title: Distinctive roles of the two ATP-binding sites in ClpA, the ATPase component of protease Ti in Escherichia coli.
abstract: ClpA is the ATPase component of the ATP-dependent protease Ti (Clp) in Escherichia coli and contains two ATP-binding sites. A ClpA variant (referred to as ClpAT) carrying threonine in place of the 169th methionine has recently been shown to be highly soluble but indistinguishable from the wild-type, 84-kDa ClpA in its ability to hydrolyze ATP and to support the casein-degrading activity of ClpP. Therefore, site-directed mutagenesis was performed to generate mutations in either of the two ATP-binding sites of ClpAT (i.e. to replace the Lys220 or Lys501 with Thr). ClpAT/K220T hydrolyzed ATP and supported the ClpP-mediated proteolysis 10-50% as well as ClpAT depending on ATP concentration, while ClpAT/K501T was unable to cleave ATP or to support the proteolysis. Without ATP, ClpAT and both of its mutant forms behaved as trimeric molecules as analyzed by gel filtration on a Sephacryl S-300 column. With 0.5 mM ATP, ClpAT and ClpAT/K501T became hexamers, but ClpAT/K220T remained trimeric. With 2 mM ATP, however, ClpAT/K220T also behaved as a hexamer. These results suggest that the first ATP-binding site of ClpA is responsible for hexamer formation, while the second is essential for ATP hydrolysis. When trimeric ClpAT/K220T was incubated with the same amount of hexameric ClpAT/K501T (i.e. at 0.5 mM ATP) and then subjected to gel filtration as above, a majority of ClpAT/K220T ran together with ClpAT/K501T as hexameric molecules. Furthermore, ClpAT/K501T in the mixture strongly inhibited the ability of ClpAT/K220T to cleave ATP and to support the ClpP-mediated proteolysis. Similar results were obtained in the presence of 2 mM ATP and also with the mixture with ClpAT. On the other hand, the ATPase activity of the mixture of ClpAT and ClpAT/K220T was significantly higher than the sum of that of each protein, particularly in the presence of 2 mM ATP, although its ability to support the proteolysis by ClpP remained unchanged. These results suggest that a rapid exchange of the subunits, possibly as a trimeric unit, occurs between the ClpAT proteins in the presence of ATP and leads to the formation of mixed hexameric molecules.
authors.affiliation: Department of Molecular Biology, College of Natural Sciences, Seoul National University, Korea.
authors.forename: J H
authors.initials: JH
authors.lastname: Seol
authors.forename: S H
authors.initials: SH
authors.lastname: Baek
authors.forename: M S
authors.initials: MS
authors.lastname: Kang
authors.forename: D B
authors.initials: DB
authors.lastname: Ha
authors.forename: C H
authors.initials: CH
authors.lastname: Chung
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D001704:Biopolymers; D017931:DNA Primers; D049071:Endopeptidase Clp; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009154:Mutation; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*
chemical_list: D001704:Biopolymers; D017931:DNA Primers; D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; D012697:Serine Endopeptidases; C483568:ClpA protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases