title: Interaction of E. coli Hsp90 with DnaK Involves the DnaJ Binding Region of DnaK.
abstract: 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 Hsp90Ecand Hsp70, DnaK, requires that the two chaperones directly interact. We used molecular docking to model the interaction of Hsp90Ecand 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 Hsp90Ec. We then made substitution mutants in DnaK residues suggested by the model to interact with Hsp90Ec. Of the 12 mutants tested, 11 were defective or partially defective in their ability to interact with Hsp90Ecin 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 Hsp90Ecbut 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 Hsp90Ec, and this interaction is functionally important. Moreover, the region of DnaK that we found to be necessary for Hsp90Ecbinding includes residues that are also involved in J-protein binding, suggesting a functional interplay among DnaK, DnaK cochaperones, and Hsp90Ec.
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Andrea N
authors.initials: AN
authors.lastname: Kravats
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: doyles@mail.nih.gov.
authors.forename: Shannon M
authors.initials: SM
authors.lastname: Doyle
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Joel R
authors.initials: JR
authors.lastname: Hoskins
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Olivier
authors.initials: O
authors.lastname: Genest
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Erin
authors.initials: E
authors.lastname: Doody
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: wickners@mail.nih.gov.
authors.forename: Sue
authors.initials: S
authors.lastname: Wickner
mesh_terms: D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D062105:Molecular Docking Simulation; D011485:Protein Binding; D025941:Protein Interaction Mapping; D020798:Two-Hybrid System Techniques
chemical_list: D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; C489352:HtpG protein, E coli; C063842:dnaK protein, E coli
keywords: CbpA; Hsp40; HtpG; molecular chaperone; protein remodeling