(492.7) Structural and Biophysical Mechanisms Driving Differential Hormone Response in Steroid Receptors

Poster Board Number: A135

Sabab Khan (Pennsylvania State University, University Park), Sean Braet (Pennsylvania State University, University Park), Stephen Koehler (Pennsylvania State University, University Park), Elizabeth Elacqua (Pennsylvania State University, University Park), Ganesh Anand (Pennsylvania State University, University Park), Denise Okafor (Pennsylvania State University, University Park, Pennsylvania State University, University Park)

Steroid receptors (SRs) belong to the superfamily of ligand-activated transcription factors that regulate a myriad of biological processes upon binding of cholesterol-derived hormones. A poorly understood aspect of this transcriptional regulation is how diverse steroid hormones modulate the ligand binding domain (LBD) of steroid receptors to achieve selective transcriptional outcomes. Here we investigate hormone specificity in the ancestral steroid receptor 2 (ancSR2) which is transcriptionally activated by 3-ketosteroid hormones and unresponsive to 3-hydroxylated, A-ring aromatized steroids (i.e., estrogens). Previous work uncovered evolutionarily conserved ligand sensing-residues in the binding pocket of LBD which also enables hormone discrimination. Specifically, the L42 (Helix3)-M75 (Helix5) interaction was predicted to enable discrimination between 3-ketosteroids and estrogens. To further probe the role of these ligand sensors in mediating differential hormone responses, we mutated M75 to modulate the Helix3-Helix5 interaction and determined how transcriptional function, protein structure and dynamics are impacted. Specifically, we have combined site-directed mutagenesis with biophysical experiments, cell-based assays, and molecular dynamics (MD) simulations. While our secondary structure measurements reveal minor impacts on structure, MD simulations reveal that M75 mutations modulate the status of the H3-H5 interaction, subsequently affecting ancSR2 conformations in a hormone-dependent manner. Additionally, hydrogen deuterium exchange-mass spectrometry (HDX-MS) measurements reveal conformational effects at regions distant from the mutation site that may modulate ancSR2 inter-residue interaction networks for differential hormone response. The differential hormone response is supported in luciferase reporter assays as well as ligand binding assays. These studies will provide biophysical and structural insight into how steroid receptors achieve hormone-specific transcriptional responses.

CDO thanks Burroughs Wellcome Fund Career Award