Heat shock protein 70 kDa (Hsp70) plays a central role in maintaining protein homeostasis. It cooperates with cochaperone Hsp40, which stimulates Hsp70 ATPase activity and presents protein substrates to Hsp70 to assist refolding. The mechanism by which Hsp40 regulates the intramolecular and intermolecular changes of Hsp70 is still largely unknown. Here, by bulk and single-molecule FRET, we report the conformational dynamics of Hsp70 and its regulation by Hsp40 as well as the kinetics of the multistep Hsp70–Hsp40 functional cycle. We show that Hsp40 modulates the conformations of ATP-bound Hsp70 to a domain-undocked ATPase-stimulated state, and facilitates the formation of a heterotetrameric Hsp70–Hsp40 complex. Our findings provide insights into the functional mechanism of this core chaperone machinery.
Hsp70 is a conserved molecular chaperone that plays an indispensable role in regulating protein folding, translocation, and degradation. The conformational dynamics of Hsp70 and its regulation by cochaperones are vital to its function. Using bulk and single-molecule fluorescence resonance energy transfer (smFRET) techniques, we studied the interdomain conformational distribution of human stress-inducible Hsp70A1 and the kinetics of conformational changes induced by nucleotide and the Hsp40 cochaperone Hdj1. We found that the conformations between and within the nucleotide- and substrate-binding domains show heterogeneity. The conformational distribution in the ATP-bound state can be induced by Hdj1 to form an “ADP-like” undocked conformation, which is an ATPase-stimulated state. Kinetic measurements indicate that Hdj1 binds to monomeric Hsp70 as the first step, then induces undocking of the two domains and closing of the substrate-binding cleft. Dimeric Hdj1 then facilitates dimerization of Hsp70 and formation of a heterotetrameric Hsp70–Hsp40 complex. Our results provide a kinetic view of the conformational cycle of Hsp70 and reveal the importance of the dynamic nature of Hsp70 for its function.