Perturbation Response Scanning Reveals Key Residues for Allosteric Control in Hsp70.

Abstract

Hsp70 molecular chaperones play an important role in maintaining cellular homeostasis, and are implicated in a wide array of cellular processes including protein recovery from aggregates, cross membrane protein translocation, and protein biogenesis. Hsp70 consists of two domains, a nucleotide binding domain (NBD) and a substrate binding domain (SBD), each of which communicates via an allosteric mechanism such that the protein interconverts between two functional states, an ATP bound open conformation and an ADP bound closed conformation. The exact mechanism for interstate conversion is not as yet fully understood. However, the ligand bound state of the NBD and SBD as well as interactions with co-chaperones such as DnaJ and nucleotide exchange factor (NEF) are thought to play crucial regulatory roles. In this study, we apply Perturbation Response Scanning (PRS) method in combination with molecular dynamics simulations as a computational tool for the identification of allosteric hot residues in the large multi-domain Hsp70 protein. We find evidence in support of the hypothesis that substrate binding triggers ATP hydrolysis, and that the ADP-substrate complex favours interstate conversion to the closed state. Furthermore, our data is in agreement with the proposal of there being an allosterically active intermediate state between the open and closed states and vice versa, as we find evidence that ATP binding to the closed structure and peptide binding to the open structure allosterically "activates" the respective complexes. We conclude our analysis by showing how our PRS data fits the current opinion for Hsp70's conformational cycle and present several allosteric hot residues that may provide a platform for further studies to gain additional insight into Hsp70 allostery.