The long-term goal of this proposal is to elucidate the molecular basis for the ligand-dependent activation of nuclear receptors (NRs). NRs are involved in regulation of gene transcription in development, reproduction, homeostasis and metabolism. NRs have a common domain structure consisting of domain A-F, and the ligand-binding domain (LBD; E-domain) is responsible for ligand-dependent activation. We will use the human estrogen receptor (ER) as a model system. ER has two subtypes, alpha and beta, and ERalpha is a therapeutic target of estrogen-responsive breast tumors. Antiestrogens competitively bind to ERalpha-LBD and induce an inactive conformation, thereby inhibiting the ER function. A series of crystal structures of ER-LBD have dramatically enhanced our understanding of the mechanism of ER function, but there are still important issues that need to be addressed. These include (1) classification of LBD conformations induced by different ligands and mutations, (2) roles of conformational dynamics in the response of ER-LBD to its ligands, (3) effects of the other domains of ER and enhancer DNA elements on the conformational changes of the LBD. The lack of studies of the solution conformation of ER, mainly due to its large size, has hindered progress in these areas. We will employ an innovative approach to address these issues. We will engineer novel, antibody-like binding proteins, termed "monobodies", that recognize specific ER conformations. Monobodies are small and stable, and function inside the cell because they lack disulfide bonds. We will generate combinatorial libraries of monobodies and select monobodies that specifically bind to a given conformation of ER using a yeast two-hybrid system. The specific aims of this project are (1) to continue engineering monobodies that bind to ER, (2) to characterize interactions of monobodies with ER using biochemical methods, (3) to characterize the conformational changes of the ER E and F domains (ER-EF) induced by its ligands and mutations, using a collection of monobodies, (4) to determine the effects of other domains on the conformations of ER-EF, and (5) to determine the crystal structures of monobody-ER complexes. Our unique approach will make it possible to probe conformational changes of ER in the yeast nucleus and to correlate them with high-resolution crystal structures. We expect that results from this project will fill a large gap that presently exists between out knowledge gained from the static