The Hsp90 Chaperone Machinery
2008; Elsevier BV; Volume: 283; Issue: 27 Linguagem: Inglês
10.1074/jbc.r800007200
ISSN1083-351X
AutoresSebastian K. Wandinger, Klaus Richter, Johannes Büchner,
Tópico(s)Toxin Mechanisms and Immunotoxins
ResumoHsp90 was originally identified as one of several conserved heat shock proteins. Like the other major classes of heat shock proteins, Hsp90 exhibits general protective chaperone properties, such as preventing the unspecific aggregation of non-native proteins (1Wiech H. Buchner J. Zimmermann R. Jakob U. Nature. 1992; 358: 169-170Crossref PubMed Scopus (417) Google Scholar). However, Hsp90 seems to be more selective than the other promiscuous general chaperones, as it preferentially interacts with a specific subset of the proteome (2Picard D. CMLS Cell. Mol. Life Sci. 2002; 59: 1640-1648Crossref PubMed Scopus (645) Google Scholar). Another specific feature of Hsp90 is its regulatory role of inducing conformational changes in folded, native-like substrate proteins that lead to their activation or stabilization (3Jakob U. Lilie H. Meyer I. Buchner J. J. Biol. Chem. 1995; 270: 7288-7294Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). Recently, the three-dimensional structures of full-length Hsp90 from Escherichia coli, yeast, and the endoplasmic reticulum were solved (4Shiau A.K. Harris S.F. Southworth D.R. Agard D.A. Cell. 2006; 127: 329-340Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 5Ali 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 (683) Google Scholar, 6Dollins D.E. Warren J.J. Immormino R.M. Gewirth D.T. Mol. Cell. 2007; 28: 41-56Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 7Pearl L.H. Prodromou C. Annu. Rev. Biochem. 2006; 75: 271-294Crossref PubMed Scopus (875) Google Scholar). Together with sequence data, these showed that, although Hsp90 maintained its general domain structure from bacteria to man, distinct changes seem to have adapted Hsp90 to the more complex protein environment of the eukaryotic cell. Concomitant with the occurrence of a long charged linker connecting the N- 3The abbreviations used are: N-domain, N-terminal domain; M-domain, middle domain; SHR, steroid hormone receptor; TPR, tetratricopeptide repeat; PPIase, peptidylprolyl cis/trans-isomerase; eNOS, epithelial nitric-oxide synthase. 3The abbreviations used are: N-domain, N-terminal domain; M-domain, middle domain; SHR, steroid hormone receptor; TPR, tetratricopeptide repeat; PPIase, peptidylprolyl cis/trans-isomerase; eNOS, epithelial nitric-oxide synthase. and M-domains, the eukaryotic protein exhibits an extension of the C-terminal domain, which includes the conserved amino acid motif MEEVD at the C terminus (8Chen S. Sullivan W.P. Toft D.O. Smith D.F. Cell Stress Chaperones. 1998; 3: 118-129Crossref PubMed Scopus (166) Google Scholar). This region serves as the major interaction site for a cohort of co-chaperones (Table 1) (9Richter K. Meinlschmidt B. Buchner J. Buchner J. Kiefhaber T Protein Folding Handbook. Wiley-VCH Verlag GmbH & Co., Weinheim, Germany2005: 768-829Crossref Scopus (1) Google Scholar), which apparently support Hsp90 in the folding and activation of its substrate proteins in eukaryotes. In this review, we summarize the current knowledge on the functional principles of this molecular machine, including the ATP-driven chaperone cycle of Hsp90 and its regulation by co-chaperones and post-translational modifications.TABLE 1Selected Hsp90 cofactors Open table in a new tab Hsp90 is a flexible dimer. Each monomer consists of three domains: the N-domain, connected by a long linker sequence (in eukaryotes) to an M-domain, which is followed by a C-terminal dimerization domain (Fig. 1). The N-domain possesses a deep ATP-binding pocket (10Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. Cell. 1997; 90: 65-75Abstract Full Text Full Text PDF PubMed Scopus (1098) Google Scholar), where ATP is bound in an unusual kinked manner. ATP hydrolysis by Hsp90 is rather slow: Hsp90 from yeast hydrolyzes one molecule of ATP every 1 or 2 min (11Panaretou 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 (610) Google Scholar, 12Scheibel T. Weikl T. Buchner J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1495-1499Crossref PubMed Scopus (229) Google Scholar), and human Hsp90 hydrolyzes one molecule of ATP every 20 min (0.04 min–1) (13McLaughlin S.H. Smith H.W. Jackson S.E. J. Mol. Biol. 2002; 315: 787-798Crossref PubMed Scopus (210) Google Scholar). The ATPase activity is essential for the function of Hsp90 in yeast (11Panaretou 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 (610) Google Scholar, 14Obermann W.M. Sondermann H. Russo A.A. Pavletich N.P. Hartl F.U. J. Cell Biol. 1998; 143: 901-910Crossref PubMed Scopus (479) Google Scholar). The slow hydrolysis suggests that complex conformational rearrangements of Hsp90 are coupled to the ATPase reaction and that these represent the rate-limiting step of the enzyme. The first steps of these conformational changes were elucidated recently in detail (15Richter 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): upon ATP binding, a short segment of the N-domain called the “ATP lid” changes its position and flaps over the binding pocket (Fig. 1, steps 2 and 3). This releases a short N-terminal segment from its original position (16Richter K. Reinstein J. Buchner J. J. Biol. Chem. 2002; 277: 44905-44910Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In a subsequent reaction, this segment binds to the respective N-domain of the other subunit in the dimer, producing a strand-swapped, transiently dimerized N-terminal conformation (step 3) (5Ali 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 (683) Google Scholar, 15Richter 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). These N-terminal rearrangements result in further conformational changes throughout the entire Hsp90 dimer leading to a twisted and compacted dimer, in which N- and M-domains associate and the distance between M-domains is shortened by 40 Å (5Ali 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 (683) Google Scholar). The association of N- and M-domains completes the active site of this “split ATPase” (step 4). Recently, a similar progression of steps was shown to occur also for the endoplasmic homolog Grp94 (17Frey S. Leskovar A. Reinstein J. Buchner J. J. Biol. Chem. 2007; 282: 35612-35620Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), mitochondrial TRAP1 (6Dollins D.E. Warren J.J. Immormino R.M. Gewirth D.T. Mol. Cell. 2007; 28: 41-56Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 18Leskovar, A., Wegele, H., Werbeck, N. D., Buchner, J., and Reinstein, J. (2008) 283, 11677–11688Google Scholar), and human Hsp90 (19Richter 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 (102) Google Scholar). Therefore, the scenario outlined above seems to be the ubiquitously conserved ATPase mechanism for Hsp90. Interestingly, the unusual way in which ATP is bound by Hsp90 is perfectly mimicked by some natural compounds, such as geldanamycin and radicicol. These are highly specific and potent inhibitors of the Hsp90 ATPase (20Roe S.M. Prodromou C. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. J. Med. Chem. 1999; 42: 260-266Crossref PubMed Scopus (871) Google Scholar), blocking the maturation of substrate proteins and eventually resulting in their degradation (21Whitesell L. Mimnaugh E.G. De Costa B. Myers C.E. Neckers L.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8324-8328Crossref PubMed Scopus (1310) Google Scholar). As several Hsp90 substrate proteins are kinases, which can be deregulated in the development of cancer, derivatives of Hsp90 inhibitors are currently being investigated as anticancer therapeutics at the stage of clinical trials (22Sharp S. Workman P. Adv. Cancer Res. 2006; 95: 323-348Crossref PubMed Scopus (269) Google Scholar). Current models assume that the conformational changes associated with ATP hydrolysis are required for reaching or maintaining an activated state of a substrate protein. In well studied examples such as the SHRs, several cofactors interact with Hsp90 in a sequential manner to assemble a functional chaperone machinery (23Pratt W.B. Toft D.O. Exp. Biol. Med. (Maywood). 2003; 228: 111-133Crossref PubMed Scopus (1239) Google Scholar, 24Smith D.F. Mol. Endocrinol. 1993; 7: 1418-1429Crossref PubMed Scopus (251) Google Scholar). The basis for this ordered succession of different assemblies can now be rationalized, as it turned out that several Hsp90 cofactors display a strong binding preference for specific Hsp90 conformations. The loading of an SHR onto Hsp90 requires the cooperation of Hsp90 with the chaperone Hsp70 and its cofactor Hsp40 (25Smith D.F. Sullivan W.P. Marion T.N. Zaitsu K. Madden B. McCormick D.J. Toft D.O. Mol. Cell. Biol. 1993; 13: 869-876Crossref PubMed Scopus (246) Google Scholar). Moreover, both chaperones become physically linked by an adaptor protein called Hop/Sti1 (Table 1). This co-chaperone binds via small helical TPR domains to the C-terminal ends of Hsp70 and Hsp90 (26Scheufler 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 (991) Google Scholar). It seems that Hsp70 stabilizes the SHR in a conformation that can be recognized and bound by Hsp90. However, experimental evidence for this notion is largely lacking. How the substrate in this complex is transferred from Hsp70 to Hsp90 is also still unclear. It might be that the bridging by Hop/Sti1 selects for Hsp90 molecules in a conformation competent for substrate binding in addition to increasing the local concentration of Hsp70 and Hsp90. For the progression of the chaperone cycle, empty Hsp70 and Hop/Sti1 have to dissociate, and other co-chaperones such as specific PPIases and p23/Sba1 enter the complex (Table 1) (24Smith D.F. Mol. Endocrinol. 1993; 7: 1418-1429Crossref PubMed Scopus (251) Google Scholar). These PPIases also possess a TPR domain, which binds to the C-terminal end of Hsp90. The second cofactor, p23/Sba1, associates with the N-terminally dimerized conformation of Hsp90 (27Prodromou 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, 28Richter K. Walter S. Buchner J. J. Mol. Biol. 2004; 342: 1403-1413Crossref PubMed Scopus (119) Google Scholar), making it likely that the dramatic conformational rearrangement from the open to the closed state of Hsp90 occurs at this stage of the Hsp90 chaperone cycle (Fig. 1, steps 3 and 4). This closed conformation is metastable and upon ATP hydrolysis returns to the open state (Fig. 1, step 5) (28Richter K. Walter S. Buchner J. J. Mol. Biol. 2004; 342: 1403-1413Crossref PubMed Scopus (119) Google Scholar). The bound substrate protein dissociates in turn from Hsp90, permitting a new round of the cycle. The first steps of the cycle for the maturation of signaling kinases is a variation of the scheme described above. Here, the kinase-specific Hsp90 cofactor Cdc37 seems to associate with substrate kinases in their inactive forms first. This complex may then be loaded onto Hsp90 (29Kimura Y. Rutherford S.L. Miyata Y. Yahara I. Freeman B.C. Yue L. Morimoto R.I. Lindquist S. Genes Dev. 1997; 11: 1775-1785Crossref PubMed Scopus (175) Google Scholar). The following steps are less clear yet. It may be that also for kinases, Hsp70, Hsp40, and Hop/Sti1 are additionally required (30Caplan A.J. Mandal A.K. Theodoraki M.A. Trends Cell Biol. 2007; 17: 87-92Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Because Cdc37 partially inhibits the ATPase of Hsp90 (31Siligardi G. Panaretou B. Meyer P. Singh S. Woolfson D.N. Piper P.W. Pearl L.H. Prodromou C. J. Biol. Chem. 2002; 277: 20151-20159Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), it is reasonable to speculate that a transient stalling of the ATPase facilitates substrate transfer onto Hsp90 in general. Up to now, more than a dozen distinct Hsp90 cofactors have been identified (9Richter K. Meinlschmidt B. Buchner J. Buchner J. Kiefhaber T Protein Folding Handbook. Wiley-VCH Verlag GmbH & Co., Weinheim, Germany2005: 768-829Crossref Scopus (1) Google Scholar). Their large number is not paralleled by other chaperone systems. Most bind Hsp90 with submicromolar affinities (Table 1). The major class of these is the TPR domain-containing cofactors, which include the proteins Hop/Sti1, PP5/Ppt1, and the large PPIases, among others (Table 1). Some of these cofactors could specifically facilitate the activation of a certain set of substrate proteins. In this context, the cofactor Unc45 has been shown to participate in early muscle development during the assembly of myosin filaments (32Barral J.M. Hutagalung A.H. Brinker A. Hartl F.U. Epstein H.F. Science. 2002; 295: 669-671Crossref PubMed Scopus (197) Google Scholar), and Xap2/AIP has been found in complex with the protein HbX from the hepatitis B virus (33Kuzhandaivelu N. Cong Y.S. Inouye C. Yang W.M. Seto E. Nucleic Acids Res. 1996; 24: 4741-4750Crossref PubMed Scopus (94) Google Scholar) or the endogenous aryl hydrocarbon receptor (34Meyer B.K. Perdew G.H. Biochemistry. 1999; 38: 8907-8917Crossref PubMed Scopus (175) Google Scholar). It remains to be seen whether these cofactors are really highly specialized or whether they are just the first substrates identified. Interestingly, in yeast, only two cofactors of the Hsp90 system are essential for viability (in addition to Hsp90). These are Cdc37 and Cns1 (29Kimura Y. Rutherford S.L. Miyata Y. Yahara I. Freeman B.C. Yue L. Morimoto R.I. Lindquist S. Genes Dev. 1997; 11: 1775-1785Crossref PubMed Scopus (175) Google Scholar, 35Dolinski K.J. Cardenas M.E. Heitman J. Mol. Cell. Biol. 1998; 18: 7344-7352Crossref PubMed Google Scholar, 36Marsh J.A. Kalton H.M. Gaber R.F. Mol. Cell. Biol. 1998; 18: 7353-7359Crossref PubMed Google Scholar). Cns1 has been shown to associate with both Hsp90 and Hsp70; however, due to the presence of a single TPR domain, no ternary complexes can be formed (37Hainzl O. Wegele H. Richter K. Buchner J. J. Biol. Chem. 2004; 279: 23267-23273Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The function of Cns1 remains elusive. Hsp90 is embedded into several control mechanisms that influence its activity. As mentioned above, the ATPase activity is intrinsically decelerated due to slow conformational changes of the lid segment within the N-domains. In addition to this, the ATPase activity of Hsp90 is regulated by several cofactors. Another set of cofactors modulates substrate processing without changing the ATPase activity. Finally, the activity of Hsp90 is also regulated by post-translational modifications. Several of the cofactors of Hsp90 modulate its ATPase activity by interacting preferentially with a specific conformation of Hsp90. p23/Sba1 binds to the ATPase domain and stabilizes the N-terminally dimerized conformation at the late stage of the ATPase cycle (Fig. 1) (28Richter K. Walter S. Buchner J. J. Mol. Biol. 2004; 342: 1403-1413Crossref PubMed Scopus (119) Google Scholar, 38Chadli A. Bouhouche I. Sullivan W. Stensgard B. McMahon N. Catelli M.G. Toft D.O. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12524-12529Crossref PubMed Scopus (135) Google Scholar, 39Siligardi G. Hu B. Panaretou B. Piper P.W. Pearl L.H. Prodromou C. J. Biol. Chem. 2004; 279: 51989-51998Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). This positions p23/Sba1 to be part of the Hsp90 complex at the moment of hydrolysis and appears to be the reason for the decrease in the ATP turnover rate of Hsp90 in the presence of p23/Sba1. A second site of cofactor interaction resides in the M-domain, to which Aha1, the only known activator of the Hsp90 ATPase, binds. This interaction stimulates the weak ATPase activity of Hsp90 by >10-fold (40Panaretou B. Siligardi G. Meyer P. Maloney A. Sullivan J.K. Singh S. Millson S.H. Clarke P.A. Naaby-Hansen S. Stein R. Cramer R. Mollapour M. Workman P. Piper P.W. Pearl L.H. Prodromou C. Mol. Cell. 2002; 10: 1307-1318Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar). The stimulatory interaction of Aha1 with the M-domain of Hsp90 suggests a participation of this domain in the rate-limiting step of the ATPase reaction. Structural studies show that Aha1 binding remodels the M-domain around the catalytically active Arg380 and shifts the domain to a conformation resembling the closed conformation, committed for ATP hydrolysis (compare Fig. 1, step 3) (7Pearl L.H. Prodromou C. Annu. Rev. Biochem. 2006; 75: 271-294Crossref PubMed Scopus (875) Google Scholar, 41Meyer P. Prodromou C. Liao C. Hu B. Roe S.M. Vaughan C.K. Vlasic I. Panaretou B. Piper P.W. Pearl L.H. EMBO J. 2004; 23: 1402-1410Crossref PubMed Scopus (56) Google Scholar). Another option for modulating the ATPase activity of Hsp90 is implemented by the co-chaperone Sti1 in yeast. Sti1 binds to the C-terminal end of Hsp90 via its TPR domain. In addition, there is a second binding site in the N- or M-domain (42Richter 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). Binding to this second site allows Sti1 to inhibit the ATPase of Hsp90 completely (42Richter 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, 43Prodromou 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 (345) Google Scholar). Interestingly, ATP binding is not affected (42Richter 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). As in the case of p23/Sba1, Sti1 selects a specific conformation of Hsp90 for binding. Biochemical analysis suggests that Sti1 binding prevents the N-terminal dimerization and association of the N- and M-domains. (Fig. 1, step 3) (42Richter 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). In consequence, the Km value of ATP hydrolysis is not affected, but kcat is effectively reduced. This is the classical behavior of a noncompetitive inhibitor. The open state propagated by Sti1 is the acceptor state for substrate, as outlined above. Cdc37 also inhibits the ATPase activity of Hsp90 (31Siligardi G. Panaretou B. Meyer P. Singh S. Woolfson D.N. Piper P.W. Pearl L.H. Prodromou C. J. Biol. Chem. 2002; 277: 20151-20159Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 43Prodromou 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 (345) Google Scholar). Details of the respective mechanisms are still unclear, but crystallographic data provide a model in which Cdc37 binds to the ATP lid in the N-terminal nucleotide-binding site of Hsp90 and prevents the N-terminal dimerization by inserting as a dimer in between the two N-domains (44Roe S.M. Ali M.M. Meyer P. Vaughan C.K. Panaretou B. Piper P.W. Prodromou C. Pearl L.H. Cell. 2004; 116: 87-98Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Because Cdc37 is involved in the loading of Hsp90 with kinases, it is consistent with notions about the acceptor state of Hsp90 that Cdc37 also keeps Hsp90 in an open state. The deceleration of the ATPase activity probably permits this state to persist for an extended period of time. Together, these cofactors allow the adjustment of the basic conformational changes of Hsp90 that can be viewed as substrate processing steps to the specific needs of certain clients concerning binding to Hsp90 but also, in the case of p23/Sba1, its release. A further level of regulation that has gained increasing attention is covalent modifications of Hsp90, such as acetylation (45Scroggins B.T. Robzyk K. Wang D. Marcu M.G. Tsutsumi S. Beebe K. Cotter R.J. Felts S. Toft D. Karnitz L. Rosen N. Neckers L. Mol. Cell. 2007; 25: 151-159Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar), S-nitrosylation (46Garcia-Cardena G. Fan R. Shah V. Sorrentino R. Cirino G. Papapetropoulos A. Sessa W.C. Nature. 1998; 392: 821-824Crossref PubMed Scopus (850) Google Scholar), and phosphorylation (47Sefton B.M. Beemon K. Hunter T. J. Virol. 1978; 28: 957-971Crossref PubMed Google Scholar, 48Dougherty J.J. Puri R.K. Toft D.O. J. Biol. 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Chem. 2005; 280: 33792-33799Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). As a consequence, an increase in proteasomal degradation of some Hsp90 substrates was found. HDAC6 was identified as the enzyme deacetylating Hsp90 (52Kovacs J.J. Murphy P.J. Gaillard S. Zhao X. Wu J.T. Nicchitta C.V. Yoshida M. Toft D.O. Pratt W.B. Yao T.P. Mol. Cell. 2005; 18: 601-607Abstract Full Text Full Text PDF PubMed Scopus (874) Google Scholar). In vivo, Hsp90 is acetylated at least at two sites; one was identified as Lys294 (in human Hsp90α) (45Scroggins B.T. Robzyk K. Wang D. Marcu M.G. Tsutsumi S. Beebe K. Cotter R.J. Felts S. Toft D. Karnitz L. Rosen N. Neckers L. Mol. Cell. 2007; 25: 151-159Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Furthermore, besides the ability to interact with its substrate proteins, the binding of ATP by Hsp90 was also shown to be compromised upon acetylation of Hsp90 (55Murphy P.J. Morishima Y. Kovacs J.J. Yao T.P. Pratt W.B. J. Biol. Chem. 2005; 280: 33792-33799Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 56Nimmanapalli R. O'Bryan E. Kuhn D. Yamaguchi H. Wang H.G. Bhalla K.N. Blood. 2003; 102: 269-275Crossref PubMed Scopus (85) Google Scholar). Phosphorylation of Hsp90 has already been known for 30 years (47Sefton B.M. Beemon K. Hunter T. J. Virol. 1978; 28: 957-971Crossref PubMed Google Scholar). By two-dimensional gel electrophoresis, it was found that at least four differently phosphorylated isoforms of Hsp90 exist (57Kelley P.M. Schlesinger M.J. Mol. Cell. Biol. 1982; 2: 267-274Crossref PubMed Scopus (171) Google Scholar, 58Welch W.J. Garrels J.I. Thomas G.P. Lin J.J. Feramisco J.R. J. Biol. Chem. 1983; 258: 7102-7111Abstract Full Text PDF PubMed Google Scholar). For mammalian Hsp90 proteins, the majority of Hsp90 molecules contain on average three phosphates/monomer (59Iannotti A.M. Rabideau D.A. Dougherty J.J. Arch. Biochem. 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The yeast homolog dephosphorylates Hsp90 in a specific manner only when bound to Hsp90 (62Wandinger S.K. Suhre M.H. Wegele H. Buchner J. EMBO J. 2006; 25: 367-376Crossref PubMed Scopus (124) Google Scholar). Dephosphorylation of Hsp90 is important for its in vivo function, as in a ppt1 deletion strain, the maturation of several substrate proteins was found to be impaired (62Wandinger S.K. Suhre M.H. Wegele H. Buchner J. EMBO J. 2006; 25: 367-376Crossref PubMed Scopus (124) Google Scholar). It will be a highly rewarding task to understand Hsp90 regulation at the level of post-translational modifications in detail, as this allows the cell to exert fine-tuned control over the Hsp90 chaperone machinery. The situation is even more complicated than described, as numerous co-chaperones of Hsp90 are regulated by modifications such as phosphorylation as well (63Cox M.B. Riggs D.L. Hessling M. Schumacher F. Buchner J. Smith D.F. Mol. 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A first step toward understanding the basis for the differences in the Hsp90 dependence of v-Src and c-Src is the finding that the intrinsic stabilities of the two Src forms differ, with v-Src being a highly unstable protein (70Falsone S.F. Leptihn S. Osterauer A. Haslbeck M. Buchner J. J. Mol. Biol. 2004; 344: 281-291Crossref PubMed Scopus (41) Google Scholar). Systematic variations of Hsp90-interacting kinases have further added to the notion that it is not a specific binding element but rather the stability of the protein that seems to be important for binding (71Hartson S.D. Ottinger E.A. Huang W. Barany G. Burn P. Matts R.L. J. Biol. Chem. 1998; 273: 8475-8482Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Whether this is the general scheme remains to be seen, as there is evidence that a specific loop (“activation loop”) in the kinase might participate in governing the interaction with Hsp90 (72Terasawa K. Yoshimatsu K. Iemura S. Natsume T. Tanaka K. Minami Y. Mol. Cell. Biol. 2006; 26: 3378-3389Crossref PubMed Scopus (35) Google Scholar). Regarding identification of the substrate-binding site, Hsp90 is certainly lagging behind other chaperones such as Hsp70 and GroE, for which we have a clear idea of this region. For Hsp90, experimental evidence exists for binding sites in all three domains of the protein (12Scheibel T. Weikl T. Buchner J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1495-1499Crossref PubMed Scopus (229) Google Scholar, 73Young J.C. Schneider C. Hartl F.U. FEBS Lett. 1997; 418: 139-143Crossref PubMed Scopus (132) Google Scholar, 74Vaughan 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 (250) Google Scholar). However, recent advances have begun to shed light on substrate binding. Determination of full-length Hsp90 crystal structures of open and closed conformations is a major achievement in this context (4Shiau A.K. Harris S.F. Southworth D.R. Agard D.A. Cell. 2006; 127: 329-340Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 5Ali 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 (683) Google Scholar, 6Dollins D.E. Warren J.J. Immormino R.M. Gewirth D.T. Mol. Cell. 2007; 28: 41-56Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). In analogy to chaperones like GroE and derived from homology of Hsp90 to DNA-binding topoisomerases (75Dutta R. Inouye M. Trends Biochem. Sci. 2000; 25: 24-28Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar), it was speculated that the substrate protein may be encapsulated by the Hsp90 dimer. This notion is not in agreement with the aforementioned structures, in which there is not enough space to accommodate a substrate protein between the two Hsp90 monomers. The first direct view of the structure of an Hsp90·substrate complex comes from electron microscopy and image processing of Hsp90 in complex with the kinase Cdk4 and the co-chaperone Cdc37. In this assembly, the substrate kinase is bound in a highly asymmetric fashion to the M-domain and probably the N-domain of one subunit, whereas the co-chaperone Cdc37 resides between the N-domains (74Vaughan 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 (250) Google Scholar). Whether this association is the same for all kinases and whether this can be generalized for other substrate proteins remain to be determined. Although the basic features of ATP turnover and associated conformational changes of Hsp90 appear to be solved now, other characteristics of the Hsp90 machinery are not understood to date. These include substrate turnover and requirements for formation of particular substrate·co-chaperone·Hsp90 complexes. A combination of in vivo and in vitro approaches will be required to resolve these questions. Once we obtain a more detailed view of the processive power of this molecular machine, we can aim to integrate variations concerning specific substrate proteins, cofactors, and particular mechanisms of regulation and post-translational modifications into the picture. We thank Heike Eberl and Emma Simpson for critical comments on the manuscript.
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