Antidepressants target the serotonin transporter (SERT) by inhibiting serotonin reuptake. Structural and biochemical studies aiming to understand binding of small-molecules to conformationally dynamic transporters like SERT often require thermostabilizing mutations and antibodies to stabilize a specific conformation, leading to questions about relationships of these structures to the bonafide conformation and inhibitor binding poses of wild-type transporter. To address these concerns, we determined the structures of ∆N72/∆C13 and ts2-inactive SERT bound to paroxetine analogues using single-particle cryo-EM and x-ray crystallography, respectively. We synthesized enantiopure analogues of paroxetine containing either bromine or iodine instead of fluorine. We exploited the anomalous scattering of bromine and iodine to define the pose of these inhibitors and investigated inhibitor binding to Asn177 mutants of ts2-active SERT. These studies provide mutually consistent insights into how paroxetine and its analogues bind to the central substrate-binding site of SERT, stabilize the outward-open conformation, and inhibit serotonin transport.

中文翻译：

---

帕罗西汀-人血清素转运蛋白复合物的化学和结构研究

抗抑郁药通过抑制血清素再摄取来靶向血清素转运蛋白 (SERT)。旨在了解小分子与构象动态转运蛋白（如 SERT）结合的结构和生化研究通常需要热稳定突变和抗体来稳定特定构象，从而导致有关这些结构与野生型真正构象和抑制剂结合姿势的关系的问题搬运工。为了解决这些问题，我们分别使用单粒子冷冻电镜和 X 射线晶体学确定了与帕罗西汀类似物结合的 ΔN72/ΔC13 和 ts2 非活性 SERT 的结构。我们合成了含有溴或碘而不是氟的帕罗西汀的对映体纯类似物。我们利用溴和碘的异常散射来定义这些抑制剂的姿势，并研究了抑制剂与 ts2 活性 SERT 的 Asn177 突变体的结合。这些研究为帕罗西汀及其类似物如何与 SERT 的中心底物结合位点结合、稳定向外开放的构象和抑制血清素转运提供了相互一致的见解。