title: Analysis of the cooperative ATPase cycle of the AAA+ chaperone ClpB from Thermus thermophilus by using ordered heterohexamers with an alternating subunit arrangement.
abstract: The ClpB/Hsp104 chaperone solubilizes and reactivates protein aggregates in cooperation with DnaK/Hsp70 and its cofactors. The ClpB/Hsp104 protomer has two AAA+ modules, AAA-1 and AAA-2, and forms a homohexamer. In the hexamer, these modules form a two-tiered ring in which each tier consists of homotypic AAA+ modules. By ATP binding and its hydrolysis at these AAA+ modules, ClpB/Hsp104 exerts the mechanical power required for protein disaggregation. Although ATPase cycle of this chaperone has been studied by several groups, an integrated understanding of this cycle has not been obtained because of the complexity of the mechanism and differences between species. To improve our understanding of the ATPase cycle, we prepared many ordered heterohexamers of ClpB from Thermus thermophilus, in which two subunits having different mutations were cross-linked to each other and arranged alternately and measured their nucleotide binding, ATP hydrolysis, and disaggregation abilities. The results indicated that the ATPase cycle of ClpB proceeded as follows: (i) the 12 AAA+ modules randomly bound ATP, (ii) the binding of four or more ATP to one AAA+ ring was sensed by a conserved Arg residue and converted another AAA+ ring into the ATPase-active form, and (iii) ATP hydrolysis occurred cooperatively in each ring. We also found that cooperative ATP hydrolysis in at least one ring was needed for the disaggregation activity of ClpB.
authors.affiliation: From the Department of Biology, Faculty of Science and Engineering and the Institute for Integrative Neurobiology, Konan University, Okamoto 8-9-1, Kobe 658-8501, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamasaki
authors.affiliation: From the Department of Biology, Faculty of Science and Engineering and.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Oohata
authors.affiliation: From the Department of Biology, Faculty of Science and Engineering and.
authors.forename: Toshiki
authors.initials: T
authors.lastname: Nakamura
authors.affiliation: From the Department of Biology, Faculty of Science and Engineering and the Institute for Integrative Neurobiology, Konan University, Okamoto 8-9-1, Kobe 658-8501, Japan ywatanab@center.konan-u.ac.jp.
authors.forename: Yo-hei
authors.initials: YH
authors.lastname: Watanabe
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D006868:Hydrolysis; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D066329:Protein Aggregates; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016963:Thermus thermophilus / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D066329:Protein Aggregates; D021122:Protein Subunits; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases
keywords: AAA+; ATPase; ATPases Associated with Diverse Cellular Activities (AAA); ClpB; chaperone; cooperativity; cysteine-mediated cross-linking; disaggregation; hsp104; protein aggregation