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

Nuclear receptors act as ligand-inducible transcription factors. Agonist binding leads to interaction with coactivator proteins, and to the assembly of the general transcription machinery. In addition to structural information, a thorough understanding of transcriptional activation by the nuclear receptors requires the characterization of the thermodynamic parameters governing these protein/protein interactions. In this study we have quantitatively characterized the interactions of full-length baculovirus expressed human estrogen receptor α (ERα), as well as ERα hormone binding domain (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 unambiguously that the stoichiometry of the SRC-1/ERα/estradiol complex is one coactivator molecule per ERα dimer. The affinity of the estradiol or estriol bound ERα/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 anisotropy values for the ERα-SRC-1 complexes with different agonists suggest distinct conformations of the complexes depending upon agonist structure.