Abstact

Hsp70 and Hsp40 molecular chaperones form a central machinery that remodels client proteins involved in numerous biological processes. Here, we integrated cryo-electron microscopy and nuclear magnetic resonance spectroscopy to determine the architecture of the full-length Hsp70-Hsp40 machinery. The structure of the complex in a physiologically inhibited state reveals distinct regulatory mechanisms. In the active state, the Hsp40 glycine-phenylalanine (G/F)-rich region acts as a pseudo-substrate for Hsp70, directly modulating refolding. This region also maintains Hsp40 in an autoinhibited state; upon binding to Hsp70, the inhibition is disrupted, exposing a cryptic client-binding site that enables client engagement and refolding. Transitions between these states are central to controlling refolding efficiency. Disrupting either the autoinhibited state or the G/F-Hsp70 interaction impairs function and elicits a compensatory heat shock response in cells. Our findings uncover the regulatory dynamics of a fundamental chaperone system, with broad implications for understanding protein homeostasis and the cellular response to stress.