title: Coupling ATP utilization to protein remodeling by ClpB, a hexameric AAA+ protein.
abstract: ClpB and Hsp104 are members of the AAA+ (ATPases associated with various cellular activities) family of proteins and are molecular machines involved in thermotolerance. They are hexameric proteins containing 12 ATP binding sites with two sites per protomer. ClpB and Hsp104 possess some innate protein remodeling activities; however, they require the collaboration of the DnaK/Hsp70 chaperone system to disaggregate and reactivate insoluble aggregated proteins. We investigated the mechanism by which ClpB couples ATP utilization to protein remodeling with and without the DnaK system. When wild-type ClpB, which is unable to remodel proteins alone in the presence of ATP, was mixed with a ClpB mutant that is unable to hydrolyze ATP, the heterohexamers surprisingly gained protein remodeling activity. Optimal protein remodeling by the heterohexamers in the absence of the DnaK system required approximately three active and three inactive protomers. In addition, the location of the active and inactive ATP binding sites in the hexamer was not important. The results suggest that in the absence of the DnaK system, ClpB acts by a probabilistic mechanism. However, when we measured protein disaggregation by ClpB heterohexamers in conjunction with the DnaK system, incorporation of a single inactive ClpB subunit blocked activity, supporting a sequential mechanism of ATP utilization. Taken together, the results suggest that the mechanism of ATP utilization by ClpB is adaptable and can vary depending on the specific substrate and the presence of the DnaK system.
authors: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors: Joel R
authors: JR
authors: Hoskins
authors: Shannon M
authors: SM
authors: Doyle
authors: Sue
authors: S
authors: Wickner
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D049071:Endopeptidase Clp; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D046912:Multiprotein Complexes; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D046912:Multiprotein Complexes; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C070972:HsP104 protein, S cerevisiae; D000255:Adenosine Triphosphate; D049071:Endopeptidase Clp; C063842:dnaK protein, E coli; C069775:ClpB protein, E coli