title: Hydrodynamic properties and quaternary structure of the 90 kDa heat-shock protein: effects of divalent cations.
abstract: The 90 kDa heat-shock protein (Hsp90) is one of the major stress proteins whose overall structure remains unknown. In this study, we investigated the influence of divalent cations Mg(2+) and Ca(2+) on the hydrodynamic properties and quaternary structure of Hsp90. Using analytical ultracentrifugation, size-exclusion chromatography, and polyacrylamide gel electrophoresis, we showed that native Hsp90 was mostly dimeric. The Hsp90 dimer had a sedimentation coefficient, s(w,20) degrees, of 6.10 +/- 0.03 S, which slightly deviated from the hydrodynamics of a globular protein. Using chemical cross-linking and analytical ultracentrifugation, we showed that Mg(2+) and Ca(2+) induced a tertiary conformational change of Hsp90, leading to a self-association process. In the presence of divalent cations, Hsp90 existed as a mixture of monomers, dimers, and tetramers at equilibrium. Finally, to identify Hsp90 domains involved in this divalent cation-dependent self-association, we studied the oligomerization state of the N-terminal (positions 1-221) of Hsp90, the influence of an N-terminal specific ligand, geldanamycin (GA), and the effect of C-terminal truncation on the ability of Hsp90 to oligomerize in the presence of divalent cations. We previously showed that GA inhibits Hsp90 heat-induced oligomerization [Garnier, C., Protasevich, I., Gilli, R., Tsvetkov, P., Lobachov, V., Peyrot, V., Briand, C., and Makarov, A. (1998) Biochem. Biophys. Res. Commun. 249, 197-201], but now we observed that GA does not influence divalent cation-dependent oligomerization of Hsp90, suggesting another mechanism. This mechanism involved the C-terminal part of the protein since C-terminally truncated Hsp90 did not oligomerize in the presence of divalent cations.
authors.affiliation: Faculté de Pharmacie, UMR-CNRS 6032, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France.
authors.forename: Cyrille
authors.initials: C
authors.lastname: Garnier
authors.forename: Pascale
authors.initials: P
authors.lastname: Barbier
authors.forename: François
authors.initials: F
authors.lastname: Devred
authors.forename: German
authors.initials: G
authors.lastname: Rivas
authors.forename: Vincent
authors.initials: V
authors.lastname: Peyrot
mesh_terms: D000818:Animals; D002118:Calcium / Q000737:chemistry*; D002413:Cations, Divalent / Q000737:chemistry; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000737:chemistry; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000031:analogs & derivatives* / Q000737:chemistry; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry*; D008274:Magnesium / Q000737:chemistry*; D008970:Molecular Weight; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry; D012996:Solutions; D013552:Swine; D014461:Ultracentrifugation
chemical_list: D002413:Cations, Divalent; D003432:Cross-Linking Reagents; D018841:HSP90 Heat-Shock Proteins; D011994:Recombinant Proteins; D012996:Solutions; C041829:1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide methiodide; D008274:Magnesium; D005022:Ethyldimethylaminopropyl Carbodiimide; D002118:Calcium