Ligand-induced estrogen receptor (ER) binding conformations determine biological output, such as gene transcription. The most detailed information about ligand-induced orientation comes from crystal structures. While the information provided by these structures is significant and important, crystal structures are difficult to obtain, do not provide dynamic information about the complex in solution, and are not always predictive of the full pharmacological character of the bound ligand. This research focused on developing a fluorescence-based assay that will be sensitive to differences in resultant ER conformations when bound to ligands of different structure. Botanicals are of specific interest due to their unregulated consumption by women in an attempt to relieve menopausal symptoms but little is known about their mechanism of action on the two estrogen receptor subtypes, ERa and ER�. Nettles et al. have described the crystal structure of various ligand bound ER protein complexes, specifically the partial antagonist, 5,6-bis-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonic acid phenyl ester, (OBHS). The crystal structure of OBHS bound to ERa shows that it pushes on amino acid H524 and distorts the C-terminal end of helix-11; this is likely also to affect helix-12, which is thought of as a molecular switch between the transcriptionally active or inactive forms of the protein complex. Exploiting the perturbation of helix-11 by OBHS, we are able to develop an assay to measure dimer exchange rates with a multitude of botanicals and other ligands of interest, and we report on the kinetic stability of the dimers as well as the ultimate distance between the donor and acceptor fluorophores, which is predictive of the geometry of each individual ER-ligand complex. Thus, this research may elucidate the mechanism of botanical estrogens, as well as other ligands, and lead to further understanding their pharmacology.

TR-FRET assay to monitor ligand-specific estrogen receptor (ER) conformations and dimer kinetic stability

246th National Meeting (2013)

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