Investigations of the ATPase Mechanism of HSP70 Molecular Chaperones upon Substrate Binding using Protein Engineering and Computational Techniques

Abstract

Hsp70 chaperones play mportant roles in cells including protein folding, trafficking, degradation and enabling survival under stress conditions. DnaK is an Echerichia coli Hsp70 homolog comprising a 44 kDa ATPase domain (NBD) and a 25 kDa substrate-binding domain (SBD). DnaK has two nucleotide substrate-affinity states: In the ATP-bound low substrate-affinity state, substrate binding and release occur rapidly, whereas in the ADP-bound high substrate-affinity state, slower substrate binding and release kinetics are observed. Communication between the two domains is essential for chaperone function and is mediated via a conserved hydrophobic linker region (384GDVKDVLLL392). Previous studies showed that when this flexible linker interacts with the ATPase domain, which was studied by the construct containing the entire linker, DnaK(1-392), an enhanced ATPase rate is observed compared to the construct lacking the conserved 389VLLL392 linker region, DnaK(1-388). This observation suggests that structural rearrangements caused by linker docking adopt the ATPase domain in a closed conformation, leading to an enhanced, pH-dependent ATPase activity. Here, our aim is to delineate the residues that are responsible for the linker induced conformational rearrangements. In that line, using molecular dynamic simulations we identified two sets of amino acids at the lobe interface of the ATPase domain that might be critical in the stabilization of the domain in the so called “open” and “closed” conformations. We made point mutations for these sites on both DnaK(1-392) and DnaK(1-388) constructs, and studied the structural and functional effects of these residues on the ATPase domain using pH varied stability measurements by circular dichroism and activity measurements as a function of pH, respectively. Our experimental results also point to the significance of these residues in the domain rearrangements when triggered by linker binding.