Role of Hsp70 chaperone in client-protein folding elucidated by Markov state modeling and NMR restraint-assisted molecular dynamics simulations.

Abstract

Heat shock protein 70 (Hsp70) is a molecular chaperone that plays a key role in cellular processes by assisting protein folding and preventing aggregation. During client-protein folding, Hsp70 undergoes an ATP-dependent chaperone cycle involving the opening and closing of a flexible lid. Although the open-lid and closed-lid states of Hsp70 have been studied extensively, the specific role of the lid upon its interaction with client proteins remains unclear. In this study, we generated a Markov state model from coarse-grained molecular dynamics (MD) simulations of Hsp70 spanning from open-lid to closed-lid states and sampling a flexible lid-domain conformational ensemble. Starting from metastable Hsp70 conformations with varying degrees of lid opening, we performed nuclear magnetic resonance distance restraint-assisted all-atom MD simulations in explicit solvent to investigate the folding of the SH3 client protein bound to nucleotide-free Hsp70. All-atom MD simulations were performed with SH3 bound to and released from Hsp70, with nuclear magnetic resonance restraints applied to guide SH3 folding. Our results show that SH3 folds more effectively after having sampled conformational space within the closed-lid state of Hsp70. Further analysis reveals that the closed-lid state of Hsp70 interacts with SH3 via specific and highly conserved nonpolar residues, preventing the nonnative hydrophobic collapse of the SH3 client upon release from the chaperone. This study provides insights into specific atomic-level interactions that can be targeted by future experiments to better understand the molecular mechanism of Hsp70-assisted protein folding.