title: ClpB chaperone passively threads soluble denatured proteins through its central pore.
abstract: ClpB disaggregase forms a ring-shaped hexamer that threads substrate proteins through the central pore using energy from ATP. The ClpB protomer consists of an N-terminal domain, a middle domain, and two AAA+ modules. These two AAA+ modules bind and hydrolyze ATP and construct the core of the hexameric ring. Here, we investigated the roles of the two AAA+ modules in substrate threading. BAP is an engineered ClpB that can bind ClpP proteolytic chamber; substrates threaded by BAP are degraded by ClpP. We combined BAP with conserved motif mutations in two AAA+ modules and measured the steady-state rates of threading of soluble denatured proteins by these mutants over a range of substrate concentrations. By fitting the data to the Michaelis-Menten equation, k(cat) and K(m) values were determined. We found that the kinetic parameters of the substrate threading correlate with the type of mutation introduced rather than the ATPase activity of the mutant. Moreover, some mutants having no or marginal ATPase activity could thread denatured proteins significantly. These results indicate that ClpB can passively thread soluble denatured proteins.
authors.affiliation: Department of Biology, Faculty of Science and Engineering, Konan University, Okamoto 8-9-1, Kobe, 658-8501, Japan; Institute for Integrative Neurobiology, Konan University, Okamoto 8-9-1, Kobe, 658-8501, Japan.
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Nakazaki
authors.forename: Yo-Hei
authors.initials: YH
authors.lastname: Watanabe
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002364:Caseins / Q000378:metabolism; D017124:Conserved Sequence; D049071:Endopeptidase Clp / Q000737:chemistry / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005456:Fluorescent Dyes; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation
chemical_list: D002364:Caseins; D005456:Fluorescent Dyes; D000255:Adenosine Triphosphate; D049071:Endopeptidase Clp; C000596639:CLPB protein, human; D016650:Fluorescein-5-isothiocyanate