Assessment of the ATP Binding Properties of Hsp90 (1996)

Hsp90, one of the most prominent proteins in eucaryotic cells under physiological and stress conditions, chaperones protein folding reactions in an ATP-independent way. Surprisingly, ATP binding and ATPase activity of Hsp90 has been reported by several groups. To clarify this important issue, we have reinvestigated the potential ATP binding properties and ATPase activity of highly purified Hsp90 using a number of different techniques.Hsp90 was compared to the well characterized ATP-binding chaperone Hsc70 and to two control proteins, immunoglobulin G and bovine serum albumin, that are known to not bind ATP. Hsp90 behaved very similarly to the non-ATP-binding proteins and very differently from the ATP-binding protein Hsc70. Like bovine serum albumin and immunoglobulin G, Hsp90 (i) did not bind to immobilized ATP, (ii) could not be specifically photocross-linked with azido-ATP, (iii) failed to exhibit significant changes in intrinsic protein fluorescence upon ATP addition, and (iv) did not bind to three fluorescent ADP analogues. In contrast, Hsc70 strongly bound ATP and ADP, specifically cross-linked with azido-ATP, and exhibited major shifts in fluorescence upon addition of ATP. Finally, reexamination of the amino acid sequence of Hsp90 failed to reveal any significant homologies to known ATP-binding motifs. Taken together, we conclude that highly purified Hsp90 does not bind ATP. Weak ATPase activities associated with Hsp90 preparations may be due to minor impurities or kinases copurifying with Hsp90. Hsp90, one of the most prominent proteins in eucaryotic cells under physiological and stress conditions, chaperones protein folding reactions in an ATP-independent way. Surprisingly, ATP binding and ATPase activity of Hsp90 has been reported by several groups. To clarify this important issue, we have reinvestigated the potential ATP binding properties and ATPase activity of highly purified Hsp90 using a number of different techniques. Hsp90 was compared to the well characterized ATP-binding chaperone Hsc70 and to two control proteins, immunoglobulin G and bovine serum albumin, that are known to not bind ATP. Hsp90 behaved very similarly to the non-ATP-binding proteins and very differently from the ATP-binding protein Hsc70. Like bovine serum albumin and immunoglobulin G, Hsp90 (i) did not bind to immobilized ATP, (ii) could not be specifically photocross-linked with azido-ATP, (iii) failed to exhibit significant changes in intrinsic protein fluorescence upon ATP addition, and (iv) did not bind to three fluorescent ADP analogues. In contrast, Hsc70 strongly bound ATP and ADP, specifically cross-linked with azido-ATP, and exhibited major shifts in fluorescence upon addition of ATP. Finally, reexamination of the amino acid sequence of Hsp90 failed to reveal any significant homologies to known ATP-binding motifs. Taken together, we conclude that highly purified Hsp90 does not bind ATP. Weak ATPase activities associated with Hsp90 preparations may be due to minor impurities or kinases copurifying with Hsp90. INTRODUCTIONCells respond to external stresses such as a sudden increase in temperature with the synthesis of a distinct set of proteins called heat shock or stress proteins (Nover, 1991). The predominant classes of stress proteins including GroE, Hsc70, 1The abbreviations used are: Hsc70/Hsp7070-kDa heat shock proteinCScitrate synthaseGdmClguanidinium chlorideBSAbovine serum albuminIgGimmunoglobulin GFab fragmentantigen-binding fragment of antibodiesPAGEpolyacrylamide gel electrophoresisMABA-ADPN-8-(4-N′-methylanthranylaminobutyl)-8aminoadenosine diphosphateMANT-ADP3′-O-N-methyl-anthraniloyl diphosphateΣ-ADPetheno-ADP. Hsp90, and small Hsps have been implicated in protein folding as molecular chaperones (Morimoto et al., 19.Morimoto R.I. Tissi é res A. Georgopolous C.(eds) Stress Proteins in Biology and Medicine. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1994Google Scholar; Jakob and Buchner, 10.Jakob U. Buchner J. Trends Biochem. Sci. 1994; 19: 205-211Google Scholar; Buchner, 4.Buchner J. FASEB J. 1996; 10: 10-20Google Scholar). While the precise molecular mechanism of these chaperones is still under extensive investigation, the ATP dependence of chaperone-mediated protein folding is clearly the hallmark of the GroE and Hsc70 class of stress proteins. Both the ATPase activity of these proteins and its influence on assisted protein folding have been analyzed in detail (cf. Morimoto et al., 1994). In contrast, conflicting evidence exists concerning Hsp90's ATP binding properties and ATPase activity. Hsp90 is one of the most abundant proteins in the eucaryotic cell, even at physiological conditions. In complex with other effector proteins such as Hsc70 and prolyl isomerases, Hsp90 has been implicated as a molecular chaperone in the maturation of specific protein substrates such as steroid receptors and kinases in vivo (Pratt, 26.Pratt W.B. J. Biol. Chem. 1993; 268: 21455-21458Google Scholar; Smith et al., 32.Smith D.F. Sullivan W.P. Johnson J. Toft D.O. Moudgil V.K. Steroid Hormone Receptors. Birkh ä user, Boston1993: 247-258Google Scholar; Jakob and Buchner, 1994; Buchner, 1996). Interestingly, assembly of these complexes with substrate proteins has been found to be ATP-dependent (Pratt, 1993; Smith et al., 1993). However, since Hsc70 is also involved in the formation of these high molecular weight assemblies, it is not yet clear which of the Hsps is responsible for the ATP requirement. More recently, using in vitro folding and unfolding assays, it has been demonstrated that Hsp90 may be a general cytosolic chaperone under physiological (Wiech et al., 36.Wiech H. Buchner J. Zimmermann R. Jakob U. Nature. 1992; 358: 169-170Google Scholar; Shaknovich et al., 29.Shaknovich R. Shue G. Kohtz D.S. Mol. Cell Biol. 1992; 12: 5059-5068Google Scholar; Shue and Kohtz, 31.Shue G. Kohtz D.S. J. Biol. Chem. 1994; 269: 2707-2711Google Scholar) and heat shock conditions (Jakob et al., 11.Jakob U. Lilie H. Meyer I. Buchner J. J. Biol. Chem. 1995; 270: 7288-7294Google Scholar; Schumacher et al., 28.Schumacher R.J. Hurst R. Sullivan W.P. McMahon N.J. Toft D.O. Matts R.L. J. Biol. Chem. 1994; 269: 9493-9499Google Scholar). These chaperone functions as well as interactions of Hsp90 with estrogen receptors (Inano et al., 9.Inano K. Curtis S.W. Korach K.S. Omata S. Horigome T. J. Biochem. (Tokyo). 1994; 116: 759-766Google Scholar) were found to be ATP-independent. In contrast, binding of ATP to Hsp90 has been reported (Csermely and Kahn, 6.Csermely P. Kahn C.R. J. Biol. Chem. 1991; 266: 4350-4943Google Scholar). Binding of ATP was suggested to result in conformational changes of Hsp90 (Csermely et al., 37.Wiech H. Buchner J. Zimmermann M. Zimmermann R. Jakob U. J. Biol. Chem. 1993; 268: 7414-7421Google Scholar), which in turn would affect the interaction with other proteins (Kellermayer and Csermely, 14.Kellermayer M.S.Z. Csermely P. Biochem. Biophys. Res. Commun. 1995; 211: 166-174Google Scholar). In addition evidence was presented suggesting that some purified Hsp90 preparations exhibit potent peptide-stimulated ATPase activity with high turnover numbers (Nadeau et al., 20.Nadeau K. Sullivan M.A. Bradley M. Engman D.M. Walsh C.T. Protein Sci. 1992; 1: 970-979Google Scholar; 21.Nadeau K. Das A. Walsh C.T. J. Biol. Chem. 1993; 268: 1479-1487Google Scholar). However, Hsp90 purified from other species did not show ATPase activity (Wiech et al., 1993; Nardai et al., 1995).Whether Hsp90 acts in an ATP-dependent or independent way is of crucial importance for understanding the molecular mechanism of this chaperone. Therefore we have examined the ATP binding properties of Hsp90 in detail using several independent experimental approaches. By including known ATP-binding or non-ATP-binding proteins as positive and negative controls, we were able to show that several of the methods employed previously are not suited to unambiguously demonstrate ATP binding. Those methods that turned out to be reliable demonstrate that Hsp90 does not bind ATP.MATERIALS AND METHODSProteinsHsp90 and Hsc70 from bovine pancreas as well as Hsp90 from yeast and Escherichia coli were purified as described previously (Wiech et al., 1993; Jakob et al., 1995a, 12.Jakob U. Meyer I. Bügl H. André S. Bardwell J.C.A. Buchner J. J. Biol. Chem. 1995; 270: 14412-14419Google Scholar). In addition, heparin-Sepharose was used as a final step in the purification of Hsp90 (Olsson et al., 24.Olsson H. Martinez-Arias W. Drobak B.K. 1995; Scholar). The protein of bovine Hsp90 and Hsc70 were to Biochem. Scholar) using bovine serum albumin as a Hsp90 were using the of for a at (Jakob et al., and the fragment were from was from and were from and ATP was from was from of the of were analyzed by with a was at The of for the of and and for the of for the were and at a of at The for the were and at a of to the protein was to any bound by the addition of of to of protein The was on for and of was to The was at for and of the was analyzed for as described influence of Hsp90 and Hsc70 on the of at was in the or of as described previously (Jakob et al., of was used in a binding at in was with and was by the in of it on a for and it at The was and the was yeast Hsp90 or bovine Hsc70 were with protein and to with A. The protein were to the and these were for at The were at for The was on for The was with A. the was in of for on a and at the was on The was as described with high and the was on at of specifically bound protein was by the addition of with and ATP. was Finally, the was with of in A. were analyzed on were with Hsp90 or were which was with of ATP. The final was a at were on and was by a at a of addition of of the were for at and to a the gel was were as described previously et al., A. J. Biol. Chem. Scholar; et al., G. S. Buchner J. J. Biol. Chem. 1995; 270: Scholar). of purified yeast Hsp90 or were at with ATP, in In some with Hsp90, or was used of of the of were to of ATP, ADP, to the on was in and The were the to ATP and ADP with and The in was by and as a of the in of unfolding of yeast Hsp90 were by the in intrinsic The were in a in a at The fluorescence was at an of and an of The were set to and for and unfolding of yeast was by of the protein of in The protein are in the The were for at to To the influence of ATP on the of yeast Hsp90, unfolding was as described in the and of of ATP on the of Hsp90, Hsc70, and proteins were of ATP in The protein are in the The were for at to fluorescence were out in a as described are as the of protein fluorescence the of the in fluorescence were for the of ATP at using the is the fluorescence and is the fluorescence the is the ATP and is the of ATP et al., Biochem. of the Binding of ADP to were with an at the was and fluorescent were to of at the protein was and are of of of and were employed to binding of to Hsp90. The used were for the and to for Hsp90. The of and were and and the were and control binding of to the molecular chaperone from was by In to this with high to and J. for The conditions used The was and the was ADP was to the and the fluorescence was Hsp90 of from different was purified by to as by of To purified Hsp90 bound we the of Hsp90 an of suggesting that the protein were To this we analyzed the of purified yeast Hsp90 by high under conditions. could be not these demonstrate other ATP-dependent chaperones such as GroE and Hsc70, purified Hsp90 does not bound of Hsp90 the influence of ATP on the chaperone activity of Hsp90 with that of Hsc70, a known ATP-dependent we in vitro unfolding in the or of ATP with proteins. an we used the unfolding of previously that Hsp90 the unfolding and by binding to unfolding (Jakob et al., in has influence on the of by Hsp90. In contrast, of by Hsc70 is strongly by ATP. different of In the of ATP, Hsc70 by binding the In the of ATP, the of Hsc70 is due to the and of bound were previously for the chaperone GroE under heat shock conditions et al., to to immobilized ATP has been as evidence for the of Hsp90 with (Csermely and Kahn, 1991). the purified Hsp90 to immobilized ATP by Hsp90 with that the addition of the protein was to the interaction of Hsp90 with the was not to influence the specific binding of the control protein Hsc70 To specific binding of proteins to of the bound proteins was by with ATP extensive These that Hsc70 to immobilized ATP and be specifically by the addition of ATP. However, specific binding of Hsp90 to could not be The of Hsp90 was found in the and the of and to the did not the not we conclude that Hsp90 does not bind to immobilized of yeast Hsp90 and bovine Hsc70 to of yeast Hsp90 or bovine Hsc70 were with of in and these were on to of in the were analyzed by and and ATP and ATP and are by and to Hsp90 of to Hsp90 has been (Csermely and Kahn, 1991; Shaknovich et al., M. R. Buchner J. 1992; Scholar). To this specific we compared the of Hsp90 with that of the known ATP-binding protein Hsc70 and the non-ATP-binding protein with ATP was used to the of the in of to Hsp90 was under the conditions However, to we could not for binding by addition of a of ATP. that and ATP bind to different on Hsp90, or that with a ATP, or that with Hsp90. the of to Hsp90 be as a reliable of ATP binding. was by the that and fragment not photocross-linked to in a to Hsp90. In contrast, the with Hsc70 could be with of of Hsp90, and Hsc70 with of Hsp90 of or Hsc70 were with in the of of ATP. in the of and it is known that upon the of et al., the of with Hsp90 to be with or binding to an increase in the of the was for the chaperone binding of the protein as by unfolding et al., 1993; Scholar). this we analyzed the addition of on the of Hsp90. In the of an unfolding to that previously (Jakob et al., was The of did not the unfolding of Hsp90, that Hsp90 is not by of on unfolding of yeast Hsp90. unfolding of yeast Hsp90 was in the or of ATP in To unfolding of yeast Hsp90, the fluorescence at was of ATP the of it been reported that addition of ATP the intrinsic fluorescence of Hsp90 (Csermely et al., P. J. M. G. S. Kahn C.R. P. K. J. J. Biol. Chem. 1993; 268: Scholar). this by Hsp90 with the ATP-binding protein Hsc70 and the non-ATP-binding proteins and fragment concerning changes in the intrinsic fluorescence in the of found that addition of of ATP in a of the intrinsic protein fluorescence independent of the protein is due to the of ATP, the of ATP to a fluorescence is to fluorescence in the of ATP for this of the as described by et to potential specific of ATP on the intrinsic fluorescence of proteins. as in a in fluorescence for of the was with proteins Hsp90 and the non-ATP-binding protein a fluorescence at ATP from to the intrinsic fluorescence of Hsc70 by the specific binding of ATP. In the of the by upon ATP most due to interaction with of ATP on the intrinsic fluorescence of Hsp90, Hsc70, and fluorescence changes of bovine Hsc70 and yeast Hsp90 and in the of of ATP were at and and for The of ATP was as described under and ADP to of ATP to proteins does not to changes in the intrinsic fluorescence of a we in which we used fluorescent ADP to binding. specific binding in a fluorescence of the of Hsp90 were with a of the and the fluorescence was The that the fluorescence of a of the and some and to Hsp90 was In contrast, control with from show the fluorescence of to increase by some to of the ATP-dependent molecular chaperone was is of a high of for in this ADP could for binding of the in a fluorescence The increase in fluorescence of in the of Hsp90 could not be by using the ADP a minor fluorescence increase upon addition of Hsp90. the small fluorescence in the of Hsp90 could not be by ADP not of ADP to Hsp90 and of yeast Hsp90 and as well as The fluorescence of the upon addition of the protein was fluorescence addition of ADP in the of and ATPase of Hsp90 to for addition to ATP also suggested that Hsp90 may be an ATP (Nadeau et al., failed to any ATPase activity in highly purified bovine Hsp90 preparations (Wiech et al., 1993). we have reinvestigated the ATPase activity of the yeast Hsp90 which we been using for the ATP binding ATP we a ATP activity activity was that of an of the Hsc70 which a very ATPase that a potent or such as in as as in protein preparations could be responsible for this To ATPase activity be in highly purified proteins, we for ATPase activity in was found to of ATPase as highly purified Hsp90. a purified which was to be under conditions that were to for Hsp90. These that the ATPase activity of Hsp90 from species could be due to minor impurities of the protein et al., Walsh C.T. Sci. U. S. A. 1994; of Hsp90 ATP Binding of the sequence of Hsp90 the of a of the ATP binding and (Csermely and Kahn, 1991). this by the for and of ATP binding et al., M. N.J. J. 1: Scholar; et al., T. Smith T. A. J. Biol. Chem. Scholar). The used the of these proteins are known or Hsp90 was not found using this in this a which is compared to that of kinases or ATP Hsp90, and kinases or the which is of the In of proteins in this the high for the of this set of proteins the sequence or the was for the is and is a for the of an ATP binding found with a sequence et al., the of and binding in proteins with ATP binding While proteins are unambiguously binding ATP, exhibit known ATP binding activity. Hsp90 to this of proteins, it that Hsp90 an ATP binding of the has concerning the and of the highly abundant cytosolic protein Hsp90. is known that Hsp90 is involved in the of proteins, acts in with several heat shock and shock proteins in vivo and functions as a general molecular chaperone in vitro (cf. Jakob and Buchner, 1994). However, has yet been concerning the or not ATP binding a in the mechanism of Hsp90. While the in vitro of Hsp90 is clearly ATP-independent (Wiech et al., 1992; Shaknovich et al., 1992; Shue and Kohtz, 1994; Schumacher et al., 1994; Jakob et al., and ATP binding and ATPase activity of Hsp90 have been reported (Csermely Kahn, 1991; Csermely et al., 1993; et al., 1993). it is of crucial importance to the potential influence of ATP on the and of Hsp90 in have this by the binding of to purified Hsp90. The step was to any were bound to purified Hsp90. two independent Hsp90 was to be was a for binding since the to ATP binding could not be to binding we the of which previously been as evidence for specific binding of to Hsp90. that Hsp90 with substrate proteins as well as with proteins, Hsp90 be to several binding it was important to specific binding of ATP to binding and interactions of ATP with the protein on these we compared the of ATP on Hsp90 with the of ATP on known ATP-binding and non-ATP-binding proteins. of the proteins with of could be for the ATP-dependent chaperone Hsc70. The that of proteins with also in a way is in with a by and is on the formation of In addition, we with ADP in which the specific binding to a fluorescence of the independent of the of amino in the the ADP significant fluorescence changes could be upon addition of Hsp90. In contrast, the Hsc70 changes in suggested to that Hsp90 is not an ATP-binding these we were to binding of Hsp90 to immobilized ATP and we did not any of ATP on the unfolding of Hsp90. Finally, we ATPase with Hsp90. In to bovine Hsp90 (Wiech et al., ATPase activities could be in some yeast Hsp90 However, ATPase are very to the of of highly and which result in the of ATPase activities in purified protein of this is an to its ATPase even purified to different ATPase activities to the for of for ATPase activities in protein is ATPase activities be to any purified on these and the of the amino acid which did not reveal significant with known ATP-binding we the binding of ATP to Hsp90 as well as the high of ATPase activity of Hsp90, previously reported (Nadeau et al., 1993). bind with very to Hsp90 be out on negative in the of the with different this In addition, we the that binding of proteins, may the of Hsp90 to ATP binding. this a understanding of the interaction of Hsp90 with these potential this the general ATP is a for protein folding The number of does not ATP such as small and strongly this The mechanism to different of these ATP-independent of and are able to bind and in the of ATP. ATP and the are for the of proteins from the ATP-dependent chaperone under folding conditions, it was demonstrated that the of binding and are independent of ATP et al., M. Buchner J. J. Biol. Chem. 1994; 269: Scholar; et al., 1996). ATP the of for which in an of protein is et al., the of Hsp90, binding for protein to the ATP-independent binding of have been (Jakob et al., was suggested that Hsp90 with proteins. The to the be by binding the of by the of or by a folding the of folding and unfolding external to the final of the substrate of the binding of Hsp90 by ATP is not and are to the Hsp90 In with this in vitro with purified Hsp90 that ATP is not for the interaction of Hsp90 with proteins such as and (Wiech et al., 1992; Jakob et al., 1995a, Shaknovich et al., 1992; and 1995; Schumacher et al., 1994). However, the of Hsp90 as a of the chaperone is the of Hsc70 and the is known it was suggested that Hsc70 with the formation of the The mechanism of this complex formation is not yet Hsc70 to be the responsible for the ATP of this and may also be involved and Toft D.O. Mol. 1995; Scholar). the has been that Hsp90 and the functions in the folding of proteins. In this the of the ATP-independent Hsp90 would be to the Hsc70 in with would the protein in an ATP-dependent way et al., A. 1995; 268: Scholar). INTRODUCTIONCells respond to external stresses such as a sudden increase in temperature with the synthesis of a distinct set of proteins called heat shock or stress proteins (Nover, 1991). The predominant classes of stress proteins including GroE, Hsc70, 1The abbreviations used are: Hsc70/Hsp7070-kDa heat shock proteinCScitrate synthaseGdmClguanidinium chlorideBSAbovine serum albuminIgGimmunoglobulin GFab fragmentantigen-binding fragment of antibodiesPAGEpolyacrylamide gel electrophoresisMABA-ADPN-8-(4-N′-methylanthranylaminobutyl)-8aminoadenosine diphosphateMANT-ADP3′-O-N-methyl-anthraniloyl diphosphateΣ-ADPetheno-ADP. Hsp90, and small Hsps have been implicated in protein folding as molecular chaperones (Morimoto et al., 19.Morimoto R.I. Tissi é res A. Georgopolous C.(eds) Stress Proteins in Biology and Medicine. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1994Google Scholar; Jakob and Buchner, 10.Jakob U. Buchner J. Trends Biochem. Sci. 1994; 19: 205-211Google Scholar; Buchner, 4.Buchner J. FASEB J. 1996; 10: 10-20Google Scholar). While the precise molecular mechanism of these chaperones is still under extensive investigation, the ATP dependence of chaperone-mediated protein folding is clearly the hallmark of the GroE and Hsc70 class of stress proteins. Both the ATPase activity of these proteins and its influence on assisted protein folding have been analyzed in detail (cf. Morimoto et al., 1994). In contrast, conflicting evidence exists concerning Hsp90's ATP binding properties and ATPase activity. Hsp90 is one of the most abundant proteins in the eucaryotic cell, even at physiological conditions. In complex with other effector proteins such as Hsc70 and prolyl isomerases, Hsp90 has been implicated as a molecular chaperone in the maturation of specific protein substrates such as steroid receptors and kinases in vivo (Pratt, 26.Pratt W.B. J. Biol. Chem. 1993; 268: 21455-21458Google Scholar; Smith et al., 32.Smith D.F. Sullivan W.P. Johnson J. Toft D.O. Moudgil V.K. Steroid Hormone Receptors. Birkh ä user, Boston1993: 247-258Google Scholar; Jakob and Buchner, 1994; Buchner, 1996). Interestingly, assembly of these complexes with substrate proteins has been found to be ATP-dependent (Pratt, 1993; Smith et al., 1993). However, since Hsc70 is also involved in the formation of these high molecular weight assemblies, it is not yet clear which of the Hsps is responsible for the ATP requirement. More recently, using in vitro folding and unfolding assays, it has been demonstrated that Hsp90 may be a general cytosolic chaperone under physiological (Wiech et al., 36.Wiech H. Buchner J. Zimmermann R. Jakob U. Nature. 1992; 358: 169-170Google Scholar; Shaknovich et al., 29.Shaknovich R. Shue G. Kohtz D.S. Mol. Cell Biol. 1992; 12: 5059-5068Google Scholar; Shue and Kohtz, 31.Shue G. Kohtz D.S. J. Biol. Chem. 1994; 269: 2707-2711Google Scholar) and heat shock conditions (Jakob et al., 11.Jakob U. Lilie H. Meyer I. Buchner J. J. Biol. Chem. 1995; 270: 7288-7294Google Scholar; Schumacher et al., 28.Schumacher R.J. Hurst R. Sullivan W.P. McMahon N.J. Toft D.O. Matts R.L. J. Biol. Chem. 1994; 269: 9493-9499Google Scholar). These chaperone functions as well as interactions of Hsp90 with estrogen receptors (Inano et al., 9.Inano K. Curtis S.W. Korach K.S. Omata S. Horigome T. J. Biochem. (Tokyo). 1994; 116: 759-766Google Scholar) were found to be ATP-independent. In contrast, binding of ATP to Hsp90 has been reported (Csermely and Kahn, 6.Csermely P. Kahn C.R. J. Biol. Chem. 1991; 266: 4350-4943Google Scholar). Binding of ATP was suggested to result in conformational changes of Hsp90 (Csermely et al., 37.Wiech H. Buchner J. Zimmermann M. Zimmermann R. Jakob U. J. Biol. Chem. 1993; 268: 7414-7421Google Scholar), which in turn would affect the interaction with other proteins (Kellermayer and Csermely, 14.Kellermayer M.S.Z. Csermely P. Biochem. Biophys. Res. Commun. 1995; 211: 166-174Google Scholar). In addition evidence was presented suggesting that some purified Hsp90 preparations exhibit potent peptide-stimulated ATPase activity with high turnover numbers (Nadeau et al., 20.Nadeau K. Sullivan M.A. Bradley M. Engman D.M. Walsh C.T. Protein Sci. 1992; 1: 970-979Google Scholar; 21.Nadeau K. Das A. Walsh C.T. J. Biol. Chem. 1993; 268: 1479-1487Google Scholar). However, Hsp90 purified from other species did not show ATPase activity (Wiech et al., 1993; Nardai et al., 1995).Whether Hsp90 acts in an ATP-dependent or independent way is of crucial importance for understanding the molecular mechanism of this chaperone. Therefore we have examined the ATP binding properties of Hsp90 in detail using several independent experimental approaches. By including known ATP-binding or non-ATP-binding proteins as positive and negative controls, we were able to show that several of the methods employed previously are not suited to unambiguously demonstrate ATP binding. Those methods that turned out to be reliable demonstrate that Hsp90 does not bind ATP.