Substrate-binding domain conformational dynamics mediate Hsp70 allostery

Binding of ATP to the N-terminal nucleotide-binding domain (NBD) of heat shock protein 70 (Hsp70) molecular chaperones reduces the affinity of their C-terminal substrate-binding domain (SBD) for unfolded protein substrates. ATP binding to the NBD leads to docking between NBD and beta SBD and releasing of the alpha-helical lid that covers the substrate-binding cleft in the SBD. However, these structural changes alone do not fully account for the allosteric mechanism of modulation of substrate affinity and binding kinetics. Through a multipronged study of the Escherichia coli Hsp70 DnaK, we found that changes in conformational dynamics within the beta SBD play a central role in interdomain allosteric communication in the Hsp70 DnaK. ATP-mediated NBD conformational changes favor formation of NBD contacts with lynchpin sites on the beta SBD and force disengagement of SBD strand beta 8 from strand beta 7, which leads to repacking of a beta SBD hydrophobic cluster and disruption of the hydrophobic arch over the substrate-binding cleft. In turn, these structural rearrangements drastically enhance conformational dynamics throughout the entire beta SBD and particularly around the substrate-binding site. This negative, entropically driven allostery between two functional sites of the beta SBD-the NBD binding interface and the substrate-binding site-confers upon the SBD the plasticity needed to bind to a wide range of chaperone clients without compromising precise control of thermodynamics and kinetics of chaperone-client interactions.