The human estrogen receptor alpha dimer binds a single SRC-1 coactivator molecule with an affinity dictated by agonist structure

Nuclear receptors act as ligand-inducible transcription factors. Agonist bi nding leads to interaction with coactivator proteins, and to the assembly o f the general transcription machinery. In addition to structural informatio n, a thorough understanding of transcriptional activation by the nuclear re ceptors requires the characterization of the thermodynamic parameters gover ning these protein/protein interactions. In this study we have quantitative ly characterized the interactions of full-length baculovirus expressed huma n estrogen receptor alpha (ER alpha), as well as ER alpha hormone binding d omain (ERHBD) with a fragment of the coactivator protein SRC-1 (amino acid residues 570 to 780). Fluorescence anisotropy and fluorescence correlation spectroscopy of fluorescently labeled SRC-1(570-780) demonstrate unambiguou sly that the stoichiometry of the SRC-1/ER alpha /estradiol complex is one coactivator molecule per ER alpha dimer. The affinity of the estradiol or e striol bound ER alpha /SRC-1 complexes was found to be significantly higher than that observed in the presence of estrone. No binding was observed in the absence of ligand or in the presence of antagonists. Distinct anisotrop y values for the ER alpha -SRC-1 complexes with different agonists suggest distinct conformations of the complexes depending upon agonist structure. ( C) 2001 Academic Press.