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• W2053708941 abstract "hsp90, in addition to being an abundant and pivotal cytoplasmic chaperone protein, has been shown to be a weak ATPase. In an effort to characterize the ATPase activity of hsp90, we have observed marked differences in activities among various species of hsp90. Chicken or human hsp90 hydrolyzed ATP with ak cat of 0.02 min−1 and aK m greater than 300 μm. In contrast, yeast hsp90 and TRAP1, an hsp90 homologue found in mitochondria, were 10–100-fold more active as ATPases. Sedimentation studies confirmed that all are dimeric proteins. Chicken hsp90 mutants were then analyzed to identify regions within the protein that influence ATPase activity. A truncation mutant of chicken hsp90, N1–573, was found to be monomeric, and yet the catalytic efficiency (k cat/K m) was greater than 100 times that of the full-length protein (k catof 0.24 min−1 and K m of 60 μm). In contrast, an internal deletion mutant, Δ661–677, was also monomeric but failed to hydrolyze ATP. Finally, deletion of the last 30 amino acids resulted in a dimeric protein with an ATPase activity very similar to full-length hsp90. These data indicate that sequences within the last one-fourth of hsp90 regulate ATP hydrolysis. hsp90, in addition to being an abundant and pivotal cytoplasmic chaperone protein, has been shown to be a weak ATPase. In an effort to characterize the ATPase activity of hsp90, we have observed marked differences in activities among various species of hsp90. Chicken or human hsp90 hydrolyzed ATP with ak cat of 0.02 min−1 and aK m greater than 300 μm. In contrast, yeast hsp90 and TRAP1, an hsp90 homologue found in mitochondria, were 10–100-fold more active as ATPases. Sedimentation studies confirmed that all are dimeric proteins. Chicken hsp90 mutants were then analyzed to identify regions within the protein that influence ATPase activity. A truncation mutant of chicken hsp90, N1–573, was found to be monomeric, and yet the catalytic efficiency (k cat/K m) was greater than 100 times that of the full-length protein (k catof 0.24 min−1 and K m of 60 μm). In contrast, an internal deletion mutant, Δ661–677, was also monomeric but failed to hydrolyze ATP. Finally, deletion of the last 30 amino acids resulted in a dimeric protein with an ATPase activity very similar to full-length hsp90. These data indicate that sequences within the last one-fourth of hsp90 regulate ATP hydrolysis. Heat shock protein 90 (hsp90) 1hspheat shock proteinHophsp-organizing proteinDTTdithiothreitol 1hspheat shock proteinHophsp-organizing proteinDTTdithiothreitol has been demonstrated to be an important chaperone for a vast array of proteins involved in cell regulation, such as transcription factors and protein kinases (1Pratt W.B. Proc. Soc. Exp. Biol. Med. 1998; 217: 420-434Crossref PubMed Scopus (416) Google Scholar, 2Caplan A.J. Trends Cell Biol. 1999; 9: 262-268Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 3Mayer M.P. Bukau B. Curr. Biol. 1999; 9: R322-R325Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 4Richter K. Buchner J. J. Cell. Physiol. 2001; 188: 281-290Crossref PubMed Scopus (506) Google Scholar, 5Pearl L.H. Prodromou C. Curr. Opin. Struct. Biol. 2000; 10: 46-51Crossref PubMed Scopus (281) Google Scholar, 6Young J.C. Moarefi I. Hartl U. J. Cell Biol. 2001; 154: 267-273Crossref PubMed Scopus (719) Google Scholar). Interestingly, hsp90 seems to assist in the late stages of folding its substrate and may be important for modulating specific interactions such as ligand or co-factor binding or covalent modifications such as phosphorylation. hsp90 functions optimally in a multicomponent complex of chaperone proteins including hsp40, hsp70, Hop, p23, and one of a variety of immunophilins (1Pratt W.B. Proc. Soc. Exp. Biol. Med. 1998; 217: 420-434Crossref PubMed Scopus (416) Google Scholar, 2Caplan A.J. Trends Cell Biol. 1999; 9: 262-268Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 3Mayer M.P. Bukau B. Curr. Biol. 1999; 9: R322-R325Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 4Richter K. Buchner J. J. Cell. Physiol. 2001; 188: 281-290Crossref PubMed Scopus (506) Google Scholar, 5Pearl L.H. Prodromou C. Curr. Opin. Struct. Biol. 2000; 10: 46-51Crossref PubMed Scopus (281) Google Scholar, 6Young J.C. Moarefi I. Hartl U. J. Cell Biol. 2001; 154: 267-273Crossref PubMed Scopus (719) Google Scholar). heat shock protein hsp-organizing protein dithiothreitol heat shock protein hsp-organizing protein dithiothreitol Until recently, the evidence for nucleotide binding and hydrolysis by hsp90 was controversial. There is now direct evidence that hsp90 binds ADP and ATP and that the conformational state of hsp90 differs substantially depending upon which nucleotide is bound (7Sullivan W. Stensgard B. Caucutt G. Bartha B. McMahon N. Alnemri E.S. Litwack G. Toft D.O. J. Biol. Chem. 1997; 272: 8007-8012Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 8Stebbins C.E. Russo A.A. Schneider C. Rosen N. Hartl F.U. Pavletich N.P. Cell. 1997; 89: 239-250Abstract Full Text Full Text PDF PubMed Scopus (1249) Google Scholar, 9Prodromou 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 (1121) Google Scholar, 10Grenert J.P. Sullivan W.P. Fadden P. Haystead T.A.J. Clark J. Mimnaugh E. Krutzsch H. Ochel H.-J. Schulte T.W. Sausville E. Neckers L.M. Toft D.O. J. Biol. Chem. 1997; 272: 23843-23850Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar, 11Scheibel T. Neuhofen S. Weikl T. Mayr C. Reinstein J. Vogel P.D. Buchner J. J. Biol. Chem. 1997; 272: 18608-18613Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The crystallization of the amino-terminal 220 amino acids of hsp90 with either nucleotide or an hsp90-specific inhibitor, geldanamycin, revealed that hsp90 possesses a unique nucleotide-binding site that differs substantially from that of hsp70 (8Stebbins C.E. Russo A.A. Schneider C. Rosen N. Hartl F.U. Pavletich N.P. Cell. 1997; 89: 239-250Abstract Full Text Full Text PDF PubMed Scopus (1249) Google Scholar, 9Prodromou 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 (1121) Google Scholar). It has thus been proposed that hsp90 belongs to the GHKL superfamily of ATP-binding proteins that includes the DNA-repair protein MutL, DNA gyrase, topoisomerase II, and histidine kinases (12Dutta R. Inouye M. Trends Biochem. Sci. 2000; 25: 24-28Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar). The proposed mechanism of action for DNA gyrase and topoisomerase II requires transient ATP-mediated amino-terminal dimerization to form a “molecular clamp” that allows single-stranded DNA strand passage to occur. Binding of single-stranded DNA increases the ATPase activity of the enzyme by severalfold, and ATP hydrolysis is required to reset the “clamp” (13Wang J.C. Q. Rev. Biophys. 1998; 31: 107-144Crossref PubMed Scopus (301) Google Scholar, 14Lindsley J.E. Wang J.C. J. Biol. Chem. 1993; 268: 8096-8104Abstract Full Text PDF PubMed Google Scholar). It has been suggested that hsp90 may interact with ATP and its substrate (client protein) through a similar molecular clamp mechanism (4Richter K. Buchner J. J. Cell. Physiol. 2001; 188: 281-290Crossref PubMed Scopus (506) Google Scholar, 5Pearl L.H. Prodromou C. Curr. Opin. Struct. Biol. 2000; 10: 46-51Crossref PubMed Scopus (281) Google Scholar, 6Young J.C. Moarefi I. Hartl U. J. Cell Biol. 2001; 154: 267-273Crossref PubMed Scopus (719) Google Scholar). Although the binding site for adenine nucleotides has been localized to the amino terminus of hsp90, nucleotide binding has a profound impact on more distal parts of the protein. Specifically, the hsp90 co-chaperone p23 associates with hsp90 only when it is bound to ATP but not when it is bound to ADP or the ansamycin inhibitor geldanamycin (7Sullivan W. Stensgard B. Caucutt G. Bartha B. McMahon N. Alnemri E.S. Litwack G. Toft D.O. J. Biol. Chem. 1997; 272: 8007-8012Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). The two nucleotide-dependent conformational states of hsp90 are perhaps similar, therefore, to the “open” and “closed” forms of either hsp70 (15Johnson E.R. McKay D.B. Biochemistry. 1999; 38: 10823-10830Crossref PubMed Scopus (45) Google Scholar, 16Mayer M.P. Schröder H. Rüdiger S. Paal K. Laufen T. Bukau B. Nat. Struct. Biol. 2000; 7: 586-593Crossref PubMed Scopus (308) Google Scholar), topoisomerase II (13Wang J.C. Q. Rev. Biophys. 1998; 31: 107-144Crossref PubMed Scopus (301) Google Scholar, 14Lindsley J.E. Wang J.C. J. Biol. Chem. 1993; 268: 8096-8104Abstract Full Text PDF PubMed Google Scholar), or MutL (17Tran P.T. Liskay R.M. Mol. Cell. Biol. 2000; 20: 6390-6398Crossref PubMed Scopus (80) Google Scholar, 18Ban C. Yang W. Cell. 1998; 95: 541-552Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). The nucleotide binding and hydrolysis properties of the hsp90 class of molecular chaperones are not as thoroughly characterized as they are for hsp70. hsp70 binds ATP with a K m of around 50 μm and hydrolyzes it slowly with ak cat of 0.5 min−1 in the absence of co-chaperones and substrate (19Wilbank S.M. DeLuca-Flaherty C. McKay D.B. J. Biol. Chem. 1994; 269: 12893-12898Abstract Full Text PDF PubMed Google Scholar). In the presence of substrate, or the co-chaperone hsp40, hsp70 hydrolyzes ATP 10–20 times more efficiently, and the resulting hsp70-ADP complex binds substrate more tightly (20McCarty J.S. Buchberger A. Reinstein J. Bukau B. J. Mol. Biol. 1995; 249: 126-137Crossref PubMed Scopus (350) Google Scholar, 21Cyr D.M., Lu, X. Douglas M.G. J. Biol. Chem. 1992; 267: 20927-20931Abstract Full Text PDF PubMed Google Scholar, 22Jordan R. McMacken R. J. Biol. Chem. 1995; 270: 4563-4569Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Dissociation of the substrate from the hsp70-ADP complex is promoted by a nucleotide exchange factor (23Skowyra D. Wickner S. J. Biol. Chem. 1995; 270: 26282-26285Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). The mechanism of action of the nucleotide exchange factor has recently been deduced. Upon binding hsp70, the nucleotide exchange factor opens the nucleotide-binding pocket, thereby releasing ADP and stabilizing the nucleotide-free state of hsp70 (24Sondermann H. Scheufler C. Schneider C. Höhfeld J. Hartl F.-U. Moarefi I. Science. 2001; 291: 1553-1557Crossref PubMed Scopus (363) Google Scholar). Unlike either hsp70 or the DNA repair enzymes, an in vitrosubstrate binding reaction has not been discovered that affects the ATP-hydrolyzing capabilities of hsp90. Additionally, a nucleotide exchange factor has not been identified. However, based on the available crystal structure data and recognized co-factor requirements for hsp90 function as a chaperone, a hypothesis in which nucleotide and most likely co-factor and/or substrate binding alter the conformation of hsp90 is plausible. The impact that these structural alterations have on ATP hydrolysis activity is unknown. Initial studies with yeast hsp90 indicate that the full-length protein is required for maximal ATPase activity (25Prodromou 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). More specifically, although amino acids 1–221 are capable of binding nucleotide, additional residues (1–450) are needed to observe tight (i.e. committed) ATP binding, and residues beyond 450 are necessary for efficient ATP hydrolysis (26Weikl T. Muschler P. Richter K. Veit T. Reinstein J. Buchner J. J. Mol. Biol. 2000; 303: 583-592Crossref PubMed Scopus (106) Google Scholar). ATP binding has been shown to induce dimer interactions near the amino terminus of hsp90, but this conformational change requires greater than 500 amino acids of the protein (25Prodromou 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). Therefore, the full expression of ATPase activity requires regions of hsp90 beyond the minimal ATP-binding domain. To further our understanding of how structure and conformation of hsp90 correlate to ATPase activity, we have studied kinetically hsp90 proteins derived from human, chicken, yeast, Escherichia coli (HtpG), and an hsp90 homologue, TRAP1, found in human mitochondria. Within this analysis, the more complex the organism from which the hsp90 protein is derived, the less efficient the enzymatic activity. This finding predicts that hsp90 from higher eukaryotes would require co-factors to regulate ATP binding, substrate binding, and/or ATPase activity. Indeed, it has been shown previously that the hsp90 cofactor Hop inhibits the ATPase activity of hsp90 (27Prodromou 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 (355) Google Scholar), but p23, another hsp90 cofactor isolated from active chaperoning complexes (28Johnson J.L. Beito T.G. Krco C.J. Toft D.O. Mol. Cell. Biol. 1994; 14: 1956-1963Crossref PubMed Scopus (181) Google Scholar), has no discernible effect on ATPase activity (29Young J.C. Hartl F.-U. EMBO J. 2000; 19: 5930-5940Crossref PubMed Scopus (197) Google Scholar). A mutational analysis of chicken hsp90 was also performed in order to correlate hsp90 structure to ATPase activity. Our findings indicate that sequences within the carboxyl-terminal fourth of the protein profoundly affect ATPase activity. E. coli hsp90 (HtpG) was expressed in E. coli from a high copy number plasmid (30Jakob U. Meyer I. Bügl H. André S. Bardwell J.C.A. Buchner J. J. Biol. Chem. 1995; 270: 14412-14419Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), and the vector was a gift from Johannes Buchner, Institut fur Organische Chemie and Biochemie, Garching, Germany. Yeast hsp90 was purified from a yeast strain that overexpresses Hsc82 from a 2-μm URA3 overproducing plasmid (30Jakob U. Meyer I. Bügl H. André S. Bardwell J.C.A. Buchner J. J. Biol. Chem. 1995; 270: 14412-14419Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) and was provided by the laboratory of S. Lindquist, University of Chicago, Chicago, IL. TRAP1 was purified from E. coli, and its cloning and expression are detailed elsewhere (31Felts S.J. Owen B.A.L. Nguyen P.M. Trepel J. Donner D.B. Toft D.O. J. Biol. Chem. 2000; 275: 3305-3312Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Full-length chicken hsp90α, deletion mutants N1–220, N1–692, and Δ661–677 were all expressed and purified from E. coli as described previously (10Grenert J.P. Sullivan W.P. Fadden P. Haystead T.A.J. Clark J. Mimnaugh E. Krutzsch H. Ochel H.-J. Schulte T.W. Sausville E. Neckers L.M. Toft D.O. J. Biol. Chem. 1997; 272: 23843-23850Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar, 32Sullivan W.P. Toft D.O. J. Biol. Chem. 1993; 268: 20373-20379Abstract Full Text PDF PubMed Google Scholar). N1–573 was expressed in E. coli for the current experiments. Human hsp90β was expressed in Sf9 cells as described previously (7Sullivan W. Stensgard B. Caucutt G. Bartha B. McMahon N. Alnemri E.S. Litwack G. Toft D.O. J. Biol. Chem. 1997; 272: 8007-8012Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 10Grenert J.P. Sullivan W.P. Fadden P. Haystead T.A.J. Clark J. Mimnaugh E. Krutzsch H. Ochel H.-J. Schulte T.W. Sausville E. Neckers L.M. Toft D.O. J. Biol. Chem. 1997; 272: 23843-23850Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar). Overexpressed proteins were purified to near-homogeneity from cell lysates by successive chromatographies on DEAE-cellulose, heparin-Sepharose, Mono-Q, and Superdex 200. Purified proteins were aliquoted and stored at −70 °C in 20 mm Tris-HCl, pH 7.5, 50 mm KCl, 0.1 mm EDTA, 1 mm DTT, and 10% glycerol. Additional details of purification are published (7Sullivan W. Stensgard B. Caucutt G. Bartha B. McMahon N. Alnemri E.S. Litwack G. Toft D.O. J. Biol. Chem. 1997; 272: 8007-8012Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 10Grenert J.P. Sullivan W.P. Fadden P. Haystead T.A.J. Clark J. Mimnaugh E. Krutzsch H. Ochel H.-J. Schulte T.W. Sausville E. Neckers L.M. Toft D.O. J. Biol. Chem. 1997; 272: 23843-23850Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar). Chicken hsp90-α (1 mg/ml) in 20 mm HEPES, pH 7.5, 100 mm KCl, 1.0 mm DTT was digested with 10 μg/ml bovine thrombin (generously supplied by W. G. Owen, Mayo Clinic, Rochester, MN) for 0–24 h at 30 °C. Proteolysis was near completion (>95%) at 4 h, and no further proteolysis was detected after 24 h at 30 °C. Under these conditions, thrombin cleaved hsp90 at a single site, between Lys-611 and Ala-612, as determined by carboxyl-terminal sequencing. Purified hsp90 (5 μm, unless otherwise indicated) was incubated with 1 μm[α-32P]ATP (2 mCi/ml; PerkinElmer Life Sciences) at 30 °C for 0–480 min in a buffer containing 40 mmHEPES-KOH, pH 7.4, 100 mm KCl, 2 mmMgCl2, and 2 mm DTT. For ATP concentrations exceeding 1 μm, unlabeled ATP was added accordingly from a 100 mm stock of ATP-Mg, pH 7.4. Reactions were stopped by addition of an equal volume of a “stop” solution containing unlabeled AMP, ADP, ATP, and EDTA (12 mm, each). Thin layer chromatography was used to assess the extent of ATP hydrolysis. A 1:5 dilution of each ATPase reaction (2.5 μl) was spotted on cellulose PEI thin layer chromatography plates (Selecto Scientific Inc., Suwanee, GA), and ascending chromatography in 0.5m LiCl and 2 n formic acid was performed for 1 h at room temperature (33Obermann W.M.J. Sondermann H. Russo A.A. Pavletich N.P. Hartl F.U. J. Cell Biol. 1998; 143: 901-910Crossref PubMed Scopus (492) Google Scholar). After completion of chromatography, the plates were air-dried, and the radioactive spots corresponding to ADP and ATP were quantified by PhosphorImaging (Molecular Dynamics, Sunnyvale, CA). The ratio of ADP to ATP was used to calculate percent ATP hydrolysis. Each reaction was analyzed in duplicate at various time points depending upon the ATP concentration. A blank consisting of all the reaction components added to the stop solution at reaction time = 0 min was run at each concentration of ATP assayed. Kinetic data were analyzed by plotting velocity, v, in micromoles of ATP hydrolyzed/min versus μm ATP concentration, [S]. Rate constants were extracted by two methods. A primary plot of v versus [S] when fit to the Michaelis-Menten equation yielded a simple, rectangular 2 parameter hyperbola (Sigmaplot) using Equation 1. v=Vm[S]/Km+[S]Equation 1 K m and V max values were calculated directly with standard deviations. A replot of the kinetic data of v/[S] versus v, using the Eadie-Hofstee equation (Equation 2), yielded −1/K m as the slope and V max/K m as the y intercept. v/[S]=Vm/Km−v/KmEquation 2 Sedimentation equilibrium experiments were performed using the Beckman Optima XL-I analytical ultracentrifuge. All proteins were equilibrated in 20 mm HEPES, pH 7.4, 100 mm KCl, 5 mmMgCl2, and 2 mm DTT buffer using BioSpin 6 chromatography columns (Bio-Rad). Samples (300 μl) of each protein at ∼1 mg/ml (5–10 μm) were analyzed in double-sector cells fitted with sapphire windows against a reference composed of the BioSpin column equilibration buffer. To visualize the protein at the bottom of the cell, 20 μl of the immiscible fluorocarbon FC-43 was added to each sample containing hsp90 or derivative protein. All hsp90 full-length proteins and truncations were analyzed at 10,000 rpm and N1–220, N1–573, and N1–692 were also analyzed at 18,000 rpm. Full-length chicken hsp90 was also analyzed at 8,000 and 12,000 rpm at protein concentrations ranging from 0.1 to 1.5 mg/ml. Samples were either run in an An60Ti (4-hole) or An50Ti (8-hole) rotor at 20 °C. Data were obtained using Rayleigh interference optics as scans of fringe displacement (protein concentration, C)versus radial distance (r). Equilibrium was achieved when scans taken 3 h apart after an initial 24 h of centrifugation were superimposable. Primary data were fit to a self-association model using a Levenberg-Marquardt non-linear, least squares fitting routine which minimizes the x 2 residuals between the function (see below, Equation 3) and the data (XL-I UltraScan-Origin, Beckman Instruments, Fullerton, CA). The monomer-polymer model is represented by Equation 3, Cr=a1exp(Mω2(1−νρ)/2RT(r2−rm2))+a2exp(nMω2Equation 3 (1−νρ)/2RT(r2−rm2))+bWhere Cr is the concentration at radial positionr in the cell, and r the radial position at the meniscus. a 1 and a 2 are the absorbance values of the monomer and dimer, respectively, at the meniscus. M is the molecular weight of the monomer,R is the gas constant, T the temperature (in degrees Kelvin), ω is the angular velocity, ε the partial specific volume of the protein, and ρ the density of the buffer. The number of monomers in the polymer assembly is n, and b is the base-line absorbance. Fig. 1 illustrates the constructs used in this study and lists their relative ATPase activities based on k cat/K m . Each species is indicated by a uniquely shaded block to emphasize that although all proteins are hsp90 family members, there are substantial species-specific differences. Residues 1–220 are delineated by the first block for each protein and represent the most conserved region between species. TRAP1 has 18 additional amino acids at its amino terminus that are unique. Both TRAP1 and HtpG lack the “charged domain” represented by the line segment shown for chicken and yeast hsp90, comprising 57 and 53 amino acids, respectively. In addition to lacking the charged domain, HtpG and TRAP1 also lack ∼30 amino acids at the carboxyl terminus that contain a terminal MEEVD sequence also found in hsp70 proteins. Truncations or internal deletions were chosen for the following reasons. N1–220 represents the minimal ATP- or geldanamycin-binding site and has been crystallized with both geldanamycin and nucleotide (8Stebbins C.E. Russo A.A. Schneider C. Rosen N. Hartl F.U. Pavletich N.P. Cell. 1997; 89: 239-250Abstract Full Text Full Text PDF PubMed Scopus (1249) Google Scholar, 9Prodromou 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 (1121) Google Scholar). A glutathione S-transferase fusion version of N1–573 had been used previously by Chadli et al. (34Chadli 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 (136) Google Scholar) and shown to bind p23 in vitro. In addition, Weikl et al. (26Weikl T. Muschler P. Richter K. Veit T. Reinstein J. Buchner J. J. Mol. Biol. 2000; 303: 583-592Crossref PubMed Scopus (106) Google Scholar) have demonstrated that residues 1–450 are required for trapping the nucleotide by yeast hsp90, and therefore N1–573 should be fully competent to bind ATP. Amino acid 573 in the chicken occurs at the beginning of an exon boundary in the corresponding human sequence and therefore may define a stable protein folding domain (35Rebbe N.F. Hickman W.S. Ley T.J. Stafford D.W. Hickman S. J. Biol. Chem. 1989; 264: 15006-15011Abstract Full Text PDF PubMed Google Scholar). N1–692 removes the last 30 amino acids from the chicken hsp90 that are not found in either HtpG or TRAP1. Finally, the internal deletion mutant, Δ661–677, removes one of the proposed dimerization domains from the carboxyl terminus of chicken hsp90, yet Δ661–677 has been shown to confer viability in a yeast strain lacking endogenous hsp90 (36Meng X. Devin J. Sullivan W.P. Toft D. Baulieu E.-E. Catelli M.-G. J. Cell Sci. 1996; 109: 1677-1687Crossref PubMed Google Scholar). ATPase activity for each protein was assayed by incubating with [α-32P]ATP and measuring the direct conversion to [α-32P]ADP as described under “Experimental Procedures”. Full-length chicken hsp90 hydrolyzed ATP poorly with aK m for ATP of 1.5 mm and ak cat of 0.02 min−1 at 30 °C (Fig. 2). In contrast, yeast hsp90 hydrolyzed ATP at nearly 10 times this rate, whereas HtpG and TRAP1 were still more efficient ATPases (Fig. 1 and TableI). These results imply that chicken hsp90 does not hydrolyze ATP efficiently in the absence of either cofactors and/or substrate.Table IA comparison of hsp90 ATPase activitiesHsp90 protein (species or mutant)K mk catCatalytic efficiency (k cat/K mμmμm/min/μmWT (chicken, α)1530 ± 330.025 ± 0.00171.6 × 10−5WT (human, β)324 ± 680.015 ± 0.00129.6 × 10−5N1–220 (chicken, α)ND<0.002NDN1–573 (chicken, α)99 ± 180.24 ± 0.032.4 × 10−3N1–692 (chicken, α)1359 ± 110.027 ± 0.0021.9 × 10−5δ661–677 (chicken, α)ND<0.002NDTRAP1 (human, mitochondrial)33 ± 80.1 ± 0.043.0 × 10−3Hsc82 (Saccharomyces cerevisiae)511 ± 540.08 ± 0.011.5 × 10−4HtpG (E. coli)260 ± 910.1 ± 0.0123.8 × 10−4Each protein (5 μm) was assayed for ATPase activity using 1–10,000 μm ATP. k cat andK m values are presented with S.D. ND, not done; WT, wild type. Open table in a new tab Each protein (5 μm) was assayed for ATPase activity using 1–10,000 μm ATP. k cat andK m values are presented with S.D. ND, not done; WT, wild type. Somewhat surprisingly, the N1–573 construct hydrolyzed ATP over 100-fold better than the full-length protein (see Fig. 1). N1–573 hydrolyzed ATP with a K m of 100 μm and a k cat of 0.24 min−1 (Fig.3). Thus, the kinetics of this carboxyl-terminally truncated chicken protein were more efficient than those found for full-length yeast hsp90 and similar to the kinetics for the mitochondrial homologue, TRAP1 (see below and Ref. 31Felts S.J. Owen B.A.L. Nguyen P.M. Trepel J. Donner D.B. Toft D.O. J. Biol. Chem. 2000; 275: 3305-3312Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). These results are particularly interesting because all carboxyl-terminal truncations tested in the yeast protein to date have resulted in decreases of ATPase activity (26Weikl T. Muschler P. Richter K. Veit T. Reinstein J. Buchner J. J. Mol. Biol. 2000; 303: 583-592Crossref PubMed Scopus (106) Google Scholar). Likewise, our efforts to generate a similar truncation mutant in TRAP1 have resulted in proteins with substantially reduced ATPase activities. Specifically, the carboxyl-terminal truncation of TRAP1 at amino acid 529, which coincides with amino acid 573 of the chicken sequence, resulted in a decrease in ATPase activity to less than 5% of the full-length protein. A more conservative carboxyl-terminal deletion mutant, TRAP1 N1–578, was slightly more active as an ATPase but still had less than 20% of the activity of wild-type TRAP1 (data not shown). Removal of the last 30 amino acids of chicken hsp90 (N1–692) had little effect on ATPase activity (Fig. 1 and Table I). The deletion mutant Δ661–677, which had one of the proposed dimerization domains removed, showed ATPase activity that was approximately one-tenth that of the full-length chicken hsp90. Similarly, and in agreement with studies in yeast, the N1–220 truncation mutant of chicken hsp90 also had less than 1/10 the ATPase activity of the full-length protein. The observation that truncating full-length hsp90 to residue 573 dramatically enhanced ATPase activity suggested that the carboxyl terminus negatively regulates activity. To test this hypothesis, we generated two additional truncation mutants by proteolysis. Bovine thrombin cleaves chicken hsp90 between residues Lys-611 and Ala-612, as determined by carboxyl-terminal sequencing of the thrombin-cleaved product, generating a homogeneous truncation mutant of N1–611 (Fig.4 B). This mutant, which has only 38 additional carboxyl-terminal amino acids than N1–573, hydrolyzed ATP in a manner similar to full-length hsp90 and N1–692 (Fig. 4 A). During purification, the internal deletion mutant, Δ661–677, displayed an increased susceptibility to proteolysis. We mapped the susceptible site by mass spectrometry to within 1200 mass units carboxyl-terminal to residue 574. Carboxyl-terminal amino acid sequencing confirmed the proteolytic cleavage site to be after amino acid Val-584. Fortuitously, the parental and cleaved Δ661–677 proteins were separable by Mono-Q chromatography (Fig. 4 B). The cleaved product, N1–584, had an ATPase activity that was 10 times that of the full-length chicken hsp90 protein and approximately one-third that of N1–573 protein (Fig. 4 A). Taken together, these findings suggest that the amino acid sequence found between residues 574 and 611 suppresses the ATPase activity of full-length, dimeric chicken hsp90. Equilibrium sedimentation analysis was used to determine the oligomerization state of the full-length or deletion mutant hsp90 proteins in solution. To determine whether full-length chicken hsp90 behaved ideally and homogeneously in solution over the concentrations used for ATPase assays, the protein was analyzed at 8,000, 10,000, and 12,000 rpm using protein concentrations that ranged from 0.5 to 2.5 mg/ml or 2.9 to 15.7 μm, as shown in TableII. The average molecular weight determined from these measurements was 166,993 ± 890 (n = 9). The calculated molecular mass for chicken hsp90, based on primary amino acid sequence, is 84,060 (monomer) or 168,120 (dimer). Therefore, in solution, full-length chicken hsp90α exists as an unambiguous dimer over a 5-fold range of protein concentration.Table IISedimentation equilibrium analysis of chicken hsp90[Protein]SpeedM̄ w(measured)Root mean squareM̄ w(theoretical)Oligomer statusμmrpm× 10 −3Chicken, α (2.9 μm)8,000168,8543.584,058Dimer10,000162,2570.3612,000168,3670.74Chicken, α (8.9 μm)8,000168,9541.384,058Dimer10,000167,4352.5312,000162,7000.69Ch" @default.
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• W2053708941 date "2002-03-01" @default.
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• W2053708941 title "Regulation of Heat Shock Protein 90 ATPase Activity by Sequences in the Carboxyl Terminus" @default.
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• W2053708941 doi "https://doi.org/10.1074/jbc.m111450200" @default.
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