Interaction of E. coli Hsp90 with DnaK Involves the DnaJ Binding Region of DnaK

The 90-kDa heat shock protein (Hsp90) is a widely conserved and ubiquitous molecular chaperone that participates in ATP-dependent protein remodeling in both eukaryotes and prokaryotes. It functions in conjunction with Hsp70 and the Hsp70 cochaperones, an Hsp40 (J-protein) and a nucleotide exchange factor. In Escherichia coli, the functional collaboration between Hsp90_Ec and Hsp70, DnaK, requires that the two chaperones directly interact. We used molecular docking to model the interaction of Hsp90_Ec and DnaK. The top-ranked docked model predicted that a region in the nucleotide-binding domain (NBD) of DnaK interacted with a region in the middle domain of Hsp90_Ec. We then made substitution mutants in DnaK residues suggested by the model to interact with Hsp90_Ec. Of the 12 mutants tested, 11 were defective or partially defective in their ability to interact with Hsp90_Ecin vivo in a bacterial two-hybrid assay and in vitro in a bio-layer interferometry assay. These DnaK mutants were also defective in their ability to function collaboratively in protein remodeling with Hsp90_Ec but retained the ability to act with DnaK cochaperones. Taken together, these results suggest that a specific region in the NBD of DnaK is involved in the interaction with Hsp90_Ec, and this interaction is functionally important. Moreover, the region of DnaK that we found to be necessary for Hsp90_Ec binding includes residues that are also involved in J-protein binding, suggesting a functional interplay among DnaK, DnaK cochaperones, and Hsp90_Ec.