Albany 2015:Book of Abstracts
June 9-13 2015
©Adenine Press (2012)
A region in the middle domain of E. coli Hsp90 is important for collaboration with DnaK
Molecular chaperones are critical members of the cellular protein quality control system that use the energy from ATP hydrolysis to assist in protein remodeling activities. Heat shock protein 90 (Hsp90) is a widely conserved and highly abundant molecular chaperone that assists in the folding and reactivation of a diverse set of client proteins. Since many of these client proteins have been linked to cancer, inhibition of Hsp90 is of interest for cancer therapy. Hsp90 assembles as a highly flexible homodimer and undergoes large-scale structural rearrangements due to ATP binding and hydrolysis in order to remodel client proteins at various stages of folding. Several cochaperones have also been shown to interact with Hsp90 to modulate ATPase activity.
In E. coli, the DnaK chaperone system (homologous to the eukaryotic Hsp70 system) has been shown to collaborate and directly interact with Hsp90 (Hsp90Ec )(Genest et al. 2011). We identified several residues of Hsp90Ec that are important for interaction with DnaK by making random substitutions in Hsp90Ec and screening for loss of interaction with DnaK by using a bacterial two hybrid assay. Additional mutants in nearby surface exposed residues were also constructed. The Hsp90Ec variants were purified and tested in vitro for ATPase activity and client protein remodeling activity in collaboration with the DnaK system. Our results indicate that a surface exposed region on the middle domain of Hsp90Ec is important for collaboration with DnaK. In order to determine whether this region is functionally conserved, we made a homologous substitution in yeast Hsp90 (Hsp82). The wild type and mutant proteins were purified and compared in protein reactivation assays in vitro. They were also tested in ATPase assays in the absence and presence of several yeast co-chaperones and client proteins. The results indicate a lower rate of client reactivation by the Hsp82 mutant. The mutant also exhibited defective ATPase activity in the presence of some cochaperones, suggesting this region is involved in an important protein-protein interaction or a conformational change. We are currently using molecular modeling to further explore the potential direct interaction between DnaK and the middle domain region of Hsp90Ec .
O. Genest, J. Hoskins, J. Camberg, S.M. Doyle, and S. Wickner. (2011). Heat shock protein 90 from Escherichia coli collaborates with the DnaK chaperone system in client protein remodeling. Proc Natl Acad Sci USA 108, 8206-8211.