PROJECT SUMMARY/ABSTRACT Maintaining protein homeostasis is fundamental for organismal survival. Integral to this process are molecular chaperones, including the highly abundant and evolutionary conserved heat shock protein 90 (Hsp90), which facilitates the folding of hundreds of `client' proteins. Hsp90 clients are enriched in signaling molecules, such as kinases and transcription factors, which regulate cell growth and survival. Consequently, many Hsp90 clients are oncoproteins, making Hsp90 an important pharmaceutical target for cancer with Hsp90 inhibitors currently in clinical oncology trials. However, a mechanistic understanding of how Hsp90 remodels client proteins is lacking and precludes further advancements in Hsp90-targeted cancer therapies. One class of clinically important Hsp90 clients are the steroid hormone receptors (SHRs), steroid-activated transcription factors that control cell growth and development and are potent therapeutic targets for cancer. One SHR, the glucocorticoid receptor (GR), is a model Hsp90 client that goes through a `chaperone cycle', where GR binds to Hsp90, Hsp70, and a variety of co-chaperones to maintain its activity. Multiple aspects of GR function are regulated by Hsp90, including ligand binding, nuclear translocation, and chromatin binding—all essential steps in GR-dependent gene expression regulation. Understanding the mechanism by which Hsp90 regulates GR will elucidate how Hsp90 influences a myriad of clinically important signaling pathways, advancing efforts to target this master regulator for cancer therapies. To investigate how Hsp90 refolds and reactivates GR, I determined the cryo-EM structure of the native, active GR ligand binding domain (LBD) bound to Hsp90, revealing, for the first time, the mechanism of Hsp90-mediated conformational remodeling of a client. I will build on the knowledge from my structure to determine how Hsp90 regulates GR functions downstream of ligand binding. Aim 1 will determine how Hsp90 regulates GR nuclear translocation with the aid of the Hsp90 co-chaperones FK506 binding protein 51 (FKBP51) and FKBP52, which regulate GR nuclear translocation by connecting to dynein. Using cryo-EM as well as in vitro and in vivo biochemical assays, I will determine how the FKBPs incorporate with the GR-chaperone cycle, influence GR conformation, and connect GR:Hsp90 to dynein for nuclear translocation. Aim 2 will investigate how Hsp90 regulates GR binding to DNA and chromatin. Previous studies have suggested Hsp90 is inhibitory to GR DNA and chromatin binding, but the mechanism of inhibition is unknown. Using the first recombinantly purified multidomain GR, containing both the LBD and DNA binding domain (DBD), I will determine how the GR chaperone cycle modulates GR binding to DNA and chromatin substrates using cryo-EM and in vitro biochemical binding assays. This project encompasses structural biology, biochemistry, and cell-based assays, while bridging the protein folding and chrom...