Molecular basis and dual ligand regulation of tetrameric estrogen receptor a/14-3-3ζ protein complex.

Therapeutic strategies targeting nuclear receptors (NRs) beyond their endogenous ligand binding pocket have gained significant scientific interest driven by a need to circumvent problems associated with drug resistance and pharmacological profile. The hub protein 14-3-3 is an endogenous regulator of various NRs, providing a novel entry point for small molecule modulation of NR activity. Exemplified, 14-3-3 binding to the C-terminal F-domain of the estrogen receptor alpha (ERa), and small molecule stabilization of the ERa/14-3-3ζ protein complex by the natural product Fusicoccin A (FC-A), was demonstrated to downregulate ERa-mediated breast cancer proliferation. This presents a novel drug discovery approach to target ERa; however, structural and mechanistic insights into ERa/14-3-3 complex formation are lacking. Here, we provide an in-depth molecular understanding of the ERa/14-3-3ζ complex by isolating 14-3-3ζ in complex with an ERa protein construct comprising its ligand-binding domain (LBD) and phosphorylated F-domain. Bacterial co-expression and copurification of the ERa/14-3-3ζ complex, followed by extensive biophysical and structural characterization, revealed a tetrameric complex between the ERa homodimer and the 14-3-3ζ homodimer. 14-3-3ζ binding to ERa, and ERa/14-3-3ζ complex stabilization by FC-A, appeared to be orthogonal to ERa endogenous agonist (E2) binding, E2-induced conformational changes, and cofactor recruitment. Similarly, the ERa antagonist 4-hydroxytamoxifen inhibited cofactor recruitment to the ERa LBD while ERa was bound to 14-3-3ζ. Furthermore, stabilization of the ERa/14-3-3ζ protein complex by FC-A was not influenced by the disease-associated and 4-hydroxytamoxifen resistant ERa-Y537S mutant. Together, these molecular and mechanistic insights provide direction for targeting ERa via the ERa/14-3-3 complex as an alternative drug discovery approach. [ABSTRACT FROM AUTHOR]