ANALYSIS OF 3 DNAK MUTANT PROTEINS SUGGESTS THAT PROGRESSION THROUGH THE ATPASE CYCLE REQUIRES CONFORMATIONAL-CHANGES

DnaK, the bacterial homolog of the eukaryotic hsp70 proteins, is an AT P-dependent chaperone whose basal ATPase is stimulated by synthetic pe ptides and its cohort heat shock proteins, DnaJ and GrpE. We have used three mutant DnaK proteins, E171K D201N, and A174T (corresponding to Glu(175), ASp(206) and Ala(179) respectively, in bovine heat stable co gnate 70) to probe the ATPase cycle. All of the mutant proteins exhibi t some alteration in basal ATP hydrolysis, However, they all exhibit m ore severe defects in the regulated activities. D201N and E171K are co mpletely defective in all regulated activities of the protein and also in making the conformational change exhibited by the wt protein upon binding ATP. We suggest that the inability of D201N and E171K to achie ve the ATP activated conformation prevents both stimulation by all eff ecters and the ATP-mediated release of GrpE. In contrast, the defect o f A174T is much more specific. It exhibits normal binding and release of GrpE and normal stimulation of ATPase activity by DnaJ, However, it is defective in the synergistic activation of its ATPase by DnaJ and GrpE. We suggest that this mutant protein is specifically defective in a DnaJ/GrpE mediated conformational change in DnaK necessary for the synergistic action of DnaJ + GrpE.