Proton Transfer Mechanisms in the MmpL3 Transporter of Mycobacterium tuberculosis Studied by Computer Simulations

The mycobacterial membrane exporter MmpL3 transports trehalose monomycolates (TMM) from the cytosol to the outer membrane of Mycobacterium tuberculosis, making it a potential drug target. Proton influx is believed to drive TMM efflux, suggesting that disrupting proton transfer (PT) could be therapeutic. However, the PT mechanism and its relation to function remain unclear. Recent MmpL3 structures reveal a potential proton channel in its hydrophobic core, which also binds potential antituberculosis compounds. We investigated the PT process using hybrid quantum-mechanical/molecular-mechanical and classical molecular dynamics simulations. We show that transient water chains form in two connected transmembrane cavities that act as proton conduits. Four consecutive PT events are necessary to alter the protonation states of acidic residues in the protein core, triggering conformational changes that affect the TMM binding site. The process begins with the tandem movement of two protons through an upper cavity, protonating two aspartate residues via a classical hydronium migration. After conformational shifts, PT proceeds through a lower cavity, protonating two glutamate residues near the cytosolic opening and inducing further conformational shifts; here, PT occurs sequentially via hydronium and proton-hole migration. The cycle ends with the release of protons into the cytosol. Based on the observed conformational changes, we propose a mechanism for TMM efflux.

分枝杆菌膜转运蛋白MmpL3（mycobacterial membrane exporter MmpL3）可将单分枝菌酸海藻糖（trehalose monomycolates, TMM）从胞质转运至结核分枝杆菌（Mycobacterium tuberculosis）的外膜，因此成为极具潜力的抗结核药物靶点。学界普遍认为质子内流驱动TMM外排，这提示阻断质子转移（proton transfer, PT）或可实现结核治疗。然而，质子转移的具体机制及其与蛋白功能的关联仍不明晰。近期解析的MmpL3结构显示，其疏水核心中存在潜在质子通道，同时该区域还可结合抗结核候选化合物。本研究通过混合量子力学/分子力学（hybrid quantum-mechanical/molecular-mechanical）与经典分子动力学模拟，对该蛋白的质子转移过程展开了探究。研究发现，两个相连的跨膜腔室内会形成瞬态水链，可作为质子传导通路。要改变蛋白核心内酸性残基的质子化状态、触发影响TMM结合位点的构象变化，需依次完成四次连续的质子转移事件。该过程首先以两个质子通过上方腔室的协同移动启动，经由经典的水合氢离子迁移机制质子化两个天冬氨酸残基。构象发生偏移后，质子转移通过下方腔室继续进行，在胞质开口附近质子化两个谷氨酸残基并诱导进一步的构象变化；此阶段质子转移通过水合氢离子与空穴质子迁移依次完成。整个循环最终以质子释放至胞质告终。基于观测到的构象变化，本研究提出了一套完整的TMM外排机制。