DISTINCTIVE ROLES OF THE 2 ATP-BINDING SITES IN CLPA, THE ATPASE COMPONENT OF PROTEASE TI IN ESCHERICHIA-COLI

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 Lys(220) or Lys(501) 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/R220T 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 un changed, 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.