Probing Conformational Changes in the Estrogen Receptor: Evidence for a Partially Unfolded Intermediate Facilitating Ligand Binding and Release

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Abstract
Because the ligand bound to the ligand-binding domain (LBD) of nuclear hormone receptors is completely enveloped by protein, it is thought that the process of ligand binding or unbinding must involve a significant conformational change of this domain. We have used the intrinsic tryptophan flu- orescence of the estrogen receptor-a (ERa )o r es- trogen receptor-b (ERb) LBD, as well as bis-anili- nonaphthalenesulfonate (bis-ANS), a probe for accessible interior regions of protein, to follow the guanidine-hydrochloride (Gua-HCl)-induced un- folding of this domain. In both cases, we find that the ER-LBD unfolding follows a two-phase pro- cess. At low Gua-HCl, the ER-LBD undergoes par- tial unfolding, whereas at high Gua-HCl, this do- main undergoes a global unfolding, with bis-ANS binding preferentially to the partially unfolded state. The partially unfolded state of the ERa-LBD induced by denaturant does not bind ligand stably, but it may resemble an intermediate that this do- main accesses transiently under native conditions that allow ligands to enter or exit the ligand-bind- ing pocket. (Molecular Endocrinology 15: 421-428, 2001)

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