Cryo-EM Structures Reveal How Hsp90 and Cochaperones Regulate the Glucocorticoid Receptor

Maintaining protein homeostasis is fundamental for organismal survival. Integral to this process are molecular chaperones, including the highly conserved heat shock protein 90 (Hsp90). Hsp90 associates with many ‘client’ proteins throughout their lifetime, regulating the transition from partially unfolded, inactive states to native, active states. However, a mechanistic understanding of how Hsp90 remodels and regulates client proteins is lacking. One class of clinically important Hsp90 clients are the steroid hormone receptors (SHRs), including the model Hsp90 client, the glucocorticoid receptor (GR). GR is a ligand-activated transcription factor that constantly relies on Hsp90 to maintain its activity. In addition to facilitating folding of GR, Hsp90 regulates multiple steps in the GR activation pathway, including ligand binding, post-translation modifications (PTMs), nuclear translocation, and DNA binding. To perform these regulatory functions, Hsp90 requires the molecular chaperone Hsp70 as well as a collection of cochaperones (p23, Hop, FKBP51, FKBP52, PP5, and others). Using cryo-EM, as well as biochemical and biophysical techniques, we investigated the molecular mechanisms describing how Hsp90, Hsp70, and various cochaperones modify the conformational state of GR and regulate various steps in the GR activation pathway. We present two cryo-EM structures that reveal, for the first time, the mechanism of how Hsp90 folds a client protein. We present the GR-loading complex (GR:Hsp90:Hsp70:Hop), in which GR is stabilized in an inactive, partially unfolded conformation that cannot bind ligand, and the GR-maturation complex (GR:Hsp90:p23), in which GR is restored to a folded, ligand-bound, active state. Together, our structures reveal how Hsp70 and Hsp90 cooperate to regulate the conformational state of a client to directly control the client’s ability to bind ligand for activation. We also present three additional cryo-EM structures (GR:Hsp90:FKBP51, GR:Hsp90:FKBP52, and GR:Hsp90:PP5), which demonstrate how Hsp90 acts a regulatory platform to couple the conformational state of the client protein to distinct client fates through interactions with various cochaperones