ROLE OF THE J-DOMAIN IN THE COOPERATION OF HSP40 WITH HSP70

The Escherichia coli Hsp40 DnaJ and Hsp70 DnaK cooperate in the bindin g of proteins at intermediate stages of folding, assembly, and translo cation across membranes. Binding of protein substrates to the DnaK C-t erminal domain is controlled by ATP binding and hydrolysis in the N-te rminal ATPase domain. The interaction of DnaJ with DnaK is mediated at least in part by the highly conserved N-terminal J-domain of DnaJ tha t includes residues 2-75. Heteronuclear NMR experiments with uniformly N-15-enriched DnaJ2-75 indicate that the chemical environment of resi dues located in helix II and the flanking loops is perturbed on intera ction with DnaK or a truncated DnaK molecule, DnaK2-388. NMR signals c orresponding to these residues broaden and exhibit changes in chemical shifts in the presence of DnaK(MgADP). Addition of MgATP largely reve rsed the broadening, indicating that NMR signals of DnaJ2-75 respond t o ATP-dependent changes in DnaK. The J-domain interaction is localized to the ATPase domain of DnaK and is likely to be dominated by electro static interactions. The results suggest that the J-domain tethers Dna K to DnaJ-bound substrates, which DnaK then binds with its C-terminal peptide-binding domain.