Investigating the Residues that are Responsible for PH Dependent Activity of the ATPase Domain of DnaK

Abstract

Hsp70 proteins have essential roles in cells such as de novo folding of newly synthesized proteins, refolding or ubiquitination of denatured proteins, protein tarfficking and translocation through membranes. DnaK, Escherichia coli homolog of Hsp70 molecular chaperone, is comprised an N-terminal ATPase domain (NBD), a C-terminal substrate-binding domain (SBD) and a partially conserved hydrophobic linker that connects the domains. Substrate-binding affinities on SBD are driven by ATP-ADP conversion cycles in NBD. Allosteric communication between the two domains is provided by the conserved 389VLLL392 sequence on the linker region. Previous studies done using truncated DnaK(1-392) construct, containing the 389VLLL392 sequence, showed a pH-dependent enhanced ATPase activity, similar to the substrate-stimulated activity of the full-length protein; whereas construct lacking this sequence, DnaK(1-388) showed an activity resembling to the unstimulated-form of full-length. In the same study, it was proposed that linker binding to the ATPase domain causes a change in the slow step of the ATP hydrolysis reaction by rearranging the ATPase domain to a conformation where ADP release becomes the rate-limiting step. Here, we are investigating the molecular details of the reason of pH dependence and enhanced ATPase activity upon linker interactions with the domain using ATPase domain constructs both in in vitro and in silico. We observed with molecular dynamic simulations significant upshift in the pKa values of Asp194 and Asp201 compared to their expected pKa values as negatively charged residues, and it seems like that protonation states of these residues at different nucleotide-bound forms are important in the linker derived conformational changes leading, speculatively, variations in the Pi and ADP affinities. Our results will be discussed with overall ATPase rate as well as ADP off-rate measurements on DnaK(1-388) and DnaK(1-392) constructs.