Estrogen receptors (ERα and ERβ) are ligand-activated nuclear receptors that mediate the action of estrogens. These receptors activate transcription by similar mechanism(s), although the overall amino acid sequence identity is only 47%. In order to compare the structural and conformational features of ERα and ERβ, we monitored their intrinsic tryptophan fluorescence during thermal unfolding. The 50% unfolding temperatures (TM) of ERα and ERβ were 39±1 and 40±2°C, respectively. Estradiol had no significant effect on the TM of ERα or ERβ. In contrast, binding of the estrogen-response element increased the TM of ERα and ERβ by 10 °C. Thermal unfolding of estradiol-bound ERα and ligand-free ERβ showed two-step transitions, with the formation of intermediates that were stable between 36-48 and 34-42°C, respectively. We confirmed the presence of intermediate states during thermal unfolding by circular dichroism spectroscopy. Atomic force microscopy showed that the ERβ intermediate consisted of discrete globular particles, whereas the ERα intermediate showed a speckled appearance, with sparse well-defined particles. Fluorescence-quenching studies showed the presence of two classes of tryptophan in unliganded ERα and ERβ. Binding of estradiol to ERβ exposed its tryptophans, whereas estradiol reduced the accessibility of the tryptophans of ERα. Our results illustrate the differential effects of ligands on the unfolding of ERα and ERβ, and identify partially unfolded intermediates. Differences in the conformational flexibility and stability of ERα and ERβ may represent functional differences of ligand-bound ERs in recruiting coactivator proteins and initiating transcription.
All Science Journal Classification (ASJC) codes
- Molecular Biology