Structural and mechanistic analyses reveal collaborative regulation of HSF1 by the Hsp70-Hsp90 chaperone systems

Heat shock factor 1 (HSF1) is the master transcriptional regulator of cellular response to disrupted cytosolic protein homeostasis. Temperature change, oxidation, and other stresses drive the trimerization and activation of HSF1 to induce expression of molecular chaperones, such as heat shock proteins Hsp70 and Hsp90, which sit at the center of cellular proteostatic networks. In turn, the HSPs and co-chaperones regulate HSF1, but mechanistic details of this cycle remain largely unknown. We developed a FRET-based approach to simultaneously monitor HSF1 conformational change and oligomeric state throughout activation and inactivation. By reconstituting Hsp-HSF1 interactions in vitro, we find that monomerization of HSF1 resembles fibril disassembly through coordinated Hsp40-Hsp70 activity. We then used site-specific photocrosslinking to track HSF1 loading into Hsp90 complexes, Hsp90 cycling, and stress-induced shifts in Hsp90-HSF1 interactions. Whereas Hsp90 inhibitors force 'loading state' type Hsp90-HSF1 interactions, heat shock promotes faster Hsp90 cycling. In this reconstituted system, HSF1-Hsp90 interactions are unexpectedly strongly dependent on the co-chaperone HOP, in contrast to canonical Hsp90 clients. Combining cryo-EM structures of Hsp90-HSF1 loading state and maturation state complexes with crosslinking mass spectroscopy and biophysical experiments, we show that Hsp90 holds HSF1 in a pre-activated, extended monomer state that is primed for trimerization. We propose this state both enhances responsivity but also promotes cytoplasmic-nuclear shuttling through exposure of the NLS. Notably, Hsp90-bound HSF1 can trimerize and bind DNA, placing it on-pathway for transcriptional activation. Together, our results unify previously contradictory view on Hsp90's role in HSF1 regulation. The integrated combination of in vitro reconstitution, photocrosslinking, cryoEM and MS is an exciting new paradigm for the study of many dynamic systems including other complex proteostasis components.