PDF(Pubmed)
Abstract：
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.
摘要：
靶向超出其内源性配体结合口袋的核受体（NRs）的治疗策略已经获得了显著的科学兴趣。由于需要规避与耐药性和药理学特征相关的问题。hub蛋白14-3-3是各种NRs的内源性调节因子，为NR活性的小分子调控提供了新的切入点。例证，14-3-3与雌激素受体α（ERα）的C末端F结构域结合，以及天然产物FusicoccinA（FC-A）对ERα/14-3-3ζ蛋白复合物的小分子稳定，被证明下调ERα介导的乳腺癌增殖。这提出了一种新的靶向ERα的药物发现方法，然而,缺乏对ERα/14-3-3复合物形成的结构和机制见解。这里,我们通过将14-3-3ζ与包含其配体结合域（LBD）和磷酸化F结构域的ERα蛋白构建体分离，提供了对ERα/14-3-3ζ复合物的深入分子理解。ERα/14-3-3ζ复合物的细菌共表达和共纯化，其次是广泛的生物物理和结构表征，揭示了ERα同二聚体和14-3-3ζ同二聚体之间的四聚体复合物。14-3-3ζ与ERα结合，FC-A稳定ERα/14-3-3ζ络合物，似乎与ERα内源性激动剂（E2）结合正交，E2诱导的构象变化，和辅因子招募。同样，ERα拮抗剂4-羟基他莫昔芬抑制了对ERαLBD的辅因子募集，而ERα与14-3-3ζ结合。此外，FC-A对ERα/14-3-3ζ蛋白复合物的稳定不受疾病相关和4-羟基他莫昔芬抗性ERα-Y537S突变体的影响。一起,这些分子和机制的见解为通过ERα/14-3-3复合物作为替代药物发现方法靶向ERα提供了方向.
