Hsp90 Regulates the Activity of Wild Type p53 under Physiological and Elevated Temperatures

Abstract

The activity and structural integrity of the tumor suppressor protein p53 is of crucial importance for the prevention of cancer. p53 is a conformational flexible and labile protein, in which structured and unstructured regions function in a synergistic manner. The molecular chaperone Hsp90 is known to bind to mutant and wild type p53 in vivo. Using highly purified proteins we analyzed the interaction and the binding sites between both proteins in detail. Our results demonstrate that Hsp90 binds to a folded, native-like conformation of p53 in vitro with micromolar affinity. Specifically, the DNA-binding domain of p53 and the middle and carboxy-terminal domains of Hsp90 are responsible for this interaction, which is essential to stabilize p53 at physiological temperatures and to prevent it from irreversible thermal inactivation. Our results are in agreement with a model in which Hsp90 is required to maintain the folded, active state of p53 by a reversible interaction, thus introducing an additional level of regulation. The activity and structural integrity of the tumor suppressor protein p53 is of crucial importance for the prevention of cancer. p53 is a conformational flexible and labile protein, in which structured and unstructured regions function in a synergistic manner. The molecular chaperone Hsp90 is known to bind to mutant and wild type p53 in vivo. Using highly purified proteins we analyzed the interaction and the binding sites between both proteins in detail. Our results demonstrate that Hsp90 binds to a folded, native-like conformation of p53 in vitro with micromolar affinity. Specifically, the DNA-binding domain of p53 and the middle and carboxy-terminal domains of Hsp90 are responsible for this interaction, which is essential to stabilize p53 at physiological temperatures and to prevent it from irreversible thermal inactivation. Our results are in agreement with a model in which Hsp90 is required to maintain the folded, active state of p53 by a reversible interaction, thus introducing an additional level of regulation. The tumor suppressor protein p53 is a transcription factor involved in cell cycle regulation and the initiation of apoptotic cell death (1Hartwell L.H. Kastan M.B. Science. 1994; 266: 1821-1828Crossref PubMed Scopus (2316) Google Scholar, 2Hofseth L.J. Hussain S.P. Harris C.C. Trends Pharmacol. Sci. 2004; 25: 177-181Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 3Lane D.P. Nature. 1992; 358: 15-16Crossref PubMed Scopus (4523) Google Scholar, 4Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6759) Google Scholar). Mutated or otherwise, deactivated p53 is observed in over 50% of human cancers (5Soussi T. Legros Y. Lubin R. Ory K. Schlichtholz B. Int. J. Cancer. 1994; 57: 1-9Crossref PubMed Scopus (373) Google Scholar, 6Vogelstein B. Lane D. Levine A.J. Nature. 2000; 408: 307-310Crossref PubMed Scopus (5850) Google Scholar). It can interact with and inactivate the protein from the wild type allele (7Kern S.E. Pietenpol J.A. Thiagalingam S. Seymour A. Kinzler K.W. Vogelstein B. Science. 1992; 256: 827-830Crossref PubMed Scopus (891) Google Scholar, 8Milner J. Medcalf E.A. Cell. 1991; 65: 765-774Abstract Full Text PDF PubMed Scopus (536) Google Scholar). Wild type p53 is a labile protein with a high turnover rate; it is degraded rapidly via proteolysis and the ubiquitin-proteasome pathway (9Maki C.G. Huibregtse J.M. Howley P.M. Cancer Res. 1996; 56: 2649-2654PubMed Google Scholar). Due to this high turnover only low concentrations of p53 reside usually in the cell. However, mutated p53 is not as strongly affected by proteolysis or ubiquitination and therefore accumulates in the cell (10Soussi T. Ann. N. Y. Acad. Sci. 2000; 910: 121-137Crossref PubMed Scopus (271) Google Scholar). p53 consists of 393 residues that can be divided in four major functional domains (see Fig. 1A) (11Bargonetti J. Manfredi J.J. Chen X. Marshak D.R. Prives C. Genes Dev. 1993; 7: 2565-2574Crossref PubMed Scopus (249) Google Scholar, 12Bell S. Klein C. Muller L. Hansen S. Buchner J. J. Mol. Biol. 2002; 322: 917-927Crossref PubMed Scopus (217) Google Scholar, 13Pavletich N.P. Chambers K.A. Pabo C.O. Genes Dev. 1993; 7: 2556-2564Crossref PubMed Scopus (449) Google Scholar). As a transcription factor, p53 possesses an amino-terminal region (NTD, 1The abbreviations used are: NTD, amino-terminal domain of p53; DBD, DNA-binding domain of p53; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; EMSA, electrophoretic mobility shift assay; FITC, fluorescein 5-isothiocyanate; hHsp90, human Hsp90; NiMB, nickel-chelating magnetic beads; RD, regulatory domain of p53; yHsp90, yeast Hsp90; MES, 4-morpholineethanesulfonic acid; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay. residues 1–93) (14Fields S. Jang S.K. Science. 1990; 249: 1046-1049Crossref PubMed Scopus (659) Google Scholar), which contains a transcription activation domain and a proline-rich domain. Highly specific DNA binding is mediated by the core domain (DBD, residues 102–292) (15Kern S.E. Kinzler K.W. Bruskin A. Jarosz D. Friedman P. Prives C. Vogelstein B. Science. 1991; 252: 1708-1711Crossref PubMed Scopus (941) Google Scholar, 16Klein C. Planker E. Diercks T. Kessler H. Kunkele K.P. Lang K. Hansen S. Schwaiger M. J. Biol. Chem. 2001; 276: 49020-49027Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), which recognizes a consensus DNA sequence (17el Deiry W.S. Kern S.E. Pietenpol J.A. Kinzler K.W. Vogelstein B. Nat. Genet. 1992; 1: 45-49Crossref PubMed Scopus (1752) Google Scholar). The high affinity binding of p53 to this sequence requires the tetrameric state of p53 (17el Deiry W.S. Kern S.E. Pietenpol J.A. Kinzler K.W. Vogelstein B. Nat. Genet. 1992; 1: 45-49Crossref PubMed Scopus (1752) Google Scholar). The tetramerization domain (18Sturzbecher H.W. Brain R. Addison C. Rudge K. Remm M. Grimaldi M. Keenan E. Jenkins J.R. Oncogene. 1992; 7: 1513-1523PubMed Google Scholar), which is followed by a flexible regulatory domain (RD), is located within the carboxy-terminal domain (residues 320–393). Stress-mediated modifications in the regulatory domain, e.g. phosphorylation or binding of other proteins, transform p53 from the common inactive form to the activated form, which is able to induce the expression of target proteins or to mediate apoptosis (19Ahn J. Prives C. Nat. Struct. Biol. 2001; 8: 730-732Crossref PubMed Scopus (103) Google Scholar). Interestingly, CD spectroscopy revealed that p53 contains large unstructured regions in its native state (12Bell S. Klein C. Muller L. Hansen S. Buchner J. J. Mol. Biol. 2002; 322: 917-927Crossref PubMed Scopus (217) Google Scholar). Further analysis using NMR spectroscopy in combination with p53 fragments showed that the NTD is natively unfolded (20Dawson R. Muller L. Dehner A. Klein C. Kessler H. Buchner J. J. Mol. Biol. 2003; 332: 1131-1141Crossref PubMed Scopus (198) Google Scholar). Thus, in p53 only the DBD and the tetramerization domain are highly structured (21Cho Y. Gorina S. Jeffrey P.D. Pavletich N.P. Science. 1994; 265: 346-355Crossref PubMed Scopus (2158) Google Scholar, 22Clore G.M. Ernst J. Clubb R. Omichinski J.G. Kennedy W.M. Sakaguchi K. Appella E. Gronenborn A.M. Nat. Struct. Biol. 1995; 2: 321-333Crossref PubMed Scopus (191) Google Scholar). This classifies p53 as a member of the growing class of proteins, in which structured and unstructured regions function in a synergistic manner (20Dawson R. Muller L. Dehner A. Klein C. Kessler H. Buchner J. J. Mol. Biol. 2003; 332: 1131-1141Crossref PubMed Scopus (198) Google Scholar). For p53 this allows the interaction with a multitude of regulators (2Hofseth L.J. Hussain S.P. Harris C.C. Trends Pharmacol. Sci. 2004; 25: 177-181Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 23Slee E.A. O'Connor D.J. Lu X. Oncogene. 2004; 23: 2809-2818Crossref PubMed Scopus (238) Google Scholar, 24Nakamura Y. Cancer Sci. 2004; 95: 7-11Crossref PubMed Scopus (151) Google Scholar). It has been shown that mutated p53 interacts with the molecular chaperone Hsp90 in J. S. L. Acad. Sci. S. A. 1996; PubMed Scopus Google Scholar, L. P.D. Mol. Cell. Biol. PubMed Scopus Google Scholar, C. Chen J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Hsp90 is essential in K.A. J. S. Mol. Cell. Biol. PubMed Scopus Google Scholar), and it is of the proteins in J.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar, K.W. Mol. Cell. Biol. PubMed Scopus Google Scholar). It high between and e.g. yeast Hsp90 is to human Hsp90 J. P. PubMed Scopus Google Scholar). the function of Hsp90 as a chaperone for a large of proteins, from transcription to to be between yeast and H. Muller L. Buchner J. Pharmacol. 2004; PubMed Scopus Google Scholar). The chaperone is an which can be divided major domains (see Fig. K. P. Buchner J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, A. B. N. Acad. Sci. S. A. 2000; PubMed Scopus Google Scholar, C. R. L.H. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. T. J. Mol. Biol. PubMed Scopus Google Scholar). The amino-terminal domain of Hsp90 contains the binding C. R. L.H. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). This binds the Hsp90 and C. N. Pavletich N.P. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, C. R. L.H. J. Chem. PubMed Scopus Google Scholar), which binding and therefore the of activity in L. Trends Mol. 2002; 8: Full Text Full Text PDF PubMed Scopus Google Scholar). The domain is to the of the protein via a highly and domain R. D. Acad. Sci. S. A. 1996; PubMed Scopus Google Scholar). The function of the middle domain of Hsp90 is not It has been that this domain is involved in the activity of Hsp90 P. C. B. C. B. L.H. Mol. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, H. P. M. J. Buchner J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The is located in the carboxy-terminal region of the protein A. B. N. Acad. Sci. S. A. 2000; PubMed Scopus Google Scholar). the carboxy-terminal the binding for proteins domains domains are a common of Hsp90 as H. Muller L. Buchner J. Pharmacol. 2004; PubMed Scopus Google Scholar, K.A. K. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, W.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of the interaction of Hsp90 and p53 is As Hsp90 binds to mutated p53 J. S. L. Acad. Sci. S. A. 1996; PubMed Scopus Google Scholar, L. P.D. Mol. Cell. Biol. PubMed Scopus Google Scholar, B. J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. M. J. 2001; PubMed Scopus Google Scholar, Y. T. T. T. Y. T. H. H. M. Oncogene. PubMed Scopus Google and of Hsp90 function mutant p53 Y. Chen L. C. Lu Chen J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). NMR that the DBD of p53 binds to Hsp90 S. Acad. Sci. S. A. 2002; PubMed Scopus Google Scholar). Interestingly, Hsp90 binds to wild type p53 A. J. M. J. 2001; PubMed Scopus Google Scholar). The binding to the wild type protein to be with mutant is in that binding to Hsp90 C. Chen J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). this we analyzed the interaction of p53 and Hsp90 in detail. that Hsp90 is for the and the DNA-binding of wild type p53 at physiological and and type p53 (residues (residues (residues (residues and (residues 1–93) and purified as (12Bell S. Klein C. Muller L. Hansen S. Buchner J. J. Mol. Biol. 2002; 322: 917-927Crossref PubMed Scopus (217) Google Scholar, R. Muller L. Dehner A. Klein C. Kessler H. Buchner J. J. Mol. Biol. 2003; 332: 1131-1141Crossref PubMed Scopus (198) Google Scholar). The DNA for and of human p53 from (12Bell S. Klein C. Muller L. Hansen S. Buchner J. J. Mol. Biol. 2002; 322: 917-927Crossref PubMed Scopus (217) Google by and the of a expression by the The and for the or of the human p53 protein, amino-terminal to The of the by DNA and as protein in with the or and the P. PubMed Scopus Google Scholar). in at to an of at for with by in and by high the a with a and purified to by affinity and a a and with a and purified to by a The of both proteins, and as by of Hsp90 purified as from J. S. C. PubMed Scopus Google and Hsp90 and fragments purified from E. as H. P. M. J. Buchner J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). in a of the E. and purified as Hansen E. 1996; PubMed Scopus Google Scholar). analysis at with a of using a or a to a high The with of proteins with a of in a of of proteins by at of Hsp90 of Hsp90 using the of a and of a of in to of The from and at for of by the with the and for at The a of The fragments binding activity analyzed by as with modifications (12Bell S. Klein C. Muller L. Hansen S. Buchner J. J. Mol. Biol. 2002; 322: 917-927Crossref PubMed Scopus (217) Google Scholar, S. Lane D.P. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). specific the p53 consensus DNA binding D. Pietenpol J.A. Mol. Cell. Biol. 2001; PubMed Scopus Google with a and T. A. T. K. H. R. L. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google or Lane D.P. Cell. 1992; Full Text PDF PubMed Scopus Google used as The and to a of concentrations of p53 and of hHsp90, or native and with a CD in a with a in The in at for to followed via the in at from to with a of The for of Hsp90 and p53 native by of the the p53 and for at in the or of in MES, The by a and with as H. M. J. Buchner J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google with purified p53 and in a of The at for in in the of the or by binding to with of the with For proteins by and with the analysis of the interaction in we used nickel-chelating magnetic of and of p53 or p53 fragments at in to a of The for at of nickel-chelating magnetic and the for at The and with of in analyzed with a The in the or of p53 and p53 at p53 or at in the and of at for in in the of as a for protein the of thermal in a in the both the and to with a of analysis of the binding we an protein interaction assay. high affinity with of a of in at over recognizes residues of the NTD of p53 The with at with in at for the the with p53 or p53 fragments or fragments in PBS, in the to the the for at an of at the with The binding of or fragments with a an additional of or of p53 to the the concentrations of hHsp90, or in of PBS, to the and as the conformation of p53 the The specific for p53 R. R. Lane D.P. J. 1990; PubMed Scopus Google Scholar), or specific for natively wild type p53 B. S. J. PubMed Scopus Google Scholar), the For a with a analysis for binding used the between the and the of protein to the is the at which p53 or are with or of Wild and tumor suppressor protein p53 consists of four functional domains and purified p53 fragments and the protein to domains of the human p53 protein in The to the human wild type protein The fragments and are able to bind to the DNA not showed that wild type and form the other fragments are in to the tetramerization domain not The of fragments by CD spectroscopy not Fig. human Hsp90 yeast Hsp90 and the fragments used in this are The and of the proteins analyzed by CD spectroscopy and H. P. M. J. Buchner J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). of the and DNA-binding of type p53 is a labile protein that is and the between the of and the of DNA binding we DNA binding at The proteins for at the the and DNA at temperatures results for the binding of p53 to an irreversible of DNA binding activity and the activity the physiological of p53 not able to bind to DNA of p53 by phosphorylation of the results not this in activity with in the of we by CD showed that structural at temperatures and a at The thermal of p53 is irreversible not Fig. the thermal of wild type by CD is with its DNA binding showed that DNA binding activity with the of of Hsp90 the of the molecular chaperone Hsp90 has an the of we activity in the of Hsp90 not binding affinity for the As shown wild type p53 is not able to bind to DNA for at However, of the at in the of DNA binding showed the activity of with at a of the at is not able to p53 of a of at in a of to in to the DNA binding activity at not the of shift to a molecular of the of that the interaction between p53 and Hsp90 is a we this is specific for the molecular chaperone Hsp90 used and the in to Hsp90 in the proteins the DNA-binding of p53 at As shown in Fig. Hsp90 is able to the DNA-binding of p53 at Interestingly, the able to p53 DNA binding at which to the that are not able to p53 from in the assay. It is known for proteins, that results in this is for the protein at and by Wild type p53 rapidly and a we the of the of Hsp90 is able to the in a and it is able to the p53 Hsp90 showed at not Wild p53 to Hsp90 the interaction of wild type p53 with Hsp90 in we of with p53 using showed that p53 can be and with Hsp90 The of p53 and in the by not Interestingly, is not able to p53 in its tetrameric This be to the high between the for the and in the tetramerization domain of we of and wild type p53 at using the which recognizes residues of human It not interact with However, in the of p53 the able to the proteins that Hsp90 binds to this interaction in we of additional an the p53 to the of an This binds to residues of Hsp90 with the and to the The at not and not with As shown in Fig. is only p53 is a we of the amino-terminal to bind p53 to nickel-chelating magnetic to the at only p53 in the an additional we DNA to the p53 This used of its to bind to DNA activation not of to DNA This that p53 is in its native state and active to or not of p53 with the for p53 only R. R. Lane D.P. J. 1990; PubMed Scopus Google or for natively wild type p53 only B. S. J. PubMed Scopus Google showed that over of the used p53 is natively The that p53 is in its native state an in which p53 to the The or and only binding of the wild to p53 of a of the Hsp90 in p53 we the of the Hsp90 binding within the domain interaction in we p53 fragments to Due to the fragments e.g. and able to bind to the to the of the revealed that not interact with the However, is able to bind to p53 fragments the For that the DBD, interaction with be a we analyzed the domain interaction with the The p53 fragments that the for (residues in the NTD of to the The interaction with by only fragments the DBD able to interact with Thus, we that Hsp90 binds to a conformation of the the DBD be able to the interaction between Hsp90 and wild type p53 as a we wild type p53 and in the and of an of the DBD at temperatures and analyzed the functional state of p53 by DNA in high over and wild type p53 to that this is The DBD is able to bind to DNA at and in activity not the at in the of DBD or and DBD, the DBD the DNA binding activity of wild type the of the DBD the function of for wild type This is with a of the DBD and wild type p53 for binding to The fragments not and not with wild type p53 for the the DBD able to with p53 for binding not p53 to at temperatures as we a with the at Fig. that the DBD within the of hHsp90, is to a large Hsp90 not and active at not This is that the DBD is the specific binding for Hsp90 and that is by the specific domain of the p53 in Hsp90 of the binding in Hsp90 has been A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. Y. Y. J. 2001; PubMed Scopus Google Scholar, T. T. Buchner J. Acad. Sci. S. A. 95: PubMed Scopus Google Scholar, C. 1997; PubMed Scopus Google Scholar). the in Hsp90 we used fragments (see Fig. Hsp90 and bind to the DBD with affinity not the we and fragments to the The the domain of Hsp90 not bind to the Interestingly, both and bind to the However, the binding of the to be the binding of to the the Hsp90 fragments of binding to p53 the DNA binding activity of p53 to we wild type p53 and fragments at temperatures and analyzed the functional state of p53 by The fragments not the DNA binding activity of p53 at not temperatures only and the fragments and able to The showed the of the middle domain and the carboxy-terminal domain of Hsp90 in the interaction with the affinity of Hsp90 for we using of p53 and and hHsp90, and and As shown in Fig. binding that the between yeast and and the of the carboxy-terminal region of Hsp90 to the binding to p53 are for the interaction with The binding of Hsp90 for p53 be to be The and activity of the tumor suppressor protein p53 has a the cancer. The molecular chaperone Hsp90 to be involved in this However, only the interaction between the proteins and the functional is It has been that wild type p53 is a labile protein, and unfolded domains that function in a synergistic manner (12Bell S. Klein C. Muller L. Hansen S. Buchner J. J. Mol. Biol. 2002; 322: 917-927Crossref PubMed Scopus (217) Google Scholar, R. Muller L. Dehner A. Klein C. Kessler H. Buchner J. J. Mol. Biol. 2003; 332: 1131-1141Crossref PubMed Scopus (198) Google Scholar). The thermal of p53 results in an irreversible of DNA binding Interestingly, p53 is at physiological the for in vivo. It is that Hsp90 interacts with p53 in and that it wild type C. Chen J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. M. J. 2001; PubMed Scopus Google Scholar). As shown in the by and D. K. M. B. Muller L. A. J. A. M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), Hsp90 the DNA-binding and activity of wild type p53 physiological in vivo. in this the interaction between the proteins is specific in the native or unfolded state of wild type this we analyzed the binding of purified p53 and Hsp90 using ELISA, and that wild type p53 is able to bind to Hsp90 that the native state of This that a structured conformation of p53 is The that the DNA can be by p53 in the of Hsp90 with the that only the specific for natively p53 the protein that Hsp90 binds to a native-like state of p53 that is in with the active wild type The results of the that binding of p53 to Hsp90 is reversible and from p53 is able to bind to This is in agreement with for Hsp90 binds to highly structured and the active H. Buchner J. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). Hsp90 is able to the irreversible of DNA binding activity of This and the by and D. K. M. B. Muller L. A. J. A. M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Hsp90 is at the conformation of p53 is and its binding activity to DNA is e.g. and the are not able to this of The binding between Hsp90 and p53 is and p53 is in with its active DNA binding form binding to we that Hsp90 the of the protein at The prevention of to be the of a folded, native-like conformation of other are known to interact with unfolded regions of a protein S. Buchner J. Chem. Int. 2002; PubMed Google Scholar), the large unstructured regions in the amino-terminal region of p53 the interaction for However, using fragments of p53 we that only p53 fragments the DBD bind to The fragments able to bind to which that in native-like The fragments only the NTD, tetramerization domain, or RD, to bind to The which consists of the DBD, not only able to bind to it with the protein for Hsp90 As shown the of the DBD is the of the protein (12Bell S. Klein C. Muller L. Hansen S. Buchner J. J. Mol. Biol. 2002; 322: 917-927Crossref PubMed Scopus (217) Google Scholar). Thus, Hsp90 not bind to an to a folded, native-like of the However, we that an unfolded DBD in interact with Hsp90 as by S. Acad. Sci. S. A. 2002; PubMed Scopus Google Scholar). has been agreement a binding for in Hsp90 A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. Y. Y. J. 2001; PubMed Scopus Google Scholar, T. T. Buchner J. Acad. Sci. S. A. 95: PubMed Scopus Google Scholar, C. 1997; PubMed Scopus Google Scholar, 2004; Full Text Full Text PDF Scopus Google Scholar). This not been for Using fragments of yeast we analyzed the interaction between wild type p53 and the domains of the only yHsp90, and bind to the of binding sites for p53 within that the binding to the middle domain to be the binding to the carboxy-terminal domain of the amino-terminal domain, which is to bind to T. T. Buchner J. Acad. Sci. S. A. 95: PubMed Scopus Google Scholar, C. 1997; PubMed Scopus Google Scholar), not bind to of of fragments revealed that the affinity is by the interaction of Hsp90 with the DBD of The binding of is in the of binding to S. M. Buchner J. J. Mol. Biol. 2002; PubMed Scopus Google Scholar). Interestingly, the middle domain and to the carboxy-terminal domain of Hsp90 are to p53 from thermal we that Hsp90 is to the of wild type p53 at physiological in vitro and in thus an additional level of for this regulatory the for and M. and for the of and and for the proteins and