Summary: Steroid hormones receptors (SR) are ligand-dependent nuclear transcription factors that exhibit remarkable functional diversity in mediating cell/tissue and target gene specific responses, largely driven by conformational dynamics of the SR protein that enables it's binding of unique subsets of transcriptional co- regulatory proteins (CoRs) and DNA response elements. The progesterone receptor (PR) is the main target of progestogens that are widely used clinically. PR is expressed as two protein isoforms, an N-terminal truncated PR-A and full-length PR-B and each have distinct physiological roles dependent on the cell/tissue type. In general PR-A is a weaker transcriptional activator than PR-B, and can act to attenuate the activity of PR-B. Both isoforms are typically co-expressed in equal proportions in most normal tissues. However, PR-A to PR-B ratios have been reported to be highly variable in pathological conditions. Mechanistic basis for differences in activity of the isoforms is not well defined but is generally believed to be due to unknown differences in structural conformations. Thus, to fully understand PRs' biology requires determination of a high-resolution structure of the full-length PR isoforms and associated CoRs as a complex on target DNA and an understanding of how protein interactions within the complex and structural conformations affect activity of PR. The conformational flexibility of SRs and CoRs, coupled and their large sizes (100–300 MW), make them unsuitable to either high resolution NMR or X-ray crystallography analysis. As an alternative, this proposal will integrate complementary solution- phase techniques to determine high-resolution 3D structural models and uncover the conformational dynamics within the PR:CoR/DNA complex. Recent advances in Cryo-EM enable the determination of solution-phase structures of large conformationally heterogenous macromolecular complexes at subnanometer resolution. We will combine Cryo-EM with crosslinking mass spectrometry (XL-MS) to further refine structural Cryo-EM models and assure high resolution and with hydrogen-deuterium exchange (HDX) to map conformational dynamics and allostery within the PR:CoR/DNA complex. The overall goal of this project is to determine the highest resolution 3D structure possible of full-length PR-A and PR-B in complex with classical CoRs and novel CoRs on PR DNA response elements. Aim 1 will utilize Cryo-EM to analyze the structural features of PR-A and PR-B in complex with the classical CoRs SRC3 and p300 and with the novel CoRs TBP and JDP2 assembled on target DNA. Aim 2 will refine the Cryo-EM structure of PR:CoR/DNA complex using integrated structural modeling and XL- MS to define distance constraints and probe conformational dynamics within the PR complex by differential HDX. Aim 3 will perform functional mutagenesis studies to determine the influence of PR:SRC3/p300 interaction surfaces revealed in structural models and from XL-MS data have on...