Molecular basis and dual ligand regulation of tetrameric estrogen receptor α/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 (ERα), and small molecule stabilization of the ERα/14-3-3ζ protein complex by the natural product Fusicoccin A (FC-A), was demonstrated to downregulate ERα-mediated breast cancer proliferation. This presents a novel drug discovery approach to target ERα; however, structural and mechanistic insights into ERα/14-3-3 complex formation are lacking. Here, we provide an in-depth molecular understanding of the ERα/14-3-3ζ complex by isolating 14-3-3ζ in complex with an ERα protein construct comprising its ligand-binding domain (LBD) and phosphorylated F-domain. Bacterial co-expression and co-purification of the ERα/14-3-3ζ complex, followed by extensive biophysical and structural characterization, revealed a tetrameric complex between the ERα homodimer and the 14-3-3ζ homodimer. 14-3-3ζ binding to ERα, and ERα/14-3-3ζ complex stabilization by FC-A, appeared to be orthogonal to ERα endogenous agonist (E2) binding, E2-induced conformational changes, and cofactor recruitment. Similarly, the ERα antagonist 4-hydroxytamoxifen inhibited cofactor recruitment to the ERα LBD while ERα was bound to 14-3-3ζ. Furthermore, stabilization of the ERα/14-3-3ζ protein complex by FC-A was not influenced by the disease-associated and 4-hydroxytamoxifen resistant ERα-Y537S mutant. Together, these molecular and mechanistic insights provide direction for targeting ERα via the ERα/14-3-3 complex as an alternative drug discovery approach. 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 (ERα), and small molecule stabilization of the ERα/14-3-3ζ protein complex by the natural product Fusicoccin A (FC-A), was demonstrated to downregulate ERα-mediated breast cancer proliferation. This presents a novel drug discovery approach to target ERα; however, structural and mechanistic insights into ERα/14-3-3 complex formation are lacking. Here, we provide an in-depth molecular understanding of the ERα/14-3-3ζ complex by isolating 14-3-3ζ in complex with an ERα protein construct comprising its ligand-binding domain (LBD) and phosphorylated F-domain. Bacterial co-expression and co-purification of the ERα/14-3-3ζ complex, followed by extensive biophysical and structural characterization, revealed a tetrameric complex between the ERα homodimer and the 14-3-3ζ homodimer. 14-3-3ζ binding to ERα, and ERα/14-3-3ζ complex stabilization by FC-A, appeared to be orthogonal to ERα endogenous agonist (E2) binding, E2-induced conformational changes, and cofactor recruitment. Similarly, the ERα antagonist 4-hydroxytamoxifen inhibited cofactor recruitment to the ERα LBD while ERα was bound to 14-3-3ζ. Furthermore, stabilization of the ERα/14-3-3ζ protein complex by FC-A was not influenced by the disease-associated and 4-hydroxytamoxifen resistant ERα-Y537S mutant. Together, these molecular and mechanistic insights provide direction for targeting ERα via the ERα/14-3-3 complex as an alternative drug discovery approach. Nuclear receptors (NRs) are a family of structurally similar, mostly ligand-regulated, transcription factors that control a diverse set of biological processes, including metabolism, cell proliferation, development, and immunity (1Robinson-Rechavi M. Garcia H.E. Laudet V. The nuclear receptor superfamily.J. Cell Sci. 2003; 116: 585-586Crossref PubMed Scopus (372) Google Scholar, 2Weikum E.R. Liu X. Ortlund E.A. The nuclear receptor superfamily: a structural perspective.Protein Sci. 2018; 27: 1876-1892Crossref PubMed Scopus (198) Google Scholar). Several small molecules, including steroid hormones, fatty acids, vitamin D, and thyroid hormones, regulate NR activity by binding the NR’s ligand binding domain (LBD) (3Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schütz G. 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To gain a robust understanding of protein complex formation and the stoichiometry of binding, several biophysical assays were performed, including analytical size exclusion chromatography (SEC) and analytical ultracentrifugation (AUC). In addition, differential scanning fluorimetry (DSF), fluorescence anisotropy (FA), and hydrogen-deuterium exchange (HDX) experiments determined the role of ERα ligands E2 and 4-OHT on ERα/14-3-3 complex formation and identified 14-3-3 binding to the drug-resistant ERα-Y537S mutant. Finally, we show that the ERα/14-3-3 PPI can be stabilized by the natural product FC-A and that this PPI stabilization can be achieved independently from ERα ligand binding in both wild-type ERα and the ERα-Y537S mutant, thus presenting a potential orthogonal therapeutic strategy for targeting endocrine resistance in breast cancer. Most biochemical and structural studies on the ERα protein have focused on the ERα LBD, most often ending after helix 12 (∼ residue S554) (57Min J. Nwachukwu J.C. Min C.K. Njeri J.W. Srinivasan S. Rangarajan E.S. et al.Dual-mechanism estrogen receptor inhibitors.Proc. Natl. Acad. Sci. U. S. A. 2021; 118e2101657118Crossref Scopus (10) Google Scholar, 58Shiau A.K. Barstad D. Radek J.T. Meyers M.J. Nettles K.W. Katzenellenbogen B.S. et al.Structural characterization of a subtype-selective ligand reveals a novel mode of estrogen receptor antagonism.Nat. Struct. Biol. 2002; 9: 359-364PubMed Google Scholar). These studies exclude the 42-residue-long, intrinsically disordered, and solvent-exposed C-terminal F-domain (Fig. S4). The ERα F-domain is described to influence ERα dimerization and co-activator binding; however, as one of the least conserved regions among NRs, the structure and role of the ERα F-domain remain largely unknown (38De Vries-van Leeuwen I.J. da Costa Pereira D. Flach K.D. 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The ERα F-domain contains a penultimate threonine residue (T594) that upon phosphorylation facilitates the binding of 14-3-3 to ERα (38De Vries-van Leeuwen I.J. da Costa Pereira D. Flach K.D. Piersma S.R. Haase C. Bier D. et al.Interaction of 14-3-3 proteins with the estrogen receptor alpha F domain provides a drug target interface.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 8894-8899Crossref PubMed Scopus (92) Google Scholar). To gain mechanistic insights into the combined role of the ERα LBD and F-domain in concert with 14-3-3 binding, an ERα construct was designed comprising both domains (residues 302–595) (Fig. 2A). Since the responsible kinase is not known, native T594 phosphorylation is inaccessible. We thus introduced double-point mutations (F591R/P592R) within the ERα construct, enabling PKA phosphorylation at T594. The two-point mutations make the ERα C-terminal end closely match with the consensus sequence of known PKA substrates (RRXS/TY), thereby increasing the chances of successful phosphorylation of T594 by PKA (62Blom N. Kreegipuu A. Brunak S. PhosphoBase: a database of phosphorylation sites.Nucleic Acids Res. 1998; 26: 382-386Crossref PubMed Scopus (52) Google Scholar). Fluorescence anisotropy binding studies of 14-3-3ζ to peptide mimics of the wild-type (WT) ERα and PKA-responsive ERα sequence confirmed that the PKA-responsive point mutations did not hamper binding of ERα to 14-3-3ζ (Fig. S5). The PKA-responsive ERα (ERα(PKA)) construct was expressed and purified from E. coli; however, yielded solely in truncated ERα protein (at residues 570 and 572) as apparent from mass spectrometry analysis (Fig. S6). The truncation of the F-domain was addressed by incorporation of a C-terminal strep-tag (ERα(PKA)-strep), resulting in successful expression, purification, and PKA-mediated phosphorylation of ERα (Fig. S7). Notably, the introduction of a C-terminal strep-tag significantly changed the 14-3-3-binding site of ERα. Fluorescence anisotropy binding studies of WT ERα, ERα(PKA), and ERα(PKA)-strep phosphopeptides to 14-3-3ζ showed a 50-fold reduction in binding affinity upon introduction of the strep-tag to the C-terminal end of ERα peptide (Fig. S8). Moreover, stabilization of the ERα(PKA)-str