Chaperones of the Hsp70 family bind to unfolded or partially folded polypeptides to facilitate many cellular processes. ATP hydrolysis and substrate binding, the two key molecular activities of this chaperone, are modulated by the cochaperone DnaJ. By using both genetic and biochemical approaches, we 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 lower cleft of the ATPase domain is defined as a binding pocket for the J-domain because (i) a DnaK mutation located in this cleft (R167H) is an allele-specific suppressor of the binding defect of the DnaJ mutation, D35N and (ii) alanine substitution of two residues close to R167 in the crystal structure, N170A and T173A, significantly decrease DnaJ binding. A second binding determinant is likely to be in the substrate-binding domain because some DnaK mutations in the vicinity of the substrate-binding pocket are defective in either the affinity (G400D, G539D) or rate (D526N) of both peptide and DnaJ 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 properties of Hsp70.Molecular chaperones of the Hsp70 family are conserved proteins that modulate intracellular protein folding. By binding to unfolded or partially folded polypeptides, chaperones prevent misfolding and aggregation and promote folding, translocation, and the assembly and disassembly of multiprotein structures (1, 2). Both prokaryotes and eukaryotes have multiple Hsp70 proteins that function in diverse processes. Hsp70s have a highly conserved 44-kDa ATPase domain followed by a highly conserved 15-kDa peptide-binding domain and a less conserved 10-kDa C-terminal region. The structures of the two conserved domains have been determined separately by x-ray crystallography (3, 4). Hsp70s function in concert with a cochaperone, called DnaJ or Hsp40. For example, the Escherichia coli Hsp70 protein DnaK requires DnaJ to function in the initiation of bacteriophage DNA replication (5). DnaJ increases the ATPase activity and modulates substrate binding of Hsp70 (6) and is required for Hsp70 function in vivo. Cells contain multiple DnaJ family members, and, in some cases, a specific DnaJ is required for a particular Hsp70 to function (7). Despite the key role of DnaJ in Hsp70 function, little is known about the Hsp70 determinants that mediate binding to DnaJ. A very recent NMR study localizes one binding determinant to the ATPase domain of DnaK (8).E. coli DnaJ is comprised of a J-domain, a glycinephenylalanine rich segment, a cysteine rich segment, and a C-terminal region, of which the J-domain is the most important (9). The J-domain defines this family of proteins, and some members contain only this domain. The NMR structure of this domain has been determined (10, 11). A pri...

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