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Collaboration and direct interaction between Hsp90 and Hsp70 of E. coli

Thursday, September 15, 2016 — Poster Session III

3:30 p.m. – 5:00 p.m.
FAES Terrace
NCI
MOLBIO-3

Authors

  • AN Kravats
  • SM Doyle
  • JR Hoskins
  • O Genest
  • S Wickner

Abstract

The 90-kDa heat shock protein (Hsp90) family is a highly conserved and ubiquitous class of ATP dependent molecular chaperones found in nearly all organisms. Hsp90 functions with the Hsp70 chaperone and numerous cochaperones to remodel hundreds of client proteins, many which are linked to cancer. In E. coli, Hsp90 and DnaK (Hsp70 homolog) directly interact and function synergistically. We identified a region in the middle domain of E. coli Hsp90 important for collaboration with DnaK by randomly mutagenizing Hsp90 and screening for a loss of interaction with DnaK in vivo using a bacterial two-hybrid assay. Mutants defective in interaction were purified and tested in vitro. The Hsp90 mutants were defective in collaborating synergistically with DnaK to reactivate heat-denatured luciferase, a model substrate. In pull down assays, wild type Hsp90 directly interacted with DnaK while Hsp90 mutants were defective in direct interactions. We used molecular modeling to further explore what region of DnaK interacts with Hsp90 by docking their full-length crystal structures. The top docking model predicted that a region in the nucleotide binding domain of DnaK interacts with the experimentally identified region of Hsp90. We made DnaK mutants in this region and tested them in vitro and in vivo. In bacterial two-hybrid assays, the DnaK mutants were defective in interaction with Hsp90. In vitro the DnaK mutants were defective in direct physical interactions and also showed decreased ability to reactivate heat-denatured luciferase. Together these results provide insight into the mechanism by which DnaK regulates the chaperone activity of Hsp90.

Category: Molecular Biology and Biochemistry