DnaJ dramatically stimulates ATP hydrolysis by DnaK: Insight into targeting of Hsp70 proteins to polypeptide substrates

Most, if not all, of the cellular functions of Hsp70 proteins require the assistance of a DnaJ homologue, which accelerates the weak intrinsic ATPase activity of Hsp70 and serves as a specificity factor by binding and targeting specific polypeptide substrates for Hsp70 action. We have used presteady-state kinetics to investigate the interaction of the Escherichia coli DnaJ and DnaK proteins, and the effects of DnaJ on the ATPase reaction of DnaK, DnaJ accelerates hydrolysis of ATP by DnaK to such an extent that ATP binding by DnaK becomes rate-limiting for hydrolysis. At high concentrations of DnaK under single-turnover conditions, the rate-limiting step is a first-order process, apparently a change of DnaK conformation, that accompanies ATP binding and proceeds at 12-15 min(-1) at 25 degrees C and 1-1.5 min(-1) at 5 degrees C. By prebinding ATP to DnaK and subsequently adding DnaJ, the effects of this slow step may be bypassed, and the maximal rate-enhancement of DnaJ on the hydrolysis step is similar to 15 000-fold at 5 degrees C, The interaction of DnaJ with DnaK.ATP is likely a rapid equilibrium relative to ATP hydrolysis, and is relatively weak, with a K-D of similar to 20 mu M at 5 degrees C, and weaker still at 25 degrees C. In the presence of saturating DnaJ, the maximal rate of ATP hydrolysis by DnaK is similar to previously reported rates for peptide release from DnaK ATP. This suggests that when DnaK encounters a DnaJ-bound polypeptide or protein complex, a significant fraction of such events result in ATP hydrolysis by DnaK and concomitant capture of the polypeptide substrate in a tight complex with DnaK ADP. Furthermore, a broadly applicable kinetic mechanism for DnaJ-mediated specificity of Hsp70 action arises from these observations, in which the specificity arises largely from the acceleration of the hydrolysis step itself, rather than by DnaJ-dependent modulation of the affinity of Hsp70 for substrate polypeptides.