Neurotransmitter:sodium symporters (NSS) terminate neurotransmission through Na⁺-driven reuptake of cognate neurotransmitters. Crystallographically, whereas both substrates and inhibitors have been found to bind in the central binding (S1) site of NSS, inhibitors were found to bind to a second binding (S2) site in the extracellular vestibule (EV) of transporters for leucine (LeuT) and serotonin. On the basis of computational and experimental studies, we proposed that substrates bind to the S2 site of LeuT as well and that substrate binding to the S2 site is essential for Na⁺-coupled symport. Recent binding experiments show that substrate (l-Trp) binding in the S2 site of MhsT, another bacterial NSS, is also central to the allosteric transport mechanism. Here, we used extensive molecular dynamics simulations combined with Markov state model analysis to investigate the interaction of l-Trp with the EV of MhsT and identified potential binding poses of l-Trp as well as induced conformational changes in the EV. Our computational findings were validated by experimental mutagenesis studies and shed light on the ligand binding characteristics of the EV of NSS, which may facilitate development of allosteric ligands targeting NSS.

中文翻译：

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通过原子模拟和马尔可夫模型探索MhsT细胞外前庭中的底物结合

神经递质：钠共转运蛋白（NSS）通过Na ⁺驱动的同源神经递质的再摄取终止神经递质。晶体学上，虽然发现底物和抑制剂都结合在NSS的中央结合（S1）位点，但发现抑制剂结合到亮氨酸（LeuT）转运蛋白的细胞外前庭（EV）的第二个结合（S2）位点和血清素。在计算和实验研究的基础上，我们提出了底物也与LeuT的S2位点结合，并且底物与S2位点的结合对于Na ⁺偶联的对称运动是必不可少的。最近结合实验表明，底物（升（Trp）结合在另一个细菌NSS MhsT的S2位点，也是变构转运机制的核心。在这里，我们使用广泛的分子动力学模拟与马尔可夫状态模型分析相结合，研究了l- Trp与MhsT EV的相互作用，并确定了l- Trp的潜在结合姿势以及EV中诱导的构象变化。我们的计算结果通过实验诱变研究得到验证，并阐明了NSS EV的配体结合特征，这可能有助于靶向NSS的变构配体的发展。