Proton-coupled oligopeptide transporters (POTs) are of great pharmaceutical interest owing to their promiscuous substrate binding site that has been linked to improved oral bioavailability of several classes of drugs. Members of the POT family are conserved across all phylogenetic kingdoms and function by coupling peptide uptake to the proton electrochemical gradient. Cryo-EM structures and alphafold models have recently provided new insights into different conformational states of two mammalian POTs, SLC15A1 and SLC15A2. Nevertheless, these studies leave open important questions regarding the mechanism of proton and substrate coupling, while simultaneously providing a unique opportunity to investigate these processes using molecular dynamics (MD) simulations. Here, we employ extensive unbiased and enhanced-sampling MD to map out the full SLC15A2 conformational cycle and its thermodynamic driving forces. By computing conformational free energy landscapes in different protonation states and in the absence or presence of peptide substrate, we identify a likely sequence of intermediate protonation steps that drive inward-directed alternating access. These simulations identify key differences in the extracellular gate between mammalian and bacterial POTs, which we validate experimentally in cell-based transport assays. Our results from constant-PH MD and absolute binding free energy (ABFE) calculations also establish a mechanistic link between proton binding and peptide recognition, revealing key details underpining secondary active transport in POTs. This study provides a vital step forward in understanding proton-coupled peptide and drug transport in mammals and pave the way to integrate knowledge of solute carrier structural biology with enhanced drug design to target tissue and organ bioavailability.

中文翻译：

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哺乳动物质子偶联肽转运蛋白的机制

质子偶联寡肽转运蛋白（POT）由于其混杂的底物结合位点而具有很大的药学意义，该位点与改善几类药物的口服生物利用度有关。 POT 家族的成员在所有系统发育界中都是保守的，并通过将肽摄取与质子电化学梯度耦合来发挥作用。冷冻电镜结构和 alphafold 模型最近为两种哺乳动物 POT（SLC15A1 和 SLC15A2）的不同构象状态提供了新的见解。然而，这些研究留下了关于质子和底物耦合机制的重要问题，同时提供了利用分子动力学（MD）模拟研究这些过程的独特机会。在这里，我们采用广泛的无偏和增强采样 MD 来绘制完整的 SLC15A2 构象循环及其热力学驱动力。通过计算不同质子化状态下以及在不存在或存在肽底物的情况下的构象自由能景观，我们确定了驱动内向交替访问的中间质子化步骤的可能序列。这些模拟确定了哺乳动物和细菌 POT 之间细胞外门的关键差异，我们在基于细胞的运输测定中通过实验验证了这一差异。我们的恒定 PH MD 和绝对结合自由能 (ABFE) 计算结果还建立了质子结合和肽识别之间的机制联系，揭示了 POT 中二次主动转运的关键细节。这项研究为理解哺乳动物中的质子偶联肽和药物转运迈出了重要的一步，并为将溶质载体结构生物学知识与增强的药物设计相结合以提高目标组织和器官的生物利用度铺平了道路。