Strategies that inform on protein structural dynamics in native environments enhance our understanding of protein function and facilitate the development of new therapies to treat disease. The human estrogen receptor α (ERα) is a ligand-mediated transcription factor that is modulated by the steroid hormone 17β-estradiol (E2). Through its interaction with E2, the ERα regulates a wide-range of cellular processes including growth, differentiation, transcription and apoptosis. Structurally, the ERα ligand-binding domain (ERα-LBD) adopts a well-folded, globular conformation comprised of 11 α-helices and a short β-strand. The globular portion of the ERα-LBD is believed to be relatively static; however, the C-terminal helix 12 (H12) is dynamic and can adopt different configurations depending on what ligand occupies the LBD. For example, binding of E2 causes H12 to associate tightly with the LBD in an ‘active’ conformation that can bind transcriptional co-activators. Alternatively, binding of the selective ERα modulator tamoxifen forces H12 to associate with the coactivator-binding site of the LBD in an ‘inactive’ conformation that prevents transcriptional activation. In this study, we applied fluorescent labeling coupled with bipartite tetracysteine display to monitor ligand-mediated H12 transitions within the ERα-LBD<i>.</i> Wild-type ERα-LBDs were mutated to express a tetracysteine (C4) motif that binds a biarsenical pro-fluorescent dye (FlAsH) upon transition of H12 to a folded state. Surprisingly, binding of E2 was accompanied by a reduction in FlAsH fluorescence, indicating that the C4 motif becomes occluded when H12 is tightly associated with the LBD. Our results also show that H12 remains flexible in unliganded receptors and receptors bound to pure ERα antagonists, allowing for significant increases in FlAsH fluorescence. Such observations made it possible to determine association rates and equilibrium binding constants for FlAsH to the ERα-LBD in the presence and absence of estrogenic ligands. We anticipate that this assay will be useful for studying how estrogenic compounds influence structural dynamics of the ERα-LBD and may be applied to studying helix transitions in other members of the nuclear receptor family.

Monitoring ligand-mediated helix transitions within the human estrogen receptor α using bipartite tetracysteine display

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