Interaction of the Hsp70 molecular chaperone, DnaK, with its cochaperone DnaJ

Chaperones of the Hsp70 family bind to unfolded or partially folded polypep tides to facilitate many cellular processes. ATP hydrolysis and substrate b inding, the two key molecular activities of this chaperone, are modulated b y the cochaperone DnaJ, By using both genetic and biochemical approaches, w e provide evidence that DnaJ binds to at least two sites on the Escherichia coli Hsp70 family member DnaK: under the ATPase domain in a cleft between its two subdomains and at or near the pocket of substrate binding. The lowe r cleft of the ATPase domain is defined as a binding pocket for the J-domai n because (i) a DnaK mutation located in this cleft (R167H) is an allele-sp ecific suppressor of the binding defect of the DnaJ mutation, D35N and (ii) alanine substitution of two residues close to R167 in the crystal structur e, N170A and T173A, significantly decrease DnaJ binding. A second binding d eterminant is likely to be in the substrate-binding domain because some Dna K mutations in the vicinity of the substrate-binding pocket are defective i n either the affinity (G400D, G539D) or rate (D526N) of both peptide and Dn aJ binding to DnaK. Binding of DnaJ may propagate conformational changes to the nearby ATPase catalytic center and substrate-binding sites as well as facilitate communication between these two domains to alter the molecular p roperties of Hsp70.