Antidepressant binding site in a bacterial homologue of neurotransmitter transporters
Antidepressant binding site in a bacterial homologue of neurotransmitter transporters
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DOI：
10.1038/nature06038
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发表时间：
2007-08-23
期刊：
影响因子：
64.8
通讯作者：
Gouaux, Eric
中科院分区：
文献类型：
--
作者：
Singh, Satinder K.;Yamashita, Atsuko;Gouaux, Eric
钠耦合转运蛋白是一种无处不在的泵，它利用预先存在的钠梯度来催化跨脂质双层对必需营养素、神经递质和无机离子的热力学不利吸收(1)。这些整合膜蛋白的功能障碍与葡萄糖/半乳糖吸收不良(2)、先天性甲状腺功能减退症(3)、巴特综合征(4)、癫痫(5)、抑郁症(6)、自闭症(7)和强迫症(8)有关。钠偶联转运蛋白可被许多治疗上重要的化合物阻断，包括利尿剂 (9)、抗惊厥药 (10) 和抗抑郁药 (11)，其中许多化合物也已成为旨在探测拮抗剂结合位点和阐明转运机制的生化实验中不可或缺的工具。稳态动力学数据表明存在竞争性 (12,13) 和非竞争性 (14,15) 抑制模式。血清素转运蛋白 (SERT) 的拮抗剂解离实验还揭示了低亲和力变构位点的存在，该位点可减缓抑制剂与单独的高亲和力位点的解离 (16)。尽管取得了这些进步，但对抑制剂作用的原子水平洞察仍然难以捉摸。在这里，我们筛选了一组分子抑制LeuT（哺乳动物神经递质钠同向转运体的原核同源物）的能力，并表明三环抗抑郁药（TCA）氯米帕明非竞争性抑制底物摄取。共晶体结构表明，氯米帕明与其他两种 TCA 结合在位于基质上方约 11 埃的面向细胞外的前庭和两个钠离子中，显然将细胞外门稳定在闭合构象中。解离速率测定表明，氯米帕明降低了亮氨酸从 LeuT 解离的速率，并强化了我们的观点，即该 TCA 通过减缓底物释放来抑制 LeuT。我们的结果代表了钠偶联转运蛋白非竞争性抑制的分子观点，并定义了新抑制剂的合理设计原则。
Sodium-coupled transporters are ubiquitous pumps that harness pre-existing sodium gradients to catalyse the thermodynamically unfavourable uptake of essential nutrients, neurotransmitters and inorganic ions across the lipid bilayer(1). Dysfunction of these integral membrane proteins has been implicated in glucose/galactose malabsorption(2), congenital hypothyroidism(3), Bartter's syndrome(4), epilepsy(5), depression(6), autism(7) and obsessive-compulsive disorder(8). Sodium-coupled transporters are blocked by a number of therapeutically important compounds, including diuretics(9), anticonvulsants(10) and antidepressants(11), many of which have also become indispensable tools in biochemical experiments designed to probe antagonist binding sites and to elucidate transport mechanisms. Steady-state kinetic data have revealed that both competitive(12,13) and noncompetitive(14,15) modes of inhibition exist. Antagonist dissociation experiments on the serotonin transporter (SERT) have also unveiled the existence of a low-affinity allosteric site that slows the dissociation of inhibitors from a separate high-affinity site(16). Despite these strides, atomic-level insights into inhibitor action have remained elusive. Here we screen a panel of molecules for their ability to inhibit LeuT, a prokaryotic homologue of mammalian neurotransmitter sodium symporters, and show that the tricyclic antidepressant (TCA) clomipramine noncompetitively inhibits substrate uptake. Cocrystal structures show that clomipramine, along with two other TCAs, binds in an extracellular-facing vestibule about 11 angstrom above the substrate and two sodium ions, apparently stabilizing the extracellular gate in a closed conformation. Off-rate assays establish that clomipramine reduces the rate at which leucine dissociates from LeuT and reinforce our contention that this TCA inhibits LeuT by slowing substrate release. Our results represent a molecular view into noncompetitive inhibition of a sodium-coupled transporter and define principles for the rational design of new inhibitors.