The Charged Linker Region Is an Important Regulator of Hsp90 Function
2009; Elsevier BV; Volume: 284; Issue: 34 Linguagem: Inglês
10.1074/jbc.m109.031658
ISSN1083-351X
AutoresOtmar Hainzl, Maria Claribel Lapina, Johannes Büchner, Klaus Richter,
Tópico(s)Bacillus and Francisella bacterial research
ResumoHsp90 is an ATP-dependent molecular chaperone which assists the maturation of a large set of target proteins. Members of the highly conserved Hsp90 family are found from bacteria to higher eukaryotes, with homologues in different organelles. The core architecture of Hsp90 is defined by the N-terminal ATP binding domain followed by the middle domain and the C-terminal dimerization domain. A long, highly charged linker between the N-terminal domain and the middle domain is a feature characteristic for Hsp90s of eukaryotic organisms. We set out to clarify the function of this linker by studying the effects of deletions in this region in vivo and in vitro. Here we show that increasing deletions in the charged linker region lead to defects ranging from mild temperature sensitivity to a lethal phenotype. The lethal deletion variants investigated in this study still exhibit ATPase activity. Deletion of the charged linker ultimately causes a loss of Hsp90 regulation by co-chaperones, as the sensitivity for Aha1-mediated ATPase acceleration declines, and binding of p23/Sba1 is lost in non-viable deletion constructs. In vivo client assays additionally demonstrated that the deletion of the linker had a pronounced effect on the ability of Hsp90 to facilitate client activation. A partial reconstruction of the linker sequence showed that the supplementation by artificial sequences can rescue the functionality of Hsp90 and restore the conformational flexibility of the protein, required for the processing of client proteins. Hsp90 is an ATP-dependent molecular chaperone which assists the maturation of a large set of target proteins. Members of the highly conserved Hsp90 family are found from bacteria to higher eukaryotes, with homologues in different organelles. The core architecture of Hsp90 is defined by the N-terminal ATP binding domain followed by the middle domain and the C-terminal dimerization domain. A long, highly charged linker between the N-terminal domain and the middle domain is a feature characteristic for Hsp90s of eukaryotic organisms. We set out to clarify the function of this linker by studying the effects of deletions in this region in vivo and in vitro. Here we show that increasing deletions in the charged linker region lead to defects ranging from mild temperature sensitivity to a lethal phenotype. The lethal deletion variants investigated in this study still exhibit ATPase activity. Deletion of the charged linker ultimately causes a loss of Hsp90 regulation by co-chaperones, as the sensitivity for Aha1-mediated ATPase acceleration declines, and binding of p23/Sba1 is lost in non-viable deletion constructs. In vivo client assays additionally demonstrated that the deletion of the linker had a pronounced effect on the ability of Hsp90 to facilitate client activation. A partial reconstruction of the linker sequence showed that the supplementation by artificial sequences can rescue the functionality of Hsp90 and restore the conformational flexibility of the protein, required for the processing of client proteins. Hsp90 3The abbreviations used are: Hsp90heat shock protein 90HSP82the S. cerevisiae gene for Hsp90Hsp90 ΔLinkeryeast Hsp90 after deletion of the charged region (amino acids 211–259)HtpGHsp90 from E. coli5′-FOA5′-fluoroorotic acidFRETfluorescence resonance energy transferGRglucocorticoid receptorSPRsurface plasmon resonancewtwild typeATPγSadenosine 5′-O-(thiotriphosphate)MP-PNPadenosine 5′-(β,γ-imino)triphosphateTPRtetratricopeptide repeat. 3The abbreviations used are: Hsp90heat shock protein 90HSP82the S. cerevisiae gene for Hsp90Hsp90 ΔLinkeryeast Hsp90 after deletion of the charged region (amino acids 211–259)HtpGHsp90 from E. coli5′-FOA5′-fluoroorotic acidFRETfluorescence resonance energy transferGRglucocorticoid receptorSPRsurface plasmon resonancewtwild typeATPγSadenosine 5′-O-(thiotriphosphate)MP-PNPadenosine 5′-(β,γ-imino)triphosphateTPRtetratricopeptide repeat. is an ATP-dependent molecular chaperone present in the cytosol of eubacteria and eucaryotes. It regulates the maturation and activation of numerous proteins involved in signal transduction, cell cycle control, hormone signaling, and transcription (1Pearl L.H. Prodromou C. Annu. Rev. Biochem. 2006; 75: 271-294Crossref PubMed Scopus (885) Google Scholar, 2Picard D. Cell. Mol. Life Sci. 2002; 59: 1640-1648Crossref PubMed Scopus (652) Google Scholar, 3Pratt W.B. Morishima Y. Murphy M. Harrell M. Handb. Exp. Pharmacol. 2006; 172: 111-138Crossref PubMed Scopus (5) Google Scholar). Recent crystal structures of HtpG from Escherichia coli and Hsp90 from Saccharomyces cerevisiae show that the overall structural organization of the proteins is highly conserved (4Dollins D.E. Warren J.J. Immormino R.M. Gewirth D.T. Mol. Cell. 2007; 28: 41-56Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 5Shiau A.K. Harris S.F. Southworth D.R. Agard D.A. Cell. 2006; 127: 329-340Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 6Ali M.M. Roe S.M. Vaughan C.K. Meyer P. Panaretou B. Piper P.W. Prodromou C. Pearl L.H. Nature. 2006; 440: 1013-1017Crossref PubMed Scopus (696) Google Scholar). Both prokaryotic and eukaryotic Hsp90 proteins consist of an N-terminal ATP binding domain, a middle domain involved in client protein binding, and a C-terminal dimerization domain. During the ATPase cycle, large conformational changes in the Hsp90 dimer lead to the transient dimerization of the N-terminal domains and their association with the middle domains (7Prodromou C. Panaretou B. Chohan S. Siligardi G. O'Brien R. Ladbury J.E. Roe S.M. Piper P.W. Pearl L.H. EMBO J. 2000; 19: 4383-4392Crossref PubMed Google Scholar, 8Weikl T. Muschler P. Richter K. Veit T. Reinstein J. Buchner J. J. Mol. Biol. 2000; 303: 583-592Crossref PubMed Scopus (103) Google Scholar, 9Richter K. Reinstein J. Buchner J. J. Biol. Chem. 2002; 277: 44905-44910Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 10Richter K. Soroka J. Skalniak L. Leskovar A. Hessling M. Reinstein J. Buchner J. J. Biol. Chem. 2008; 283: 17757-17765Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 11Leskovar A. Wegele H. Werbeck N.D. Buchner J. Reinstein J. J. Biol. Chem. 2008; 283: 11677-11688Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 12Frey S. Leskovar A. Reinstein J. Buchner J. J. Biol. Chem. 2007; 282: 35612-35620Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Despite the conservation of the basic molecular architecture and the ATPase mechanism, there are major differences between Hsp90 from prokaryotes and eukaryotes. The most striking difference is the emergence of a large set of co-chaperones in eucaryotes that bind to Hsp90 and seem to modulate and expand its properties (13Wandinger S.K. Richter K. Buchner J. J. Biol. Chem. 2008; 283: 18473-18477Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar). Furthermore, in contrast to procaryotes, Hsp90 is an essential protein in eucaryotes (14Bardwell J.C. Craig E.A. J. Bacteriol. 1988; 170: 2977-2983Crossref PubMed Scopus (139) Google Scholar, 15Borkovich K.A. Farrelly F.W. Finkelstein D.B. Taulien J. Lindquist S. Mol. Cell. Biol. 1989; 9: 3919-3930Crossref PubMed Scopus (533) Google Scholar) and it had been shown in S. cerevisiae that ATP hydrolysis by Hsp90 is required for sustaining its essential function (16Obermann W.M. Sondermann H. Russo A.A. Pavletich N.P. Hartl F.U. J. Cell Biol. 1998; 143: 901-910Crossref PubMed Scopus (487) Google Scholar, 17Panaretou B. Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1998; 17: 4829-4836Crossref PubMed Scopus (613) Google Scholar, 18Richter K. Moser S. Hagn F. Friedrich R. Hainzl O. Heller M. Schlee S. Kessler H. Reinstein J. Buchner J. J. Biol. Chem. 2006; 281: 11301-11311Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Finally, eukaryotic Hsp90 contains additional structural elements compared with prokaryotic Hsp90; that is, a long charged linker between the N terminus and the middle domain and a conserved motif (MEEVD) at the extended C-terminal end of the eucaryotic protein. Although it has been established that the C-terminal motif is the interaction site for TPR domain containing co-chaperones (19Chen S. Sullivan W.P. Toft D.O. Smith D.F. Cell Stress Chaperones. 1998; 3: 118-129Crossref PubMed Scopus (166) Google Scholar, 20Prodromou C. Siligardi G. O'Brien R. Woolfson D.N. Regan L. Panaretou B. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1999; 18: 754-762Crossref PubMed Scopus (349) Google Scholar, 21Scheufler C. Brinker A. Bourenkov G. Pegoraro S. Moroder L. Bartunik H. Hartl F.U. Moarefi I. Cell. 2000; 101: 199-210Abstract Full Text Full Text PDF PubMed Scopus (1000) Google Scholar), the function of the charged linker is less than clear. In general terms, it seems to contribute to the flexibility of the protein as the successful crystallization of Hsp90 from S. cerevisiae required the linker to be shortened and modified (6Ali M.M. Roe S.M. Vaughan C.K. Meyer P. Panaretou B. Piper P.W. Prodromou C. Pearl L.H. Nature. 2006; 440: 1013-1017Crossref PubMed Scopus (696) Google Scholar). Hallmarks of this linker region are its low sequence complexity and the high amount of charged amino acids. In yeast Hsp90, the motif (D/E)(D/E)(D/E)KK is repeated five times in the linker region (22Farrelly F.W. Finkelstein D.B. J. Biol. Chem. 1984; 259: 5745-5751Abstract Full Text PDF PubMed Google Scholar). Various secondary structure prediction programs canonically predict the charged linker region as predominantly in a coiled-coil conformation (23Combet C. Blanchet C. Geourjon C. Deléage G. Trends Biochem. Sci. 2000; 25: 147-150Abstract Full Text Full Text PDF PubMed Scopus (1413) Google Scholar, 24Pollastri G. McLysaght A. Bioinformatics. 2005; 21: 1719-1720Crossref PubMed Scopus (381) Google Scholar). In a previous complementation study, it was shown that the charged linker is dispensable for the essential function of Hsp90 in S. cerevisiae, as a construct lacking residues 211–259 was still able to support cell proliferation (25Louvion J.F. Warth R. Picard D. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 13937-13942Crossref PubMed Scopus (96) Google Scholar). In this study we constructed and analyzed a set of variants of Hsp90 in which the linker region was successively shortened, with a view to determine its contribution to the mechanism of Hsp90 in vivo and in vitro.DISCUSSIONThe molecular chaperone Hsp90 is an ATP-driven machine whose conformational changes are required to perform a complex reaction cycle (31Hessling M. Richter K. Buchner J. Nat. Struct. Mol. Biol. 2009; 16: 287-293Crossref PubMed Scopus (249) Google Scholar). Comparing prokaryotic and eukaryotic Hsp90 homologues, two specific regions obviously differ strongly. One is a flexible linker region connecting the N-terminal (ATPase) and middle domains, and the other one is a C-terminal extension, which serves as the primary binding site for TPR domain-containing co-chaperones. Both regions are dispensable in yeast (25Louvion J.F. Warth R. Picard D. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 13937-13942Crossref PubMed Scopus (96) Google Scholar), which raised questions about their function. Especially, the function of the linker sequence remained enigmatic. We deleted this sequence to a different extent, including one version that did not have any linker sequence and two versions which had artificial amino acid stretches to connect the two domains. Our results highlight a minimal linker length required to retain viability. Interestingly, the full deletion of the linker, although leaving Hsp90 structurally intact and preserving ATP binding, does reduce ATP hydrolysis to values similar to those known from the isolated N-terminal domain/middle domain construct (53Wegele H. Muschler P. Bunck M. Reinstein J. Buchner J. J. Biol. Chem. 2003; 278: 39303-39310Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Hsp90 activity can be rescued when an artificial linker is introduced, implying that one function of the linker region is to contribute conformational freedom for connection of the N and M domains. It might well be that conformational changes at the N-M interface require this conformational space to rearrange productively during the ATPase cycle. This alone appears to be not sufficient given the observation that the amino acids 266–272 are required for purposes, which cannot be fully supplemented fully by a Gly-Ser stretch as judged by the lower ATP turnover of 211–272SUP compared with 211–266SUP and differences in Aha1 interaction. Analysis of x-ray structures of HtpG and Grp94 (4Dollins D.E. Warren J.J. Immormino R.M. Gewirth D.T. Mol. Cell. 2007; 28: 41-56Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 5Shiau A.K. Harris S.F. Southworth D.R. Agard D.A. Cell. 2006; 127: 329-340Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar) suggests that these amino acid participate in the formation of a β-sheet-like structure with the N-terminal domain. However, this is difficult to decide based on the crystal structures, as both proteins were strongly mutated within the linker region to allow crystallization. Nevertheless, the participation of this sequence in a β-sheet structure could explain the requirement for certain amino acids within this region.The analysis of the co-chaperone interaction with the Hsp90 linker deletion variants allowed us to further scrutinize the function of the linker region. Some co-chaperones can be used as conformational sensors as they associate with specific states of Hsp90 during the ATPase cycle (45Richter K. Muschler P. Hainzl O. Reinstein J. Buchner J. J. Biol. Chem. 2003; 278: 10328-10333Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 46Meyer P. Prodromou C. Liao C. Hu B. Mark R.S. Vaughan C.K. Vlasic I. Panaretou B. Piper P.W. Pearl L.H. EMBO J. 2004; 23: 511-519Crossref PubMed Scopus (192) Google Scholar, 51Richter K. Walter S. Buchner J. J. Mol. Biol. 2004; 342: 1403-1413Crossref PubMed Scopus (119) Google Scholar). The interaction with Sti1 was not significantly influenced by the linker deletions. This may be explained with the relative flexibility of the adaptor protein Sti1, which could compensate for a changed domain orientation in the charged linker constructs of Hsp90. The data for the p23/Sba1 interaction on the other hand suggest that the conformational changes leading to the N-terminal-dimerized state of Hsp90 seem to be inhibited by the linker deletion. This conformation is induced upon ATP binding (7Prodromou C. Panaretou B. Chohan S. Siligardi G. O'Brien R. Ladbury J.E. Roe S.M. Piper P.W. Pearl L.H. EMBO J. 2000; 19: 4383-4392Crossref PubMed Google Scholar). The conformational changes leading to this state are rate-limiting during the hydrolysis cycle (9Richter K. Reinstein J. Buchner J. J. Biol. Chem. 2002; 277: 44905-44910Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Apparently, the deletion of the linker slows the repositioning of the N and the M domain at the N-M interface. Interestingly, also the ability of Aha1 to stimulate the ATP turnover is strongly reduced in the linker variants or even absent in the maximum deletion construct. Our data indicate that the Aha1-induced ATPase stimulation is only possible with unconstrained N-M interactions. It is reasonable to assume that if this interaction is sterically inhibited, Aha1 can no longer exert stimulatory effects even though Aha1 binding itself is not affected.To date, the ability to hydrolyze ATP was believed to be an essential prerequisite for in vivo functionality of Hsp90 wt as ATPase-defective mutants are not able to replace Hsp90 in an Hsp90-deficient background (16Obermann W.M. Sondermann H. Russo A.A. Pavletich N.P. Hartl F.U. J. Cell Biol. 1998; 143: 901-910Crossref PubMed Scopus (487) Google Scholar, 17Panaretou B. Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1998; 17: 4829-4836Crossref PubMed Scopus (613) Google Scholar). Our results indicate that ATP hydrolysis alone is not sufficient for viability. Also, conformationally restricted but ATPase-active Hsp90 versions are unable to replace Hsp90 wt. The unrestricted motility of the N-M interface seems required for in vivo functions even more than for the ATP turnover. This is true even for the smallest deletions used in this study, which are not affected in their ATP turnover yet, but their chaperone effect on known in vivo clients of Hsp90, like the GR, luciferase, and v-Src kinase, is diminished. The severely compromised yeast strains carrying the Hsp90 linker constructs show a reduced ability to interact with Hsp90 clients, consistent with the view that the charged linker region is involved in Hsp90 chaperone function (54Scheibel T. Siegmund H.I. Jaenicke R. Ganz P. Lilie H. Buchner J. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 1297-1302Crossref PubMed Scopus (97) Google Scholar). The recent structure of the kinase Cdk4 in complex with Hsp90-Cdc37 implies client interactions, which span the N-terminal part of Hsp90 and the middle domain (55Vaughan C.K. Gohlke U. Sobott F. Good V.M. Ali M.M. Prodromou C. Robinson C.V. Saibil H.R. Pearl L.H. Mol. Cell. 2006; 23: 697-707Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). It might very well be that the conformational flexibility required at the N-M interface is even more pronounced in the case of client binding as compared with ATP hydrolysis. The strongly extended linker region, which can be found in all eukaryotic Hsp90 proteins, might therefore result from the need of a highly flexible contact surface that is able to integrate various client proteins into the Hsp90 dimer structure. Hsp90 3The abbreviations used are: Hsp90heat shock protein 90HSP82the S. cerevisiae gene for Hsp90Hsp90 ΔLinkeryeast Hsp90 after deletion of the charged region (amino acids 211–259)HtpGHsp90 from E. coli5′-FOA5′-fluoroorotic acidFRETfluorescence resonance energy transferGRglucocorticoid receptorSPRsurface plasmon resonancewtwild typeATPγSadenosine 5′-O-(thiotriphosphate)MP-PNPadenosine 5′-(β,γ-imino)triphosphateTPRtetratricopeptide repeat. 3The abbreviations used are: Hsp90heat shock protein 90HSP82the S. cerevisiae gene for Hsp90Hsp90 ΔLinkeryeast Hsp90 after deletion of the charged region (amino acids 211–259)HtpGHsp90 from E. coli5′-FOA5′-fluoroorotic acidFRETfluorescence resonance energy transferGRglucocorticoid receptorSPRsurface plasmon resonancewtwild typeATPγSadenosine 5′-O-(thiotriphosphate)MP-PNPadenosine 5′-(β,γ-imino)triphosphateTPRtetratricopeptide repeat. is an ATP-dependent molecular chaperone present in the cytosol of eubacteria and eucaryotes. It regulates the maturation and activation of numerous proteins involved in signal transduction, cell cycle control, hormone signaling, and transcription (1Pearl L.H. Prodromou C. Annu. Rev. Biochem. 2006; 75: 271-294Crossref PubMed Scopus (885) Google Scholar, 2Picard D. Cell. Mol. Life Sci. 2002; 59: 1640-1648Crossref PubMed Scopus (652) Google Scholar, 3Pratt W.B. Morishima Y. Murphy M. Harrell M. Handb. Exp. Pharmacol. 2006; 172: 111-138Crossref PubMed Scopus (5) Google Scholar). Recent crystal structures of HtpG from Escherichia coli and Hsp90 from Saccharomyces cerevisiae show that the overall structural organization of the proteins is highly conserved (4Dollins D.E. Warren J.J. Immormino R.M. Gewirth D.T. Mol. Cell. 2007; 28: 41-56Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 5Shiau A.K. Harris S.F. Southworth D.R. Agard D.A. Cell. 2006; 127: 329-340Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 6Ali M.M. Roe S.M. Vaughan C.K. Meyer P. Panaretou B. Piper P.W. Prodromou C. Pearl L.H. Nature. 2006; 440: 1013-1017Crossref PubMed Scopus (696) Google Scholar). Both prokaryotic and eukaryotic Hsp90 proteins consist of an N-terminal ATP binding domain, a middle domain involved in client protein binding, and a C-terminal dimerization domain. During the ATPase cycle, large conformational changes in the Hsp90 dimer lead to the transient dimerization of the N-terminal domains and their association with the middle domains (7Prodromou C. Panaretou B. Chohan S. Siligardi G. O'Brien R. Ladbury J.E. Roe S.M. Piper P.W. Pearl L.H. EMBO J. 2000; 19: 4383-4392Crossref PubMed Google Scholar, 8Weikl T. Muschler P. Richter K. Veit T. Reinstein J. Buchner J. J. Mol. Biol. 2000; 303: 583-592Crossref PubMed Scopus (103) Google Scholar, 9Richter K. Reinstein J. Buchner J. J. Biol. Chem. 2002; 277: 44905-44910Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 10Richter K. Soroka J. Skalniak L. Leskovar A. Hessling M. Reinstein J. Buchner J. J. Biol. Chem. 2008; 283: 17757-17765Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 11Leskovar A. Wegele H. Werbeck N.D. Buchner J. Reinstein J. J. Biol. Chem. 2008; 283: 11677-11688Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 12Frey S. Leskovar A. Reinstein J. Buchner J. J. Biol. Chem. 2007; 282: 35612-35620Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Despite the conservation of the basic molecular architecture and the ATPase mechanism, there are major differences between Hsp90 from prokaryotes and eukaryotes. The most striking difference is the emergence of a large set of co-chaperones in eucaryotes that bind to Hsp90 and seem to modulate and expand its properties (13Wandinger S.K. Richter K. Buchner J. J. Biol. Chem. 2008; 283: 18473-18477Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar). Furthermore, in contrast to procaryotes, Hsp90 is an essential protein in eucaryotes (14Bardwell J.C. Craig E.A. J. Bacteriol. 1988; 170: 2977-2983Crossref PubMed Scopus (139) Google Scholar, 15Borkovich K.A. Farrelly F.W. Finkelstein D.B. Taulien J. Lindquist S. Mol. Cell. Biol. 1989; 9: 3919-3930Crossref PubMed Scopus (533) Google Scholar) and it had been shown in S. cerevisiae that ATP hydrolysis by Hsp90 is required for sustaining its essential function (16Obermann W.M. Sondermann H. Russo A.A. Pavletich N.P. Hartl F.U. J. Cell Biol. 1998; 143: 901-910Crossref PubMed Scopus (487) Google Scholar, 17Panaretou B. Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1998; 17: 4829-4836Crossref PubMed Scopus (613) Google Scholar, 18Richter K. Moser S. Hagn F. Friedrich R. Hainzl O. Heller M. Schlee S. Kessler H. Reinstein J. Buchner J. J. Biol. Chem. 2006; 281: 11301-11311Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Finally, eukaryotic Hsp90 contains additional structural elements compared with prokaryotic Hsp90; that is, a long charged linker between the N terminus and the middle domain and a conserved motif (MEEVD) at the extended C-terminal end of the eucaryotic protein. Although it has been established that the C-terminal motif is the interaction site for TPR domain containing co-chaperones (19Chen S. Sullivan W.P. Toft D.O. Smith D.F. Cell Stress Chaperones. 1998; 3: 118-129Crossref PubMed Scopus (166) Google Scholar, 20Prodromou C. Siligardi G. O'Brien R. Woolfson D.N. Regan L. Panaretou B. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1999; 18: 754-762Crossref PubMed Scopus (349) Google Scholar, 21Scheufler C. Brinker A. Bourenkov G. Pegoraro S. Moroder L. Bartunik H. Hartl F.U. Moarefi I. Cell. 2000; 101: 199-210Abstract Full Text Full Text PDF PubMed Scopus (1000) Google Scholar), the function of the charged linker is less than clear. In general terms, it seems to contribute to the flexibility of the protein as the successful crystallization of Hsp90 from S. cerevisiae required the linker to be shortened and modified (6Ali M.M. Roe S.M. Vaughan C.K. Meyer P. Panaretou B. Piper P.W. Prodromou C. Pearl L.H. Nature. 2006; 440: 1013-1017Crossref PubMed Scopus (696) Google Scholar). Hallmarks of this linker region are its low sequence complexity and the high amount of charged amino acids. In yeast Hsp90, the motif (D/E)(D/E)(D/E)KK is repeated five times in the linker region (22Farrelly F.W. Finkelstein D.B. J. Biol. Chem. 1984; 259: 5745-5751Abstract Full Text PDF PubMed Google Scholar). Various secondary structure prediction programs canonically predict the charged linker region as predominantly in a coiled-coil conformation (23Combet C. Blanchet C. Geourjon C. Deléage G. Trends Biochem. Sci. 2000; 25: 147-150Abstract Full Text Full Text PDF PubMed Scopus (1413) Google Scholar, 24Pollastri G. McLysaght A. Bioinformatics. 2005; 21: 1719-1720Crossref PubMed Scopus (381) Google Scholar). In a previous complementation study, it was shown that the charged linker is dispensable for the essential function of Hsp90 in S. cerevisiae, as a construct lacking residues 211–259 was still able to support cell proliferation (25Louvion J.F. Warth R. Picard D. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 13937-13942Crossref PubMed Scopus (96) Google Scholar). In this study we constructed and analyzed a set of variants of Hsp90 in which the linker region was successively shortened, with a view to determine its contribution to the mechanism of Hsp90 in vivo and in vitro. heat shock protein 90 the S. cerevisiae gene for Hsp90 yeast Hsp90 after deletion of the charged region (amino acids 211–259) Hsp90 from E. coli 5′-fluoroorotic acid fluorescence resonance energy transfer glucocorticoid receptor surface plasmon resonance wild type adenosine 5′-O-(thiotriphosphate) adenosine 5′-(β,γ-imino)triphosphate tetratricopeptide repeat. heat shock protein 90 the S. cerevisiae gene for Hsp90 yeast Hsp90 after deletion of the charged region (amino acids 211–259) Hsp90 from E. coli 5′-fluoroorotic acid fluorescence resonance energy transfer glucocorticoid receptor surface plasmon resonance wild type adenosine 5′-O-(thiotriphosphate) adenosine 5′-(β,γ-imino)triphosphate tetratricopeptide repeat. DISCUSSIONThe molecular chaperone Hsp90 is an ATP-driven machine whose conformational changes are required to perform a complex reaction cycle (31Hessling M. Richter K. Buchner J. Nat. Struct. Mol. Biol. 2009; 16: 287-293Crossref PubMed Scopus (249) Google Scholar). Comparing prokaryotic and eukaryotic Hsp90 homologues, two specific regions obviously differ strongly. One is a flexible linker region connecting the N-terminal (ATPase) and middle domains, and the other one is a C-terminal extension, which serves as the primary binding site for TPR domain-containing co-chaperones. Both regions are dispensable in yeast (25Louvion J.F. Warth R. Picard D. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 13937-13942Crossref PubMed Scopus (96) Google Scholar), which raised questions about their function. Especially, the function of the linker sequence remained enigmatic. We deleted this sequence to a different extent, including one version that did not have any linker sequence and two versions which had artificial amino acid stretches to connect the two domains. Our results highlight a minimal linker length required to retain viability. Interestingly, the full deletion of the linker, although leaving Hsp90 structurally intact and preserving ATP binding, does reduce ATP hydrolysis to values similar to those known from the isolated N-terminal domain/middle domain construct (53Wegele H. Muschler P. Bunck M. Reinstein J. Buchner J. J. Biol. Chem. 2003; 278: 39303-39310Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Hsp90 activity can be rescued when an artificial linker is introduced, implying that one function of the linker region is to contribute conformational freedom for connection of the N and M domains. It might well be that conformational changes at the N-M interface require this conformational space to rearrange productively during the ATPase cycle. This alone appears to be not sufficient given the observation that the amino acids 266–272 are required for purposes, which cannot be fully supplemented fully by a Gly-Ser stretch as judged by the lower ATP turnover of 211–272SUP compared with 211–266SUP and differences in Aha1 interaction. Analysis of x-ray structures of HtpG and Grp94 (4Dollins D.E. Warren J.J. Immormino R.M. Gewirth D.T. Mol. Cell. 2007; 28: 41-56Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 5Shiau A.K. Harris S.F. Southworth D.R. Agard D.A. Cell. 2006; 127: 329-340Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar) suggests that these amino acid participate in the formation of a β-sheet-like structure with the N-terminal domain. However, this is difficult to decide based on the crystal structures, as both proteins were strongly mutated within the linker region to allow crystallization. Nevertheless, the participation of this sequence in a β-sheet structure could explain the requirement for certain amino acids within this region.The analysis of the co-chaperone interaction with the Hsp90 linker deletion variants allowed us to further scrutinize the function of the linker region. Some co-chaperones can be used as conformational sensors as they associate with specific states of Hsp90 during the ATPase cycle (45Richter K. Muschler P. Hainzl O. Reinstein J. Buchner J. J. Biol. Chem. 2003; 278: 10328-10333Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 46Meyer P. Prodromou C. Liao C. Hu B. Mark R.S. Vaughan C.K. Vlasic I. Panaretou B. Piper P.W. Pearl L.H. EMBO J. 2004; 23: 511-519Crossref PubMed Scopus (192) Google Scholar, 51Richter K. Walter S. Buchner J. J. Mol. Biol. 2004; 342: 1403-1413Crossref PubMed Scopus (119) Google Scholar). The interaction with Sti1 was not significantly influenced by the linker deletions. This may be explained with the relative flexibility of the adaptor protein Sti1, which could compensate for a changed domain orientation in the charged linker constructs of Hsp90. The data for the p23/Sba1 interaction on the other hand suggest that the conformational changes leading to the N-terminal-dimerized state of Hsp90 seem to be inhibited by the linker deletion. This conformation is induced upon ATP binding (7Prodromou C. Panaretou B. Chohan S. Siligardi G. O'Brien R. Ladbury J.E. Roe S.M. Piper P.W. Pearl L.H. EMBO J. 2000; 19: 4383-4392Crossref PubMed Google Scholar). The conformational changes leading to this state are rate-limiting during the hydrolysis cycle (9Richter K. Reinstein J. Buchner J. J. Biol. Chem. 2002; 277: 44905-44910Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Apparently, the deletion of the linker slows the repositioning of the N and the M domain at the N-M interface. Interestingly, also the ability of Aha1 to stimulate the ATP turnover is strongly reduced in the linker variants or even absent in the maximum deletion construct. Our data indicate that the Aha1-induced ATPase stimulation is only possible with unconstrained N-M interactions. It is reasonable to assume that if this interaction is sterically inhibited, Aha1 can no longer exert stimulatory effects even though Aha1 binding itself is not affected.To date, the ability to hydrolyze ATP was believed to be an essential prerequisite for in vivo functionality of Hsp90 wt as ATPase-defective mutants are not able to replace Hsp90 in an Hsp90-deficient background (16Obermann W.M. Sondermann H. Russo A.A. Pavletich N.P. Hartl F.U. J. Cell Biol. 1998; 143: 901-910Crossref PubMed Scopus (487) Google Scholar, 17Panaretou B. Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1998; 17: 4829-4836Crossref PubMed Scopus (613) Google Scholar). Our results indicate that ATP hydrolysis alone is not sufficient for viability. Also, conformationally restricted but ATPase-active Hsp90 versions are unable to replace Hsp90 wt. The unrestricted motility of the N-M interface seems required for in vivo functions even more than for the ATP turnover. This is true even for the smallest deletions used in this study, which are not affected in their ATP turnover yet, but their chaperone effect on known in vivo clients of Hsp90, like the GR, luciferase, and v-Src kinase, is diminished. The severely compromised yeast strains carrying the Hsp90 linker constructs show a reduced ability to interact with Hsp90 clients, consistent with the view that the charged linker region is involved in Hsp90 chaperone function (54Scheibel T. Siegmund H.I. Jaenicke R. Ganz P. Lilie H. Buchner J. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 1297-1302Crossref PubMed Scopus (97) Google Scholar). The recent structure of the kinase Cdk4 in complex with Hsp90-Cdc37 implies client interactions, which span the N-terminal part of Hsp90 and the middle domain (55Vaughan C.K. Gohlke U. Sobott F. Good V.M. Ali M.M. Prodromou C. Robinson C.V. Saibil H.R. Pearl L.H. Mol. Cell. 2006; 23: 697-707Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). It might very well be that the conformational flexibility required at the N-M interface is even more pronounced in the case of client binding as compared with ATP hydrolysis. The strongly extended linker region, which can be found in all eukaryotic Hsp90 proteins, might therefore result from the need of a highly flexible contact surface that is able to integrate various client proteins into the Hsp90 dimer structure. The molecular chaperone Hsp90 is an ATP-driven machine whose conformational changes are required to perform a complex reaction cycle (31Hessling M. Richter K. Buchner J. Nat. Struct. Mol. Biol. 2009; 16: 287-293Crossref PubMed Scopus (249) Google Scholar). Comparing prokaryotic and eukaryotic Hsp90 homologues, two specific regions obviously differ strongly. One is a flexible linker region connecting the N-terminal (ATPase) and middle domains, and the other one is a C-terminal extension, which serves as the primary binding site for TPR domain-containing co-chaperones. Both regions are dispensable in yeast (25Louvion J.F. Warth R. Picard D. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 13937-13942Crossref PubMed Scopus (96) Google Scholar), which raised questions about their function. Especially, the function of the linker sequence remained enigmatic. We deleted this sequence to a different extent, including one version that did not have any linker sequence and two versions which had artificial amino acid stretches to connect the two domains. Our results highlight a minimal linker length required to retain viability. Interestingly, the full deletion of the linker, although leaving Hsp90 structurally intact and preserving ATP binding, does reduce ATP hydrolysis to values similar to those known from the isolated N-terminal domain/middle domain construct (53Wegele H. Muschler P. Bunck M. Reinstein J. Buchner J. J. Biol. Chem. 2003; 278: 39303-39310Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Hsp90 activity can be rescued when an artificial linker is introduced, implying that one function of the linker region is to contribute conformational freedom for connection of the N and M domains. It might well be that conformational changes at the N-M interface require this conformational space to rearrange productively during the ATPase cycle. This alone appears to be not sufficient given the observation that the amino acids 266–272 are required for purposes, which cannot be fully supplemented fully by a Gly-Ser stretch as judged by the lower ATP turnover of 211–272SUP compared with 211–266SUP and differences in Aha1 interaction. Analysis of x-ray structures of HtpG and Grp94 (4Dollins D.E. Warren J.J. Immormino R.M. Gewirth D.T. Mol. Cell. 2007; 28: 41-56Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 5Shiau A.K. Harris S.F. Southworth D.R. Agard D.A. Cell. 2006; 127: 329-340Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar) suggests that these amino acid participate in the formation of a β-sheet-like structure with the N-terminal domain. However, this is difficult to decide based on the crystal structures, as both proteins were strongly mutated within the linker region to allow crystallization. Nevertheless, the participation of this sequence in a β-sheet structure could explain the requirement for certain amino acids within this region. The analysis of the co-chaperone interaction with the Hsp90 linker deletion variants allowed us to further scrutinize the function of the linker region. Some co-chaperones can be used as conformational sensors as they associate with specific states of Hsp90 during the ATPase cycle (45Richter K. Muschler P. Hainzl O. Reinstein J. Buchner J. J. Biol. Chem. 2003; 278: 10328-10333Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 46Meyer P. Prodromou C. Liao C. Hu B. Mark R.S. Vaughan C.K. Vlasic I. Panaretou B. Piper P.W. Pearl L.H. EMBO J. 2004; 23: 511-519Crossref PubMed Scopus (192) Google Scholar, 51Richter K. Walter S. Buchner J. J. Mol. Biol. 2004; 342: 1403-1413Crossref PubMed Scopus (119) Google Scholar). The interaction with Sti1 was not significantly influenced by the linker deletions. This may be explained with the relative flexibility of the adaptor protein Sti1, which could compensate for a changed domain orientation in the charged linker constructs of Hsp90. The data for the p23/Sba1 interaction on the other hand suggest that the conformational changes leading to the N-terminal-dimerized state of Hsp90 seem to be inhibited by the linker deletion. This conformation is induced upon ATP binding (7Prodromou C. Panaretou B. Chohan S. Siligardi G. O'Brien R. Ladbury J.E. Roe S.M. Piper P.W. Pearl L.H. EMBO J. 2000; 19: 4383-4392Crossref PubMed Google Scholar). The conformational changes leading to this state are rate-limiting during the hydrolysis cycle (9Richter K. Reinstein J. Buchner J. J. Biol. Chem. 2002; 277: 44905-44910Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Apparently, the deletion of the linker slows the repositioning of the N and the M domain at the N-M interface. Interestingly, also the ability of Aha1 to stimulate the ATP turnover is strongly reduced in the linker variants or even absent in the maximum deletion construct. Our data indicate that the Aha1-induced ATPase stimulation is only possible with unconstrained N-M interactions. It is reasonable to assume that if this interaction is sterically inhibited, Aha1 can no longer exert stimulatory effects even though Aha1 binding itself is not affected. To date, the ability to hydrolyze ATP was believed to be an essential prerequisite for in vivo functionality of Hsp90 wt as ATPase-defective mutants are not able to replace Hsp90 in an Hsp90-deficient background (16Obermann W.M. Sondermann H. Russo A.A. Pavletich N.P. Hartl F.U. J. Cell Biol. 1998; 143: 901-910Crossref PubMed Scopus (487) Google Scholar, 17Panaretou B. Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1998; 17: 4829-4836Crossref PubMed Scopus (613) Google Scholar). Our results indicate that ATP hydrolysis alone is not sufficient for viability. Also, conformationally restricted but ATPase-active Hsp90 versions are unable to replace Hsp90 wt. The unrestricted motility of the N-M interface seems required for in vivo functions even more than for the ATP turnover. This is true even for the smallest deletions used in this study, which are not affected in their ATP turnover yet, but their chaperone effect on known in vivo clients of Hsp90, like the GR, luciferase, and v-Src kinase, is diminished. The severely compromised yeast strains carrying the Hsp90 linker constructs show a reduced ability to interact with Hsp90 clients, consistent with the view that the charged linker region is involved in Hsp90 chaperone function (54Scheibel T. Siegmund H.I. Jaenicke R. Ganz P. Lilie H. Buchner J. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 1297-1302Crossref PubMed Scopus (97) Google Scholar). The recent structure of the kinase Cdk4 in complex with Hsp90-Cdc37 implies client interactions, which span the N-terminal part of Hsp90 and the middle domain (55Vaughan C.K. Gohlke U. Sobott F. Good V.M. Ali M.M. Prodromou C. Robinson C.V. Saibil H.R. Pearl L.H. Mol. Cell. 2006; 23: 697-707Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). It might very well be that the conformational flexibility required at the N-M interface is even more pronounced in the case of client binding as compared with ATP hydrolysis. The strongly extended linker region, which can be found in all eukaryotic Hsp90 proteins, might therefore result from the need of a highly flexible contact surface that is able to integrate various client proteins into the Hsp90 dimer structure. This work was supported by grants from the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie (to J. B. and K. R.).
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