title: Allosteric communication between the nucleotide binding domains of caseinolytic peptidase B.
abstract: ClpB is a hexameric chaperone that solubilizes and reactivates protein aggregates in cooperation with the Hsp70/DnaK chaperone system. Each of the identical protein monomers contains two nucleotide binding domains (NBD), whose ATPase activity must be coupled to exert on the substrate the mechanical work required for its reactivation. However, how communication between these sites occurs is at present poorly understood. We have studied herein the affinity of each of the NBDs for nucleotides in WT ClpB and protein variants in which one or both sites are mutated to selectively impair nucleotide binding or hydrolysis. Our data show that the affinity of NBD2 for nucleotides (K(d) = 3-7 μm) is significantly higher than that of NBD1. Interestingly, the affinity of NBD1 depends on nucleotide binding to NBD2. Binding of ATP, but not ADP, to NBD2 increases the affinity of NBD1 (the K(d) decreases from ≈160-300 to 50-60 μm) for the corresponding nucleotide. Moreover, filling of the NBD2 ring with ATP allows the cooperative binding of this nucleotide and substrates to the NBD1 ring. Data also suggest that a minimum of four subunits cooperate to bind and reactivate two different aggregated protein substrates.
authors.affiliation: Biophysics Unit (Consejo Superior de Investigaciones Científicas-Universidad del País Vasco/Euskal Herriko Unibertsitatea) and Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country (Universidad del País Vasco/Euskal Herriko Unibertsitatea), Bilbao, Spain.
authors.forename: José Ángel
authors.initials: JÁ
authors.lastname: Fernández-Higuero
authors.forename: Sergio P
authors.initials: SP
authors.lastname: Acebrón
authors.forename: Stefka G
authors.initials: SG
authors.lastname: Taneva
authors.forename: Urko
authors.initials: U
authors.lastname: Del Castillo
authors.forename: Fernando
authors.initials: F
authors.lastname: Moro
authors.forename: Arturo
authors.initials: A
authors.lastname: Muga
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D049071:Endopeptidase Clp; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D009711:Nucleotides; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D049071:Endopeptidase Clp; C069775:ClpB protein, E coli