DnaK, a heat-shock protein 70 (Hsp70) homologue in Escherichia coli, possesses a single tryptophan residue in its ATPase domain. Changes in the intrinsic fluorescence of DnaK offer a simple means not only to follow the binding of ATP and of ADP plus the co-chaperone GrpE to the ATPase domain, but also to investigate the kinetics of peptide binding to the substrate-binding domain of ATP·DnaK and GrpE-liganded ADP·DnaK. Addition of ATP or of ADP plus GrpE to nucleotide-free DnaK resulted in a similar decrease in intrinsic fluorescence, indicating similar open conformations of the ATPase domain under these two conditions. Binding of peptide increased the intrinsic fluorescence of both ATP·DnaK and ADP·DnaK·GrpE and rendered their spectra similar to the spectrum of ADP·DnaK with closed conformation of the ATPase domain. These results, together with the differential kinetics of peptide binding to ADP·DnaK on the one hand, and to ATP·DnaK or ADP·DnaK·GrpE on the other, suggest that ligands for either domain, i.e. ATP or ADP plus GrpE for the ATPase domain and peptides for the substrate-binding domain, shift the conformational equilibrium of both domains of DnaK towards the open and closed forms, respectively, in a concerted and parallel manner.
Interdomain communication in the molecular chaperone DnaK
