Roles of the two ATP binding sites of ClpB from Thermus thermophilus.
As a member of molecular chaperone Hsp100/Clp family, TClpB from Thermus thermophilus has two nucleotide binding domains, NBD1 and NBD2, in a single polypeptide, each containing WalkerA and WalkerB consensus motifs. To probe their roles, mutations were introduced into the WalkerA or WalkerB motifs of each or both of the NBDs. The results are as follows. 1) For each of the NBDs, the ability of nucleotide binding is lost by mutations in the WalkerA motif but is retained by mutations in the WalkerB motif. 2) Each NBD has a casein-stimulatable small basic ATPase activity that is lost when the WalkerB motif is mutated. 3) TClpB assembles into a uniform 580-kDa oligomer when ATP is present at 55 degrees C, and only the mutants in the WalkerA motif in NBD1 fail to assemble, indicating that ATP binding to NBD1 but not hydrolysis is necessary and sufficient for the assembly. 4) Chaperone function of TClpB was lost when the WalkerA motif in each of the NBDs was mutated. Mutants in the WalkerB motifs of each NBD retained some chaperone activity.
Watanabe
Yo-hei
YH
Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259, Yokohama 226-8503, Japan.
Motohashi
Ken
K
Yoshida
Masasuke
M
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D003432:Cross-Linking Reagents; D049071:Endopeptidase Clp; D005976:Glutaral; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016963:Thermus thermophilus
D003432:Cross-Linking Reagents; D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D049071:Endopeptidase Clp; C095049:ClpB protein, Leishmania; D005976:Glutaral