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 states of the NBD and SBD as well as interactions with cochaperones such as DnaJ and nucleotide exchange factor are thought to play crucial regulatory roles. In this study, we apply the 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 multidomain Hsp70 protein. We find evidence in support of the hypothesis that substrate binding triggers ATP hydrolysis and that the ADP-substrate complex favors interstate conversion to the closed state. Furthermore, our data are in agreement with the proposal that there is 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 "activate" the respective complexes. We conclude our analysis by showing how our PRS data fit the current opinion on the Hsp70 conformational cycle and present several allosteric hot residues that may provide a platform for further studies to gain additional insight into Hsp70 allostery.

Unraveling the Motions behind Enterovirus 71 Uncoating

“…More specifically, PRS determines single residues that play a role in activating the conformational change in proteins. The method has previously been used to locate allosteric regions (47,48) and explore protein-ligand interactions (49). PRS has also been applied to an enzyme in complex with its inhibitor to map sites responsible for inhibited conformations (50).…”

The Hsp70 interdomain linker is a dynamic switch that enables allosteric communication between two structured domains

“…Their approach identified several candidate residue interaction patterns that may be important in allostery. Finally, Penkler et al (15) used MD to study structural perturbations brought about by the binding and unbinding of nucleotides and substrates. They report the residues that undergo the largest, induced structural perturbations and infer that these are probably critical to allostery.…”

Allosteric Modulation of Conformational Dynamics in Human Hsp90α: A Computational Study

“…Perturbation response scanning (PRS) is a computational technique used to predict the relative response of all residues in a protein, to an external force perturbation, such as ligand binding, at a single residue. The theory of PRS has been thoroughly described in previous studies (Atilgan & (1) Atilgan, 2009;Atilgan et al, 2010;Penkler et al, 2017); the algorithm is available in the MD-TASK software suite (Brown et al, 2017a). In brief, PRS utilizes linear response theory (LRT) to approximate the shift in coordinates (R 1 ) of a given protein conformation (R 0 ) in response to a perturbation of the Hamiltonian (Yilmaz & Atilgan, 2000;Ikeguchi et al, 2005;Atilgan & Atilgan, 2009).…”

“…In each case only the maximum C i is recorded for each residue. We have previously discussed and shown the reproducibility of this approach with large multi-domain proteins with complex rotational and translational conformational restructuring (Penkler et al, 2017).…”

“…PRS is a technique used to assess the allosteric influence each residue has on all other residues when externally perturbed. Several previous studies have demonstrated the use of PRS as an effective approach for the identification of functionally important sites that are potentially involved in modulating binding-region motions (Atilgan & Atilgan, 2009;Atilgan et al, 2010;General et al, 2014;Abdizadeh et al, 2015;Abdizadeh & Atilgan, 2016;Penkler et al, 2017), and PRS analysis has been previously shown to be an efficient method for determining the allosteric potential of large multi-domain proteins (General et al, 2014;Penkler et al, 2017). Briefly, PRS uses linear response theory to predict whole protein displacements in response to the insertion of external forces at single residue sites (see equation (2).…”