. Kashi Y. Furtak K. Horwich A.L. Nature. 1994; 371: 614-619Crossref PubMed Scopus (569) Google Scholar); typically, the rate is reduced. In a GroEL·GroES complex the downward movement around Pro137 impedes dissociation of ADP, locking the chaperonin complex in the nucleotide-bound state. It is conceivable that NEM·GroEL adopts a conformation that mimics part of this movement and thereby leads to an increase in nucleotide affinity. This conformational switch could bring Cys138 closer to the nucleotide-binding site, explaining earlier data from cross-linking experiments with ATPγS that had placed Cys138 in the vicinity of the ATP phosphate groups (32Bochkareva E.S. Horovitz A. Girshovich A.S. J. Biol. Chem. 1994; 269: 44-46Abstract Full Text PDF PubMed Google Scholar). Remarkably, despite its higer affinity for nucleotides, the affinity of NEM-GroEL for GroES is reduced compared with wild-type GroEL. This result demonstrates that a tighter binding of ATP or ADP to the chaperonin does not automatically correlate with better binding of the chaperonin cofactor as expected. As a consequence of the weak interaction of NEM-GroEL with GroES, the modified chaperonin is compromised in its ability to promote protein folding at 25 °C. Substrate proteins are likely to make contact with GroEL at several of the seven subunits in the cylindrical ring. A coordinated release of substrate from all these sites during ATP hydrolysis would give the protein time to partially fold within the cavity until the high affinity ADP state is regenerated. Without GroES, nucleotide binding and ATP hydrolysis by the individual GroEL subunits result in nonproductive release of folding intermediates into the bulk solution. For smaller proteins that can fold relatively easily, such as DHFR, release per se may be sufficient. In contrast, for those substrates that are more likely to engage in non-productive intermolecular interactions and are prone to misfolding, such as rhodanese, full coordination of the conformational changes in the individual subunits and the presence of GroES is required for a productive release into the chaperonin cavity. Unsynchronized release of substrate protein, as in NEM-GroEL, would shorten the time window for folding, because at any given time a binding site in NEM-GroEL would be competent for (re)binding the substrate. GroES binding to NEM-GroEL may facilitate coordinated release at least to such a degree that is sufficient for the release and folding of the weakly binding substrate DHFR, whose reactivation consequently becomes GroES-dependent. However, for tight binding substrate proteins like rhodanese, the GroES interaction with NEM-GroEL is still too weak to promote their efficient displacement into the cavity. At 37 °C, several effects may contribute to the partially restored chaperonin activity of NEM-GroEL. First, folding of substrate proteins is expected to occur with faster kinetics, which might enable the folding proteins to escape an untimely rebinding to NEM-GroEL. Second, the conformational changes induced by NEM modification may be less pronounced at 37 °C, or may affect the chaperonin conformation less than at 25 °C due to an increased flexibility of the chaperonin structure. Third, and most importantly, there is a strong improvement in GroES binding to NEM-GroEL at higher temperatures. A firmly anchored GroES is required to displace the substrate protein from its binding site at GroEL which partially overlaps with that of the smaller cofactor. In fact, it appears that this aspect of GroES function is the most critical one, more important than a precise regulation of the GroEL ATPase activity (35Hayer-Hartl M.K. Weber F. Hartl F.U. EMBO J. 1996; 15: 6111-6121Crossref PubMed Scopus (132) Google Scholar). High affinity binding of GroES to the apical domain after nucleotide binding to the central domain would be made possible by the intermediate domain, coupling not only these two events, but also ATP hydrolysis to productive protein release. This work was begun in the laboratory of Dr. Ulrich Hartl to whom I am grateful for initial support. I also thank Drs. Carol Robinson and Sheena Radford for performing mass spectrometry analysis of the NEM-modified GroEL form, and Dr. John Biggins for critically reading the manuscript.