title: Dissection of the contribution of individual domains to the ATPase mechanism of Hsp90.
abstract: Hsp90 is a dimeric, ATP-regulated molecular chaperone. Its ATPase cycle involves the N-terminal ATP binding domain (amino acids (aa) 1-272) and, in addition, to some extent the middle domain (aa 273-528) and the C-terminal dimerization domain (aa 529-709). To analyze the contribution of the different domains and the oligomeric state on the progression of the ATPase cycle of yeast Hsp90, we created deletion constructs lacking either the C-terminal or both the C-terminal and the middle domain. To test the effect of dimerization on the ATPase activity of the different constructs, we introduced a Cys residue at the C-terminal ends of the constructs, which allowed covalent dimerization. We show that all monomeric constructs tested exhibit reduced ATPase activity and a decreased affinity for ATP in comparison with wild type Hsp90. The covalently linked dimers lacking only the C-terminal domain hydrolyze ATP as efficiently as the wild type protein. Furthermore, this construct is able to trap the ATP molecule similar to the full-length protein. This demonstrates that in the ATPase cycle, the C-terminal domain can be replaced by a cystine bridge. In contrast, the ATPase activity of the artificially linked N-terminal domains remains very low and bound ATP is not trapped. Taken together, we show that both the dimerization of the N-terminal domains and the association of the N-terminal with the middle domain are important for the efficiency of the ATPase cycle. These reactions are synergistic and require Hsp90 to be in the dimeric state.
authors.affiliation: Institut für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching, Germany.
authors.forename: Harald
authors.initials: H
authors.lastname: Wegele
authors.forename: Paul
authors.initials: P
authors.lastname: Muschler
authors.forename: Melanie
authors.initials: M
authors.lastname: Bunck
authors.forename: Jochen
authors.initials: J
authors.lastname: Reinstein
authors.forename: Johannes
authors.initials: J
authors.lastname: Buchner
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D005800:Genes, Fungal; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion
chemical_list: D018841:HSP90 Heat-Shock Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D003545:Cysteine