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

Abstract

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...

The Roles of the Two Zinc Binding Sites in DnaJ

“…The first interaction, which has been recognized for many years, seems to occur between the N-terminal J-domain of DnaJ and the Nterminal ATPase domain of DnaK. The second interaction site was proposed to be between an unidentified site in DnaJ and the C-terminal substrate binding site in DnaK (35). It appears that we have succeeded in identifying that this required additional interaction site in class I DnaJ homologues is mediated by the highly conserved zinc center II.…”

“…7). The requirement for such additional interactions between DnaJ and DnaK-substrate protein complexes have been suggested before in DnaK suppressor studies that showed that DnaJ binds to at least two sites in DnaK (35). The first interaction, which has been recognized for many years, seems to occur between the N-terminal J-domain of DnaJ and the Nterminal ATPase domain of DnaK.…”

“…(ii) DnaJ⌬ZnII mutant protein might be incapable of effectively stimulating ATP hydrolysis of the DnaK-ATP-substrate protein complexes, or (iii) DnaJ⌬ZnII mutant protein might fail to provide a necessary second DnaK interaction site, whose existence has been proposed previously and, which might be necessary for a catalytically active DnaK/DnaJ/GrpE complex (34,35).…”

Allosteric signal transmission in the nucleotide-binding domain of 70-kDa heat shock protein (Hsp70) molecular chaperones

“…Indeed, single point mutations of the linker and hydrophobic cleft residues perturb the NBD ATPase activity and disturb its regulation by the SBD and cochaperones (2,16,32,33). The fact that the hydrophobic cleft is the binding site for the Hsp70 cochaperone DnaJ, which regulates the rate of ATP hydrolysis (2,34,35), indicates that cochaperone regulation of ATPase activity most likely occurs through the same allosteric networks (i.e., via the linker and the hydrophobic cleft). Intriguingly, the hydrophobic cleft comprises a sector of coevolved residues in the NBD, which is important for stabilizing the interdomain interfaces and mediating allosteric communication between the NBD and SBD (36).…”

Analysis of σ 32 mutants defective in chaperone-mediated feedback control reveals unexpected complexity of the heat shock response

“…The following proteins were purified essentially as described: RNA polymerase (38), His-tagged 32 (18), GroEL, GroES (39), DnaK, DnaJ, and GrpE (40). Misfolded proteins were removed from chaperone preparations as described (16).…”