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• W2049805211 abstract "Heat shock protein 90 (Hsp90) plays a central role in signal transduction and has emerged as a promising target for anti-cancer therapeutics, but its molecular mechanism is poorly understood. At physiological concentration, Hsp90 predominantly forms dimers, but the function of full-length monomers in cells is not clear. Hsp90 contains three domains: the N-terminal and middle domains contribute directly to ATP binding and hydrolysis and the C domain mediates dimerization. To study the function of Hsp90 monomers, we used a single-chain strategy that duplicated the C-terminal dimerization domain. This novel monomerization strategy had the dual effect of stabilizing the C domain to denaturation and hindering intermolecular association of the ATPase domain. The resulting construct was predominantly monomeric at physiological concentration and did not function to support yeast viability as the sole Hsp90. The monomeric construct was also defective at ATP hydrolysis and the activation of a kinase and steroid receptor substrate in yeast cells. The ability to support yeast growth was rescued by the addition of a coiled-coil dimerization domain, indicating that the parental single-chain construct is functionally defective because it is monomeric. Heat shock protein 90 (Hsp90) plays a central role in signal transduction and has emerged as a promising target for anti-cancer therapeutics, but its molecular mechanism is poorly understood. At physiological concentration, Hsp90 predominantly forms dimers, but the function of full-length monomers in cells is not clear. Hsp90 contains three domains: the N-terminal and middle domains contribute directly to ATP binding and hydrolysis and the C domain mediates dimerization. To study the function of Hsp90 monomers, we used a single-chain strategy that duplicated the C-terminal dimerization domain. This novel monomerization strategy had the dual effect of stabilizing the C domain to denaturation and hindering intermolecular association of the ATPase domain. The resulting construct was predominantly monomeric at physiological concentration and did not function to support yeast viability as the sole Hsp90. The monomeric construct was also defective at ATP hydrolysis and the activation of a kinase and steroid receptor substrate in yeast cells. The ability to support yeast growth was rescued by the addition of a coiled-coil dimerization domain, indicating that the parental single-chain construct is functionally defective because it is monomeric. Among heat shock proteins, Hsp90 is unusual because it is not required for the proper folding of most cellular proteins (1Nathan D.F. Vos M.H. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12949-12956Crossref PubMed Scopus (312) Google Scholar) and instead is disproportionately linked to a select group of proteins required for receiving, transducing, and responding to environmental signals. The list of Hsp90 substrates continues to grow and includes over 40 kinases (2Zhao R. Davey M. Hsu Y.C. Kaplanek P. Tong A. Parsons A.B. Krogan N. Cagney G. Mai D. Greenblatt J. Boone C. Emili A. Houry W.A. Cell. 2005; 120: 715-727Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar) and many steroid hormone receptors (3Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1537) Google Scholar). Biochemical studies demonstrate that Hsp90 along with a handful of co-chaperones is required for many hormone receptors (including glucocorticoid, androgen, estrogen, and progesterone) to bind steroid ligand (3Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1537) Google Scholar, 4Smith D.F. Stensgard B.A. Welch W.J. Toft D.O. J. Biol. Chem. 1992; 267: 1350-1356Abstract Full Text PDF PubMed Google Scholar). V-src, the transforming tyrosine kinase from Rous sarcoma virus, was the first kinase to be identified as an Hsp90 substrate and is one of the most extensively studied. The transforming phenotype of v-src can be reversed with polyketide benzoquinone ansamycins including geldanamycin (GA), 2The abbreviations used are: GA, geldanamycin; Cm, concentration midpoint; GPD, glyceraldehyde 3-phosphate dehydrogenase; GR, glucocorticoid receptor; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; WT, wild-type. which directly bind to Hsp90 (5Uehara Y. Hori M. Takeuchi T. Umezawa H. Mol. Cell. Biol. 1986; 6: 2198-2206Crossref PubMed Scopus (234) Google Scholar) and blocks the association of v-src and Hsp90 (6Whitesell 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 (1332) Google Scholar). Stimulated by these observations, Hsp90 has emerged as a promising molecular target for anti-cancer therapeutics (7Whitesell L. Lindquist S.L. Nat. Rev. Cancer. 2005; 5: 761-772Crossref PubMed Scopus (1975) Google Scholar). Recently, Hsp90 activation of the Chk1 kinase has been reconstituted in vitro and has been shown to require a set of co-chaperones that partially differ from those required for hormone receptors (8Arlander S.J. Felts S.J. Wagner J.M. Stensgard B. Toft D.O. Karnitz L.M. J. Biol. Chem. 2006; 281: 2989-2998Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). As with hormone receptors, the molecular mechanism of Hsp90 activation of v-src and other kinases remains to be determined. How does Hsp90 dimerization contribute to its function in cells? Structurally, Hsp90 contains an N-terminal domain that binds nucleotide, a middle domain, and a C-terminal dimerization domain (Fig. 1). Truncations of Hsp90 that lack the C domain are largely monomeric (9Richter K. Muschler P. Hainzl O. Buchner J. J. Biol. Chem. 2001; 276: 33689-33696Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) and do not support yeast viability (10Louvion J.F. Warth R. Picard D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13937-13942Crossref PubMed Scopus (97) Google Scholar). These results do not demonstrate that dimerization of Hsp90 is essential because the C domain may contribute other functional properties in addition to dimerization. The deletion of a loop from the C domain (582-601) that is largely solvent exposed (11Ali 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 (719) Google Scholar) prevents yeast viability (10Louvion J.F. Warth R. Picard D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13937-13942Crossref PubMed Scopus (97) Google Scholar), suggesting that dimerization is not the only essential role of the C domain. In addition, a point mutation (A587T) in this same loop impairs the chaperoning of glucocorticoid receptor (GR) in yeast (12Nathan D.F. Lindquist S. Mol. Cell. Biol. 1995; 15: 3917-3925Crossref PubMed Scopus (370) Google Scholar). Because Hsp90 activity requires co-chaperones and Hsp90 binds promiscuously to about 10% of the yeast proteome (2Zhao R. Davey M. Hsu Y.C. Kaplanek P. Tong A. Parsons A.B. Krogan N. Cagney G. Mai D. Greenblatt J. Boone C. Emili A. Houry W.A. Cell. 2005; 120: 715-727Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar), it is important to study Hsp90 function in cells where all endogenous binding partners are present. To rigorously test how dimerization affects Hsp90 function in cells requires a full-length Hsp90 monomer. The ATPase activity of Hsp90 is required for cellular function, although the molecular mechanism that couples ATP hydrolysis to substrate activation is poorly understood. Consistent with its role in signal transduction, Hsp90 is required for viability in all eukaryotes that have been tested (yeast, flies, and worms) (13Borkovich K.A. Farrelly F.W. Finkelstein D.B. Taulien J. Lindquist S. Mol. Cell. Biol. 1989; 9: 3919-3930Crossref PubMed Scopus (540) Google Scholar, 14Birnby D.A. Link E.M. Vowels J.J. Tian H. Colacurcio P.L. Thomas J.H. Genetics. 2000; 155: 85-104Crossref PubMed Google Scholar, 15Yue L. Karr T.L. Nathan D.F. Swift H. Srinivasan S. Lindquist S. Genetics. 1999; 151: 1065-1079Crossref PubMed Google Scholar). Structural and biochemical studies demonstrate that Hsp90 is a “split” ATPase with catalytic amino acids from both the N (Glu33) and M (Arg380) domains contributing to hydrolysis (11Ali 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 (719) Google Scholar, 16Chadli 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, 17Meyer P. Prodromou C. Hu B. Vaughan C. Roe S.M. Panaretou B. Piper P.W. Pearl L.H. Mol. Cell. 2003; 11: 647-658Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar). Mutation of either of these catalytic amino acids impairs ATPase activity and does not support yeast viability as the sole Hsp90. Hsp90 point mutations that perturb the ATPase activity from 2 to 400% of the wild-type level can support yeast growth under non-stressful conditions (18Prodromou 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). Thus, cell viability can tolerate a wide range of Hsp90 ATPase levels. Dimerization of Hsp90 has been implicated in ATP hydrolysis. NM constructs of yeast Hsp90 lacking the C-terminal dimerization domain have concentration-dependent specific ATPase activity (9Richter K. Muschler P. Hainzl O. Buchner J. J. Biol. Chem. 2001; 276: 33689-33696Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). These results indicate that the N and/or M domains can mediate weak dimerization that leads to increased ATPase activity. Consistent with this analysis, the disulfide cross-linking of truncated Hsp90 constructs increases their ATPase activity (19Wegele H. Muschler P. Bunck M. Reinstein J. Buchner J. J. Biol. Chem. 2003; 278: 39303-39310Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). These studies raise the possibility that full-length Hsp90 monomers may be inactive for ATP hydrolysis. Studies with heterodimers made up of one full-length Hsp90 subunit and one C-domain, indicate that full-length/C-domain Hsp90 heteromers have about 30% the ATPase activity of wild-type (9Richter K. Muschler P. Hainzl O. Buchner J. J. Biol. Chem. 2001; 276: 33689-33696Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), well within the 2 to 400% range previously observed to support yeast viability (18Prodromou 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). These observations have stimulated the current work to determine whether Hsp90 monomers are functional in cells. We have used a single-chain strategy to design a full-length NMCC Hsp90 monomer (Fig. 1) and analyzed its function in yeast cells. To our knowledge, this is a novel approach to making full-length monomers and relative to traditional strategies that mutagenize the dimer interface, has the advantage of stabilizing the dimerization domain to denaturation. We find that NMCC monomers do not function to support yeast growth, the activation of a kinase nor a hormone receptor substrate in yeast. Appending a coiled-coil dimerization motif to NMCC rescues dimerization and function, indicating that the parental NMCC construct is defective only because dimerization is impaired. Whereas Hsp90 monomers do not support yeast viability, wild-type Hsp90 monomers form transiently, on the same time scale as ATP hydrolysis, suggesting that they may play a role in the Hsp90 chaperone cycle. NMCC Hsp90 will provide a tool to further explore the biochemical properties of Hsp90 monomers and their role in the chaperone cycle. Construction of Hsp90 Variants—All Hsp90 constructs used in these studies were generated from the yeast HSP82 gene. To aid in protein purification and Western blot detection, a His6 tag (GGHHHHHHGGH) was appended to the N terminus of Hsp90 constructs. Constructs were cloned into pACYC for bacterial expression and into p414GPD for expression in yeast. The C-terminal domain of Hsp90 contains amino acids 542-709. Single-chain constructs of the C-terminal domain were constructed with amino acids 537-680 from HSP82 followed by the Gly-rich linkers outlined in Fig. 2 then by amino acids 542-709 from HSP82. The NMCC construct contains amino acids 1-680 from HSP82 followed by the 15-amino acid linker in Fig. 2, then amino acids 542-709 from HSP82. The NMCCcoil construct contains a coiled-coil sequence (GGGTSSVKELEDKNEELLSEIAHLKNEVARLKKLVGERTG) inserted after amino acid 678 of the second C domain of NMCC. Protein Production—All Hsp90 constructs were expressed in BLR(DE3) cells induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside at 30 °C for 5 h. Cells were harvested by centrifugation and resuspended in Wash Buffer (50 mm potassium phosphate, pH 8, 300 mm potassium chloride, 20 mm imidazole). Cell lysis was accomplished by treatment with lysozyme and sonication. After pelleting cell debris, lysates were incubated with Ni2+-nitrilotriacetic acid-agarose (Qiagen). After rinsing the nickel resin extensively with Wash Buffer, Hsp90 protein was competitively eluted with Elution Buffer (200 mm imidazole, pH 7.5). EDTA was added to 10 mm to eluates that were subsequently dialyzed into Buffer A (20 mm potassium phosphate, pH 6.8, 1 mm EDTA). Protein samples were further purified by anion exchange chromatography using a Q Sepharose HP column (GE Healthcare) eluted with a linear gradient from 0 to 500 mm potassium chloride in Buffer A. As a final purification, protein samples were subjected to size exclusion chromatography using a Sephacryl S300 column (GE Healthcare) in Buffer B (20 mm potassium phosphate, pH 6.8, 1 mm EDTA, 100 mm potassium chloride). Proteins were concentrated in Buffer B to ∼10 mg/ml using Amicon Ultra concentrators (Millipore). Protein concentrations were determined spectroscopically using extinction coefficients (m−1 cm−1) based on amino acid composition using the program Sednterp (Amgen) at 280 nm: WT (54,050), NMCC and NMCCcoil (64,860), C domain (10,810), and C-linker-C constructs (21,620). Circular Dichroism—CD measurements were acquired on a Jasco A-810 spectropolarimeter equipped with a Peltier temperature control unit and an autotitrator. C domain spectra were acquired in a 1-mm path length cuvette at a subunit concentration of 20 μm in 20 mm potassium phosphate, pH 7, at 25 °C. All equilibrium urea titrations were performed in Buffer B at 25 °C. Protein denaturation was followed by monitoring the loss of ellipticity at 222 nm. Measurements at a subunit concentration of 2 μm were made in a cuvette with a 1-cm path length using an autotitrator with a mixing time of 600 s (determined to be more than three times greater than the time constant for unfolding at the Cm). For urea titrations at a subunit concentration of 20 μm, samples were manually mixed, allowed to equilibrate for 30 min, and measurements made in a 1-mm cuvette. Both the pre- and post-transition regions were fit to linear equations and used to replot the data as the fraction unfolded. The fraction unfolded plots were fit to two-state models with or without dimerization as appropriate and previously described (20Bowie J.U. Sauer R.T. Biochemistry. 1989; 28: 7139-7143Crossref PubMed Scopus (221) Google Scholar, 21Santoro M.M. Bolen D.W. Biochemistry. 1988; 27: 8063-8068Crossref PubMed Scopus (1607) Google Scholar) using the program Kaleidograph (Synergy Software). Analytical Ultracentrifugation—Equilibrium experiments were performed at 20 °C on a Beckman XLI instrument using absorbance optics and a Ti60 rotor. Absorbance profiles were taken at 12-h intervals, and overlapping profiles were used as a criterion for equilibration. For the C domain constructs, samples were analyzed at a subunit concentration of 50 μm in Buffer A with a rotor speed of 15,000 rpm and an equilibration time of 36 h. For full-length constructs, samples were analyzed at a subunit concentration of 12 μm in Buffer B with a rotor speed of 8,000 rpm and an equilibration time of 36 h. Absorbance profiles were fit to a single species model as previously described (22Cantor C.R. Schimmel P.R. Biophysical Chemistry. W.H. Freeman and Co., New York1980Google Scholar) using the equation, c2(x)=c2(x0)exp{[M(1-Vp)ω2/2RT](x2-x02)}(Eq. 1) where c2(x) is the concentration at a radial distance of x, x0 is a reference point, M is the molecular weight, V is the partial specific volume, ρ is the buffer density, ω is the angular velocity, R is the universal gas constant, and T is the temperature. Data were fit in Kaleidograph using buffer density and V-bar values based on the buffer and amino acid composition and determined using the Sednterp program. Analytical Size Exclusion Chromatography—100-μl samples ranging in subunit concentration from 5 to 50 μm were analyzed using Buffer B and a Superdex 200 column (GE Healthcare). Absorbance at 280 nm was used to monitor the elution profile. Enzymatically Coupled ATPase Assay—ATP hydrolysis was enzymatically linked to NADH oxidation that was monitored spectroscopically (23Norby J.G. Methods Enzymol. 1988; 156: 116-119Crossref PubMed Scopus (253) Google Scholar). Hsp90 catalyzed ATP hydrolysis to generate ADP. Pyruvate kinase was used to convert ADP and phosphenolpyruvate to ATP and pyruvate. Lactose dehydrogenase was used to convert pyruvate and NADH to lactate and NAD with a corresponding drop in absorbance at 340 nm (Δϵ340 = 6220 m−1 cm−1). ATPase assays were performed at 37 °C. Both Hsp90 protein samples and ATPase components were pre-warmed, then mixed. Using a Bio50 Spectrophotometer equipped with a Peltier temperature control unit (Cary) and a 1-cm path length cuvette, absorbance at 340 nm was measured at 15-s intervals (avoids photobleaching of NADH) for 10 min. The final concentration of ATPase components was 20 mm Tris, pH 7.5, 5 mm MgCl2, 100 mm KCl, 1 mm ATP (Sigma), 0.17 mm NADH (Sigma), 0.67 mm phosphoenolpyruvate (Sigma), 0.01 mg/ml pyruvate kinase (Sigma), and 0.02 mg/ml lactose dehydrogenase (EMD Biosciences). The rate of NADH oxidation with 0.3 mm ADP was more than an order of magnitude greater than the highest rate observed with Hsp90 demonstrating that ATPase components are not rate-limiting. Rates were determined by fitting the change in absorbance versus time to a linear model, converting absorbance units to the amount of NADH oxidized and normalizing to the concentration of Hsp90. Fluorescent GA Binding—BODIPY-GA was synthesized as described (24Llauger-Bufi L. Felts S.J. Huezo H. Rosen N. Chiosis G. Bioorg. Med. Chem. Lett. 2003; 13: 3975-3978Crossref PubMed Scopus (48) Google Scholar). BODIPY-GA concentration was determined by absorbance in methanol using an extinction coefficient of 80,000 m−1 cm−1 at 506 nm (25Lakowicz J.R. Principles of Fluorescence Spectroscopy.Second Ed. Kluwer Academic/Plenum Publishers, Boston, MA1999Crossref Google Scholar) and was in close agreement with the weighed mass of BODIPY-GA used. 3 μm BODIPY-GA was preincubated in assay buffer (20 mm Tris, pH 7.5, 5 mm MgCl2, 100 mm KCl, 0.01% Nonidet P-40) with 10 mm dithiothreitol for 4 h at room temperature to convert GA to the high-affinity hydroquinone form (26Maroney A.C. Marugan J.J. Mezzasalma T.M. Barnakov A.N. Garrabrant T.A. Weaner L.E. Jones W.J. Barnakova L.A. Koblish H.K. Todd M.J. Masucci J.A. Deckman I.C. Galemmo Jr., R.A. Johnson D.L. Biochemistry. 2006; 45: 5678-5685Crossref PubMed Scopus (49) Google Scholar). Samples were prepared in assay buffer with 5 mm dithiothreitol containing 1 μm reduced BODIPY-GA and wild-type or NMCC Hsp90 ranging from 0.1 to 3.3 μm. These concentrations of Hsp90 and BODIPY-GA are both well above the 5 nm Kd determined for human Hsp90 and BODIPY-GA (26Maroney A.C. Marugan J.J. Mezzasalma T.M. Barnakov A.N. Garrabrant T.A. Weaner L.E. Jones W.J. Barnakova L.A. Koblish H.K. Todd M.J. Masucci J.A. Deckman I.C. Galemmo Jr., R.A. Johnson D.L. Biochemistry. 2006; 45: 5678-5685Crossref PubMed Scopus (49) Google Scholar). Samples were equilibrated for 10 min at room temperature and fluorescence anisotropy measurements were made in a 0.3-cm path length cuvette on a PTI QM-4SE spectrofluorometer with excitation set to 488 nm and emission at 510 nm. Sole Hsp90 in Yeast—The haploid Saccharomyces cerevisiae strains iG170Da (1Nathan D.F. Vos M.H. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12949-12956Crossref PubMed Scopus (312) Google Scholar) and ECU82a (12Nathan D.F. Lindquist S. Mol. Cell. Biol. 1995; 15: 3917-3925Crossref PubMed Scopus (370) Google Scholar) are derivatives of W303 in which both endogenous Hsp90 genes, HSP82 and HSC82, are knocked out. In strain iG170Da, the temperature-sensitive G170D mutant of HSP82 is chromosomally integrated and expressed from a GPD promoter. In strain ECU82a, wild-type HSC82 is constitutively expressed from pKAT6, a URA3 marked 2-μm (hi copy) plasmid that is amenable to negative selection. To test the ability of our Hsp90 constructs to support yeast viability, we generated them in p414GPD (27Mumberg D. Muller R. Funk M. Gene (Amst.). 1995; 156: 119-122Crossref PubMed Scopus (1594) Google Scholar), a TRP marked CEN plasmid with a strong constitutive promoter. Lithium acetate was used to introduce our Hsp90 constructs into iG170Da and ECU82a and plating in the absence of tryptophan was used to select for transformants. Transformants were grown in liquid media lacking tryptophan to an A600 of 0.7, serially diluted, and plated under permissive or non-permissive conditions. All samples were grown and plated in synthetic media lacking tryptophan and with 2% dextrose as a sugar source. For strain iG170Da, 25 °C was permissive and 37 °C was non-permissive. Strain ECU82a was grown at 25 °C, in the absence or presence of 5-fluoroorotic acid, which selects for loss of the pKAT6 plasmid. To determine the expression level of our Hsp90 constructs in ECU82a, liquid cultures in minimal media were grown to a cell density of 5 × 107 cells/ml determined with a hemacytometer. Cells were lysed by vortexing with glass beads and resuspension in SDS. Proteins from the lysis of 107 cells were separated by SDS-PAGE and the expression level quantified by Western blotting against the His6 epitope tag. A standard curve was generated using purified Hsp90 added to lysates from cells that did not express any epitope-tagged Hsp90. V-src Assay—Plasmid Y316v-srcv5 was generated from Y316v-src (12Nathan D.F. Lindquist S. Mol. Cell. Biol. 1995; 15: 3917-3925Crossref PubMed Scopus (370) Google Scholar) and contains v-src with a C-terminal v5 epitope tag (GKPIPNPLLGLDST) under a galactose-regulated promoter on a URA3-marked CEN plasmid. Lithium acetate was used to introduce Y316v-srcv5 into iG170Da cells and plating in the absence of uracil was used to select for transformants. To these cells we introduced our Hsp90 constructs on p414GPD plasmids. On plates, cells were grown with dextrose as the sugar source to prevent v-src expression. Liquid cultures were grown in synthetic raffinose media without uracil and tryptophan at 25 °C to a cell density of about 5 × 106 cells/ml (determined with a hemocytometer). Cells were pelleted and resuspended in media with either 2% raffinose or galactose as the sugar source pre-warmed to 38 °C (to inactivate G170D Hsp90). Cells were grown in a shaking incubator at 35 °C for 6 h. Cell pellets were collected by centrifugation, washed once with water, and frozen at -80 °C. The frozen cell pellets were lysed by vortexing with glass beads in Src Lysis Buffer (50 mm Tris, pH 7.5, 5 mm EDTA, 0.2 mm sodium orthovanadate to inhibit dephosphorylation, 1 mm phenylmethylsulfonyl fluoride) followed by addition of SDS to 2%. Protein concentration in these SDS lysates was assessed using the BCA assay (Pierce). Samples (2 μg of protein) were subject to SDS-PAGE and phosphotyrosine levels quantified by Western blot analysis with antibody 4G10 (Upstate) in the presence of 0.1% Tween 20. The level of v-src was quantified by Western blot analysis (20 μg of protein/lane) with α-v5 antibody (Invitrogen). GR Assay—P2A/GRGZ (12Nathan D.F. Lindquist S. Mol. Cell. Biol. 1995; 15: 3917-3925Crossref PubMed Scopus (370) Google Scholar) is a 2-μm ADE2 plasmid that contains rat glucocorticoid receptor expressed from the constitutive GPD promoter and the β-galactosidase reporter under the control of three glucocorticoid response elements. We introduced P2A/GRGZ together with our p414GPD Hsp90 constructs into iG170Da cells. Cells were grown in synthetic dextrose media lacking tryptophan and adenine at 25 °C to a cell density of about 5 × 106 cells/ml. Cells were pelleted by centrifugation and resuspended in media prewarmed to 38 °C (to inactivate G170D Hsp90). 15-ml cultures were grown in a shaking incubator at 35 °C for 5 min. Deoxycorticosterone dissolved in ethanol was added to final concentrations of 0, 0.08, 0.4, 2, 10, or 50 μm (final concentration of ethanol was 0.1% in all cases). Cells were grown for a further 60 min at 35 °C and collected by centrifugation. After washing once with water, cell pellets were frozen at -80 °C. Cells were lysed by vortexing with glass beads in βGal Lysis Buffer (100 mm potassium phosphate, pH 7.3, 2 mm magnesium acetate, 1 mm phenylmethylsulfonyl fluoride). After removing cell debris, protein concentration in the lysates was determined using the BCA assay (Pierce). A total of 10 μg of lysate was reacted for 15 min in a total volume of 80 μl of 1 mg/ml o-nitrophenyl β-d-galactoside (Sigma) in βGal Lysis Buffer. The reaction was stopped by adding 80 μl of 1 m sodium carbonate. Reporter activity was quantified by monitoring the absorbance at 420 nm. GR assays were repeated three times starting from fresh yeast colonies. C Domain Has Marginal Stability to Urea Denaturation—The goal of our engineering efforts is to generate a natively folded full-length Hsp90 monomer that has one ATPase site. We considered traditional dimer disruption strategies, but were concerned that mutations at the dimer interface might destabilize the C domain causing it to misfold. To address this concern, we monitored the stability of the C domain. The stability of a protein is a measure of its capacity to tolerate destabilizing mutations and still fold (28Bornberg-Bauer E. Chan H.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10689-10694Crossref PubMed Scopus (198) Google Scholar). We analyzed the stability of the isolated C domain to urea-induced unfolding. In the absence of urea, the circular dichroism spectrum of the C domain has minima at 208 and 222 nm indicating that it forms a folded structure with significant helical content (supplemental Fig. S1). We used CD to monitor the transition of the isolated C domain from folded to unfolded as a function of urea concentration (Fig. 1B). The urea concentration midpoint (Cm) of denaturation increases with higher C domain concentration (2.38 m urea at 2 μm and 2.74 m urea at 20 μm). The dependence of Cm on protein concentration indicates that dimerization and protein folding are coupled. At concentrations of Hsp90 (9Richter K. Muschler P. Hainzl O. Buchner J. J. Biol. Chem. 2001; 276: 33689-33696Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) where monomers become populated (the midpoint of association is 60 nm), our results indicate that the C domain would be marginally stable to denaturation. Taking these observations into account, we decided upon a monomerization strategy that would enhance the stability of the C domain (Fig. 1C). Duplication of the C domain in NMCC should both increase the stability of the C domain to denaturation and disfavor intermolecular association. Thus NMCC should have one ATPase site per molecule. Isolated Single-chain C Domain Is Stable and Monomeric—With the aim of generating a single-chain C domain that is both monomeric and stable, we generated CC constructs separated by variable length glycine-rich linkers (Fig. 2A). Based on the crystal structure of Hsp90, the flexible linkers need to span a distance of 27 Å. Whereas 8 amino acids in a fully extended conformation can span this distance, previous single-chain studies have found that a greater number of amino acids is required to avoid strain and maximize stability (29Robinson C.R. Sauer R.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5929-5934Crossref PubMed Scopus (215) Google Scholar). We made glycine-rich linkers of 11, 15, and 19 amino acids based on a proteolytically stable loop in bacteriophage gene-3 protein that had been used in a previous single-chain study (30Martin A. Baker T.A. Sauer R.T. Nature. 2005; 437: 1115-1120Crossref PubMed Scopus (301) Google Scholar). All three single-chain constructs (C11C, C15C, and C19C) expressed to high levels in Escherichia coli, were readily purified, highly soluble, and had CD spectra similar to the wild-type C domain (Fig. S1). At a subunit concentration of 2 μm, we observe that C11C, C15C, and C19C all have increased stability to urea denaturation relative to wild type (Fig. 2B). These results indicate that the effective concentration caused by the covalent single-chain linkages is greater than the concentration of wild-type protein (2 μm). The 15-amino acid linker (C15C) was the smallest link eliciting the greatest stability and was chosen for further analysis. The increased stability of C15C could be caused by either a high effective concentration of covalently linked subunits as designed, or from unanticipated oligomerization. To differentiate between these possibilities, we analyzed the solution oligomeric state of C15C using equilibrium analytical ultracentrifugation (Fig. 2C). The distribution of C15C" @default.
• W2049805211 created "2016-06-24" @default.
• W2049805211 creator A5001880132 @default.
• W2049805211 creator A5029418807 @default.
• W2049805211 date "2007-11-01" @default.
• W2049805211 modified "2023-10-14" @default.
• W2049805211 title "Dimerization of Hsp90 Is Required for in Vivo Function" @default.
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