Concurrent chaperone and protease activities of ClpAP and the requirement for the N-terminal ClpA ATP binding site for chaperone activity

ClpA, a member of the Clp/Hsp100 family of ATPases, is both an ATP-dependen t molecular chaperone and the regulatory component of ClpAP protease. We de monstrate that chaperone and protease activities occur concurrently in ClpA P complexes during a single round of RepA binding to ClpAP and ATP-dependen t release. This result was substantiated with a ClpA mutant, ClpA(K220V), c arrying an amino acid substitution in the N-terminal ATP binding site. ClpA (K220V) is unable to activate RepA, but the presence of ClpP or chemically inactivated ClpP restores its ability to activate RepA. The presence of Clp P simultaneously facilitates degradation of RepA ClpP must remain bound to ClpA(K220V) for these effects, indicating that both chaperone and proteolyt ic activities of the mutant complex occur concurrently. ClpA(K220V) itself is able to form stable complexes with RepA in the presence of a poorly hydr olyzed ATP analog, adenosine 5'-O-(thiotriphosphate), and to release RepA u pon exchange of adenosine 5'-O-(thiotriphosphate) with ATP. However, the re leased RepA is inactive in DNA binding, indicating that the N-terminal ATP binding site is essential for the chaperone activity of ClpA. Taken togethe r, these results suggest that substrates bound to the complex of the proteo lytic and ATPase components can be partitioned between release/reactivation and translocation/degradation.