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• W2073886258 abstract "In vivo activation of client proteins by Hsp90 depends on its ATPase-coupled conformational cycle and on interaction with a variety of co-chaperone proteins. For some client proteins the co-chaperone Sti1/Hop/p60 acts as a “scaffold,” recruiting Hsp70 and the bound client to Hsp90 early in the cycle and suppressing ATP turnover by Hsp90 during the loading phase. Recruitment of protein kinase clients to the Hsp90 complex appears to involve a specialized co-chaperone, Cdc37p/p50cdc37, whose binding to Hsp90 is mutually exclusive of Sti1/Hop/p60. We now show that Cdc37p/p50cdc37, like Sti1/Hop/p60, also suppresses ATP turnover by Hsp90 supporting the idea that client protein loading to Hsp90 requires a “relaxed” ADP-bound conformation. Like Sti1/Hop/p60, Cdc37p/p50cdc37 binds to Hsp90 as a dimer, and the suppressed ATPase activity of Hsp90 is restored when Cdc37p/p50cdc37 is displaced by the immunophilin co-chaperone Cpr6/Cyp40. However, unlike Sti1/Hop/p60, which can displace geldanamycin upon binding to Hsp90, Cdc37p/p50cdc37 forms a stable complex with geldanamycin-bound Hsp90 and may be sequestered in geldanamycin-inhibited Hsp90 complexes in vivo. In vivo activation of client proteins by Hsp90 depends on its ATPase-coupled conformational cycle and on interaction with a variety of co-chaperone proteins. For some client proteins the co-chaperone Sti1/Hop/p60 acts as a “scaffold,” recruiting Hsp70 and the bound client to Hsp90 early in the cycle and suppressing ATP turnover by Hsp90 during the loading phase. Recruitment of protein kinase clients to the Hsp90 complex appears to involve a specialized co-chaperone, Cdc37p/p50cdc37, whose binding to Hsp90 is mutually exclusive of Sti1/Hop/p60. We now show that Cdc37p/p50cdc37, like Sti1/Hop/p60, also suppresses ATP turnover by Hsp90 supporting the idea that client protein loading to Hsp90 requires a “relaxed” ADP-bound conformation. Like Sti1/Hop/p60, Cdc37p/p50cdc37 binds to Hsp90 as a dimer, and the suppressed ATPase activity of Hsp90 is restored when Cdc37p/p50cdc37 is displaced by the immunophilin co-chaperone Cpr6/Cyp40. However, unlike Sti1/Hop/p60, which can displace geldanamycin upon binding to Hsp90, Cdc37p/p50cdc37 forms a stable complex with geldanamycin-bound Hsp90 and may be sequestered in geldanamycin-inhibited Hsp90 complexes in vivo. The in vivo activity of heat shock protein 90 (Hsp90) 1The abbreviations used are: HSP90heat shock protein 90AMP-PNPadenosine 5′-(β,γ-imino)triphosphate depends on its association with a variety of co-chaperones that are components in a series of Hsp90-based multiprotein complexes involved in folding of client proteins (reviewed in Ref. 1Pearl L.H. Prodromou C. Adv. Protein Chem. 2002; 59: 157-185Crossref Scopus (178) Google Scholar). Authentic Hsp90 function in vitro and in vivo is also dependent on the binding and hydrolysis of ATP (2Grenert J.P. Johnson B.D. Toft D.O. J. Biol. Chem. 1999; 274: 17525-17533Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 3Panaretou B. Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1998; 17: 4829-4836Crossref PubMed Scopus (628) Google Scholar, 4Obermann W.M.J. Sondermann H. Russo A.A. Pavletich N.P. Hartl F.U. J. Cell Biol. 1998; 143: 901-910Crossref PubMed Scopus (494) Google Scholar, 5Scheibel T. Weikl T. Buchner J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1495-1499Crossref PubMed Scopus (232) Google Scholar), and this is regulated by interaction with TPR domain co-chaperones such as Sti1/Hop/p60 and immunophilins (6Prodromou 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 (356) Google Scholar). heat shock protein 90 adenosine 5′-(β,γ-imino)triphosphate Sti1/Hop/p60 is a scaffold protein implicated in mammalian cells in the recruitment of steroid hormone receptor-Hsp70-Hsp40 complexes to Hsp90 via simultaneous interaction with the C-terminal tails of Hsp70 and Hsp90 (7Smith D.F. Sullivan W.P. Marion T.N. Zaitsu K. Madden B. McCormick D.J. Toft D.O. Mol. Cell. Biol. 1993; 13: 869-876Crossref PubMed Scopus (247) Google Scholar, 8Johnson B.D. Schumacher R.J. Ross E.D. Toft D.O. J. Biol. Chem. 1998; 273: 3679-3686Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 9Chen S.Y. Prapapanich V. Rimerman R.A. Honore B. Smith D.F. Mol. Endocrinol. 1996; 10: 682-693Crossref PubMed Google Scholar, 10Scheufler C. Brinker A. Bourenkov G. Pegoraro S. Moroder L. Bartunik H. Hartl F.U. Moarefi I. Cell. 2000; 101: 199-210Abstract Full Text Full Text PDF PubMed Scopus (1016) Google Scholar, 11Russell L.C. Whitt S.R. Chen M.-S. Chinkers M. J. Biol. Chem. 1999; 274: 20060-20063Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Hop/Sti1/p60 has an additional role in regulating ATP turnover by Hsp90 (6Prodromou 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 (356) Google Scholar), locking Hsp90 into an “open” conformation and preventing progress through its ATP-dependent conformational cycle (12Prodromou 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). Although not essential, mammalian Hop/p60 increases the efficiency of steroid hormone receptor activation by Hsp90 and Hsp70 in vitro (13Morishima Y. Kanelakis K.C. Silverstein A.M. Dittmar K.D. Estrada L. Pratt W.B. J. Biol. Chem. 2000; 275: 6894-6900Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), and sti1− yeasts, although viable, are temperature-sensitive and display growth defects (14Chang H.C.J. Nathan D.F. Lindquist S. Mol. Cell. Biol. 1997; 17: 318-325Crossref PubMed Scopus (193) Google Scholar). Whereas steroid hormone receptors have been the most studied in terms of their activation by Hsp90, protein kinases form the largest coherent class of Hsp90-dependent client proteins (1Pearl L.H. Prodromou C. Adv. Protein Chem. 2002; 59: 157-185Crossref Scopus (178) Google Scholar). Recruitment of many protein kinase clients to the Hsp90 system involves a specialized co-chaperone Cdc37p (in budding yeast) or its mammalian orthologue p50cdc37 (15Perdew G.H. Wiegand H. VandenHeuvel J.P. Mitchell C. Singh S.S. Biochemistry. 1997; 36: 3600-3607Crossref PubMed Scopus (74) Google Scholar). Unlike Sti1/Hop/p60, which recruits clients to Hsp90 via interaction with Hsp70, Cdc37p/p50cdc37 can interact directly with client protein kinases via its N-terminal region (16Grammatikakis N. Lin J.-H. Grammatikakis A. Tsichlis P.N. Cochran B.H. Mol. Cell. Biol. 1999; 19: 1661-1672Crossref PubMed Scopus (229) Google Scholar) and with Hsp90 via its C terminus (17Shao J. Grammatikakis N. Scroggins B.T. Uma S. Huang W.J. Chen J.J. Hartson S.D. Matts R.L. J. Biol. Chem. 2001; 276: 206-214Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). However, it is far from clear whether all protein kinases that interact with Cdc37p/p50cdc37 are Hsp90-dependent or if all kinases that are Hsp90-dependent are recruited by Cdc37p/p50cdc37 (18Hunter T. Poon R.Y.C. Trends Cell Biol. 1997; 7: 157-161Abstract Full Text PDF PubMed Scopus (101) Google Scholar). Unlike Sti1, Cdc37p is essential for yeast viability probably due to its involvement in formation of Cdc28-cyclin complexes (19Gerber M.R. Farrell A. Deshaies R.J. Herskowitz I. Morgam D.O. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4651-4655Crossref PubMed Scopus (129) Google Scholar). Sti1/Hop/p60 and other TPR domain co-chaperones bind with mutual exclusivity to Hsp90 (20Owens-Grillo J.K. Czar M.J. Hutchison K.A. Hoffmann K. Perdew G.H. Pratt W.B. J. Biol. Chem. 1996; 271: 13468-13475Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) via the C-terminal MEEVD sequence (10Scheufler C. Brinker A. Bourenkov G. Pegoraro S. Moroder L. Bartunik H. Hartl F.U. Moarefi I. Cell. 2000; 101: 199-210Abstract Full Text Full Text PDF PubMed Scopus (1016) Google Scholar, 21Ramsey A.J. Russell L.C. Whitt S.R. Chinkers M. J. Biol. Chem. 2000; 275: 17857-17862Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), although this may not be the sole site of interaction (6Prodromou 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 (356) Google Scholar, 14Chang H.C.J. Nathan D.F. Lindquist S. Mol. Cell. Biol. 1997; 17: 318-325Crossref PubMed Scopus (193) Google Scholar, 22Chen S.Y. Sullivan W.P. Toft D.O. Smith D.F. Cell Stress Chaperones. 1998; 3: 118-129Crossref PubMed Scopus (169) Google Scholar). In contrast, Cdc37p/p50cdc37 has no detectable TPR motifs and does not require the C-terminal MEEVD of Hsp90, however, its binding is mutually exclusive with Sti1/Hop/p60 (20Owens-Grillo J.K. Czar M.J. Hutchison K.A. Hoffmann K. Perdew G.H. Pratt W.B. J. Biol. Chem. 1996; 271: 13468-13475Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 23Silverstein A.M. Grammatikakis N. Cochran B.H. Chinkers M. Pratt W.B. J. Biol. Chem. 1998; 273: 20090-20095Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), suggesting that the binding sites overlap or are at least topologically adjacent. The common ability of Sti1/Hop/p60 and Cdc37p/p50cdc37 to interact (directly or indirectly) with Hsp90 client proteins, together with their mutual exclusivity of binding to Hsp90, suggests that they may act as alternative recruitment factors for different classes of client protein. To gain further insight into this possibility, we have characterized the interaction between Cdc37p/p50cdc37 and Hsp90 and investigated the effect of Cdc37p/p50cdc37 co-chaperone binding on the inherent ATPase activity of Hsp90. The results of these studies suggest a common mechanism for recruitment of client proteins into the Hsp90 complex requiring suppression of ATP turnover by Hsp90 during the loading phase. Expression and purification of His-tagged yeast Hsp90, His-tagged cSti11 (C-terminal Hsp90-binding domain of Sti1, residues 237–589), and His-tagged Cpr6 were described previously (6Prodromou 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 (356) Google Scholar). DNA sequences encoding a truncated N-terminal domain of human p50cdc37 (sNp50, amino acid residues 30–127) and C-terminal p50cdc37 domain (Cp50, amino acid residues 128–379) were cloned in-frame with the His tag of pRSETA (NheI-XhoI). Full-length p50cdc37 cloned into pET16d was a kind gift from Nick Grammatikakis.Saccharomyces cerevisiae CDC37 was cloned in-frame with the His tag of pRSETA (NheI-XhoI). The various p50cdc37 and Cdc37p constructs were expressed in Escherichia coli BL21(DE3) pLysS by induction at 20 °C with 1 mmisopropyl-1-thio-β-d-galactopyranoside. Cells were harvested and resuspended in 60 ml of 20 mm Tris (pH 8.0) containing 100 mm NaCl (Buffer A) and lysed by sonication. The cell lysate was centrifuged (20,000 × g for 60 min at 4 °C) and the supernatant applied to a Talon metal affinity column equilibrated in Buffer A. The column was washed with Buffer A containing 10 mm imidazole (pH 8.0) and finally p50cdc37 /Cdc37p eluted with Buffer A containing 300 mm imidazole (pH 7.0). 10 mm EDTA and 1 mm dithiothreitol were added, and the eluent was concentrated with Vivaspin concentrators (Sartorius) with an appropriate molecular weight cut-off. Concentrated sample was applied to a gel filtration column (Superdex 75PG for Np50 and Cp50 and Superdex 200PG for p50cdc37 and Cdc37p), equilibrated in 20 mm Tris (pH 7.5), 500 mm NaCl, and 1 mm EDTA. Fractions containing p50cdc37 or Cdc37p were dialyzed (20 mm Tris (pH 7.5), 1 mm EDTA) and applied to a Q-Sepharose column equilibrated in the same buffer. The bound protein was eluted with a 0–1 m NaCl gradient. Fractions containing pure p50cdc37 or Cdc37p were dialyzed (20 mm Tris (pH 7.5), 1 mm EDTA) and subsequently concentrated using Vivaspin 5,000 and 30,000 molecular weight cutoff concentrators as appropriate. The protein concentrations reached are as follows: p50cdc37 = 99.7 mg ml−1, Np50 = 60 mg ml−1, Cp50 = 50 mg ml−1, Cdc37p = 131.6 mg ml−1, Hsp90 = 31.2 mg ml−1, cSti1 33.2 mg ml−1, and Cpr6 = 92.7 mg ml−1. Further treatment of proteins destined for ATPase assays and the regenerating enzyme-linked ATPase assay itself were as described previously (3Panaretou B. Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1998; 17: 4829-4836Crossref PubMed Scopus (628) Google Scholar, 6Prodromou 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 (356) Google Scholar). Each assay was repeated between 3 and 6 times, and average activities were calculated. Average ATPase activities were plotted as a percentage of the maximum average activity for Hsp90 at 37 °C. Sedimentation equilibration experiments were conducted at 15 °C in a Beckman-Optima XLI analytical centrifuge using an An-60 Ti rotor and 1.2-cm path length cells. p50cdc37, sNp50, and Cp50 were dialyzed against 20 mm Tris (pH 7.4) containing 1 mm EDTA and 25 mm NaCl. The proteins were used at concentrations of 23.6, 76.8, and 34.9 μm, respectively. Experiments were performed on 100-μl samples that were equilibrated for ∼48 h at speeds ranging from 10,000 to 50,000 rpm. Sedimentation curves measured by absorbance at 280 nm were compared with a buffer reference cell. Multiple data sets were fitted simultaneously to yield the Kd and molecular mass values quoted, using Beckman Optima XL-A/XL-I data analysis software (version 4.0). Data fitting and simulations used the following calculated parameters: buffer density at 15 °C = 1.00064 mg ml−1; monomeric molecular mass, p50cdc37 = 45,780 Da; sNp50 = 13,508 Da, and Cp50 = 30,817 Da; partial specific volumes, p50cdc37 = 0.7241; sNp50 = 0.7161 and Cp50 = 0.7271 and extinction coefficients (280 nm), p50cdc37 = 50,042, sNp50 = 16,500, and Cp50 = 26,930. CD spectra were recorded on a nitrogen-flushed Jasco J720 spectropolarimeter. For an accurate determination of Kd values, multiscanning was required for high data precision. At a scan speed of 20 nm min−1, nine scans were demanded in the 245–335 nm region for the CD titrations of Hsp90 with several co-chaperones. The concentration of the proteins were determined spectroscopically using the following molar extinction coefficients: ε(Hsp90) = 54,050m −1 cm−1, ε(Cdc37p)= 24,180 m −1 cm−1, ε(p50cdc37 ) = 50,340m −1 cm−1, ε(sNp50)= 17,190 m −1 cm−1, ε(Cp50) = 26,390 m −1cm−1, and ε(cSti1) = 34,480m −1 cm−1. All CD spectra were reported in ΔA = (AL −AR). The spectropolarimeter was calibrated with ammonium d-champor-10-sulfonate. The dissociation constant Kd was determined by analyzing the CD data using a non-linear regression analysis as described previously (6Prodromou 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 (356) Google Scholar, 24Freeman D.J. Pattenden G. Drake A.F. Siligardi G. J. Chem. Soc. Perkin Trans. 1998; 2: 129-135Crossref Scopus (68) Google Scholar). The CD titrations were conducted in a stepwise manner adding small aliquots of 5–10 μl of ligand stock solution directly into the cuvette of 1 cm path length containing an initial volume of 520 μl of Hsp90 in 20 mm Tris-HCl. Each titration was terminated upon ligand saturation that never reached a final volume greater than 15% of the initial volume, and each CD spectrum was subsequently corrected for its dilution. To achieve the highest accuracy and precision, Finnpipette PCR with a volume range of 2–20 and 20–200 μl were used to add aliquots of ligand solution in a stepwise manner to reach the desired molar ratio for the solution of Hsp90 of the appropriate concentration. For a single binding site at equilibrium, the CD, expressed in ΔA, at any wavelength is proportional to the concentration of the bound and unbound species of the host (H) and ligand (L) components (as shown in Equation 1) (24Freeman D.J. Pattenden G. Drake A.F. Siligardi G. J. Chem. Soc. Perkin Trans. 1998; 2: 129-135Crossref Scopus (68) Google Scholar).ΔA=ΔAHL+ΔAH+ΔALEquation 1 Based on the Beer's Law (ΔA = Δε.c.l, where differential molar extinction coefficient Δε is εL − εR (m −1cm−1), c is molarity, and l is the pathlength expressed in centimeters) and for a titration carried out in a 1-cm path length cell (l = 1) with the concentration at equilibrium of the host [H], ligand [L], and host-ligand complex [HL] (corresponding to the concentration of the bound species of the host and ligand), the CD of each species can be described as shown in Equations Equation 2, Equation 3, Equation 4,ΔAHL=ΔɛHL[HL]Equation 2 ΔAH=ΔɛH[H]Equation 3 ΔAL=ΔɛL[L]Equation 4 Substituting Equations Equation 2, Equation 3, Equation 4 into 1 (Equation 5),ΔA=ΔɛHL[HL]+ΔɛH[H]+ΔɛL[L]Equation 5 In the titration, the total concentration of the host and the ligand is as shown in Equations 6 and 7,[HT]=[H]+[HL]Equation 6 [LT]=[L]+[HL]Equation 7 Rearranging Equations 6 and 7 (see Equations 8 and 9),[L]=[LT]−[HL]Equation 8 [H]=[HT]−[HL]Equation 9 Substituting Equations 8 and 9 into 5 leads to EquationsEquation 10, Equation 11, Equation 12, Equation 13,ΔA=ΔɛHL[HL]+ΔɛH([HT]−[HL])+ΔɛL([LT]−[HL])Equation 10 ΔA=ΔɛHL[HL]+ΔɛH[HT]−ΔɛH[HL]+ΔɛL[LT]−ΔɛL[HL]Equation 11 ΔA=(ΔɛHL−ΔɛH−ΔɛL)[HL]+ΔɛH[HT]+ΔɛL[LT]Equation 12 ΔεHL and [HL] are unknown. From the association constant K = [HL]/[H][L], [HL] can be described as shown in Equation 13,[HL]=K[H][L]Equation 13 Substituting Equations 8 and 9 to 13 leads to EquationsEquation 14, Equation 15, Equation 16,[HL]=K{[LT]−[HL]}{[HT]−[HL]}Equation 14 K[HL ]2−(K[HT]+K[LT]+1)+K[HT][LT]=0Equation 15 [HL]={(K[HT]+K[LT]+1)±((K[HT]+K[LT]+1 )2−4KK[HT][LT] )−2}/2KEquation 16 Substituting Equations 16 into 12 leads to the final Equation17,ΔA=(ΔɛHL−ΔɛH−ΔɛL)({(K[HT]+K[LT]+1)±((K[HT]+K[LT]+1 )2−4KK[HT][LT] )−2}/2K)+H[HT]+L[LT]Equation 17 Of the two solutions, only the one with −((K[HT ] + K[LT ] + 1)2 − 4 KK[HT ][LT ])−2 fits the observed CD data. Equation 17 can be simplified if the ligand has no CD (ΔεL = 0) either because it has no absorbing chromophore in the UV region studied (25Renzoni D.A. Pugh D.J.R. Siligardi G. Das P. Morton C.J. Rossi C. Waterfield M.D. Campbell I.D. Ladbury J.E. Biochemistry. 1996; 35: 15646-15653Crossref PubMed Scopus (86) Google Scholar) or because it is non-chiral (26Siligardi G. Hussain R. Enantiomer. 1998; 3: 77-87PubMed Google Scholar) (see Equation 18).ΔA=(ΔɛHL−ΔɛH)({(K[HT]+K[LT]+1)−((K[HT]+K[LT]+1 )2−4KK[HT][LT] )−2}/2K)+ΔɛH[HT]Equation 18 The plot of the titration CD data at single wavelength versus the molar concentration of the ligand was analyzed by non-linear regression to Equation 18 as a function of ligand concentration to determine the dissociation constant Kd = 1/K. This equation is not only valid when only one component of the binary complex has intrinsic CD (one “active” CD component with bound and unbound species). It can also be applied to systems where both components show intrinsic CD, providing the data collected are transformed back to one active CD component system. This can be achieved by transforming the CD data to differential CD data (Δ(ΔA)) calculated by subtracting from each observed spectrum of the host-ligand mixture the equivalent CD contribution of the ligand. The ΔεL[LT ] component of Equation 17, which is the CD of the ligand added to the host, is subtracted, and ΔεHL − ΔεH becomes the new ΔεHL. In this way the fitted or simulated Equation 18resembles a Michaelis-Menten saturation curve rather than a change in slope of the curve generated by Equation 17. It is crucial to terminate the titration upon reaching saturation. As a rule of thumb, to achieve saturation within the ligand excess used in the CD titration, the total host concentration [HT ] should be ≥30 to 50 times the value of Kd. Obviously, for dissociation constant Kd in the order of 10−4 m, these conditions might not be achieved and hence saturation will not be reached, but as long as the plot ΔA versus ligand concentration is of parabolic type, the Kd can be still determined by Equation 18. For Kd in the region between 10−7 and 10−6 m, the plot is of Michaelis-Menten type of shape with saturation that can be achieved at 1:1 molar ratio stoichiometry. In the case of a ligand with no CD, the CD changes associated with the host plateau upon ligand saturation. In the case of a ligand with CD, the saturation is normally achieved with [LT ] ≥5–10-fold [HT ], and ΔεHLcan be calculated in a first approximation using the concentration of [HT ] = [HL] (that is all host molecules are bound to the ligand). With the best fitting achieved by either visual inspection or by Levenberg-Marquardt method, the calculated value of K can be used to calculate [HL] from Equation 16. From Beer's Law, a more accurate value of ΔεHL = ΔAHL/[HL] can be calculated and used again in Equation 18 to fit again the experimental CD data in order to determine a more accurate value of K and hence Kd. This method has been used to calculate the Kd value for 29 titrations of Hsp90 and several of its mutants with different ligands, such as ADP, ATP, AMP-PNP, Sti1, Cpr6, and geldanamycin (6Prodromou 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 (356) Google Scholar). 2G. Siligardi, B. Panaretou, P. Meyer, S. Singh, D. N. Woolfson, P. W. Piper, L. H. Pearl, and C. Prodromou, unpublished data. The Kd values determined from differential CD data were in very good agreement with those obtained from calorimetry. A similar approach has been used to determine the Kd values of binary complexes by a non-linear regression method (27Kurz L.C. Shah S. Crane B.R. Donald L.J. Duckworth H.W. Drysdale G.R. Biochemistry. 1992; 31: 7908-7914Crossref PubMed Scopus (28) Google Scholar), using the difference CD obtained by subtracting the CD of the total added concentration of both host and ligand from the observed CD of the host-ligand complex. To address the question of whether X and Y form a binary complex, three CD spectra were measured ((X), (Y), and (X +Y)). Spectrum (X + Y) is the observed spectrum of X + Y, whereas spectrum ((X) + (Y)) is the simulated spectrum calculated by adding the observed spectrum of X to that of Y. If the observed and simulated spectra are identical, one can readily infer that there is no detectable interaction between X and Y, whereas if the spectra are different, there is unambiguously a binding interaction and hence a binary complex. To address the question whether X, Y, and Z form a ternary complex, eight CD spectra were measured ((X), (Y), (Z), (X + Y), (X + Z), (Y + Z), (X + Y + Z) and (X +Z + Y)). Spectrum (X + Y) is the observed spectrum of X + Y, whereas spectrum ((X + Y) + (Z)) is the simulated spectrum calculated by adding the observed spectrum of the binary complex X + Y to the observed spectrum of Z. Difference CD spectra {(X +Y) − (X)} and {(X +Z) − (X)} were calculated by subtracting the spectrum of the total concentration of (X) from the observed spectrum of the binary complexes (X + Y) and (X + Z), respectively. The observed spectra of (X + Y + Z) and (X +Z + Y) were compared with the simulated spectra ((X) + (Y) + (Z)), ((X +Y) + (Z)), ((X + Z) + (Y)), ((X) + (Y + Z)), ((X) + {(X + Y) − (X)} + {(X + Z) − (X)}). The simulated spectrum ((X) + (Y) + (Z)) is the sum of the spectra of each component as if no binding interactions were present among the three components X, Y, and Z. The simulated spectra ((X + Y) + (Z)), ((X +Z) + (Y)), ((X) + (Y +Z)) represent each of the three independent combinations of binary complexes together with the non-interacting third component. The simulated spectrum ((X + Y) + (Z)) can be seen as the spectrum obtained when component Z does not bind to the binary complex (X + Y). The simulated spectra ((X + Z) + (Y)) and ((X) + (Y + Z)) represent the displacement of Y and X, respectively, from their binary (X + Y) complex by ligand Z. Of course, if ligand Y and Z do not interact with each other, the simulated spectrum ((X) + (Y +Z)) is therefore redundant. The simulated spectrum ((X) + {(X + Y) − (X)} + {(X + Z) − (X)}) represents the spectrum as if a ternary complex was formed but without interactions between ligands Y and Z, namely without direct contacts. The difference CD components represent the CD changes associated with each binary complex. If none of the simulated spectra superimposes the observed spectra, it excludes all the possibilities described above. There is only one possibility left, which cannot be simulated, that all three components X, Y, and Z interact with each other upon forming the ternary complex. The apparently similar roles played by Cdc37p/p50cdc37 and by Sti1/Hop/p60 in the recruitment of client proteins to the Hsp90 complex prompted us to characterize the binding of Cdc37p and p50cdc37 to Hsp90 using CD spectroscopy. As observed previously with Sti1/Hop/p60 (6Prodromou 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 (356) Google Scholar), the near-UV CD spectra obtained for mixtures of Hsp90 and Cdc37p cannot be simulated by linear combination of the individual spectra of Hsp90 and Cdc37p in isolation, indicating that Hsp90 and Cdc37p interact to form a complex (Fig. 1 A). Titration of Cdc37p into Hsp90 produces dose-dependent perturbations in the near-UV region (240–280 nm), and difference spectra (Fig. 1 B) were obtained by subtracting the spectrum of the isolated co-chaperone (n molar equivalent) from the spectrum of Hsp90 + co-chaperone (1:n) mixtures. Although saturable changes in the near-UV region were not achieved due to the relatively weak binding of Cdc37p to Hsp90, the observed signals are consistent with changes in the environment of aromatic residues due to molecular interaction. The Kd value for the interaction of Cdc37p with Hsp90 was estimated as 113 ± 4 μm (Fig. 1 C). As in the case for Cdc37p, titration of human p50cdc37 into Hsp90 also produced dose-dependent perturbations in the near-UV region (240–280 nm). The difference spectra (Fig. 2 A) showed changes that initially peaked at a molar ratio between 1:1 and 1:1.4 (Hsp90:p50cdc37) and subsequently decreased at higher p50cdc37 concentrations. The behavior of these spectra is consistent with two different association processes occurring. The first phase results from binding of p50cdc37 to Hsp90 that saturates at ≈1:1, whereas the second phase results from self-association of excess p50cdc37. This interpretation is supported by the observation that the CD spectrum of p50cdc37 is concentration-dependent, consistent with self-association (Fig. 2 B). The Kd value calculated for the interaction of p50cdc37 with Hsp90 was 2.5 μm(Fig. 2 C) and that for p50cdc37 dimerization was 9.8 ± 1.9 μm (Fig. 2 D), assuming saturation of Hsp90 by p50cdc37 at a 1:1 molar ratio. Because binding of the mammalian p50cdc37 to Hsp90 was significantly tighter than that of its yeast orthologue Cdc37p, further analysis of interactions with Hsp90 was conducted mainly with p50cdc37. To confirm and quantitatively analyze the self-association of p50cdc37 observed in the CD studies, p50cdc37 oligomerization was analyzed by equilibrium sedimentation in an analytical ultracentrifuge. Equilibrium data sets obtained were intermediate between theoretical traces calculated assuming ideal single species with molecular weights equal to one or two p50cdc37 units. These data could be fitted with high confidence to an equation describing a monomer-dimer equilibrium mixture (Fig. 3) with a Kd for the monomer-dimer transition of 5.5 μm (95% confidence limits = 4.4–7.1 μm) which is comparable with the results obtained by CD spectroscopy (Kd = 9.8 ± 1.9 μm). It has been demonstrated previously (16Grammatikakis N. Lin J.-H. Grammatikakis A. Tsichlis P.N. Cochran B.H. Mol. Cell. Biol. 1999; 19: 1661-1672Crossref PubMed Scopus (229) Google Scholar, 17Shao J. Grammatikakis N. Scroggins B.T. Uma S. Huang W.J. Chen J.J. Hartson S.D. Matts R.L. J. Biol. Chem. 2001; 276: 206-214Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) that Cdc37p/p50cdc37 possesses two separable domains as follows: an N-terminal domain capable of binding Hsp90-dependent protein kinases and a C-terminal domain that binds Hsp90 itself. To determine which domain was responsible for the inherent dimerization of p50cdc37, the separate N- and C-terminal domains of p50cdc37 were subjected to analytical centrifugation in a similar way to the full-length protein. For both domains the data could only be fitted with high confidence to an equation describing monomer-dimer equilibrium mixtures (results not shown) indicating that homodimerization is a consequence of both N to N-terminal domain and C to C-terminal domain interactions. However, dimerization of the N-terminal kinase-binding domain was very weak with a Kd estimated as 971 μm (95% confidence limits = 812–1160 μm) and that for the C-terminal domain was 167 μm (95% confidence limits = 145–192), indicating that the main dimerization interface is between the C-terminal domains. However, it should be noted that the p50cdc37 N-terminal domain used in the current studies lacks the first 29 N-terminal amino acids which, although relatively poorly conserved, could contribute to dimerization. The similar recruiting roles that Cdc37p/p50cdc37 and Sti1/Hop/p60 play in the Hsp90 system, and the fact that both are capable of dimerization and of binding to Hsp90 as dimers, led us to q" @default.
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• W2073886258 title "Regulation of Hsp90 ATPase Activity by the Co-chaperone Cdc37p/p50" @default.
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