Escherichia coli NhaA is a prototypical sodium–proton antiporter responsible for maintaining cellular ion and volume homeostasis by exchanging two protons for one sodium ion; despite two decades of research, the transport mechanism of NhaA remains poorly understood. Recent crystal structure and computational studies suggested Lys300 as a second proton-binding site; however, functional measurements of several K300 mutants demonstrated electrogenic transport, thereby casting doubt on the role of Lys300. To address the controversy, we carried out state-of-the-art continuous constant pH molecular dynamics simulations of NhaA mutants K300A, K300R, K300Q/D163N, and K300Q/D163N/D133A. Simulations suggested that K300 mutants maintain the electrogenic transport by utilizing an alternative proton-binding residue Asp133. Surprisingly, while Asp133 is solely responsible for binding the second proton in K300R, Asp133 and Asp163 jointly bind the second proton in K300A, and Asp133 and Asp164 jointly bind two protons in K300Q/D163N. Intriguingly, the coupling between Asp133 and Asp163 or Asp164 is enabled through the proton-coupled hydrogen-bonding network at the flexible intersection of two disrupted helices. These data resolve the controversy and highlight the intricacy of the compensatory transport mechanism of NhaA mutants. Alternative proton-binding site and proton sharing between distant aspartates may represent important general mechanisms of proton-coupled transport in secondary active transporters.

大肠杆菌NhaA是典型的钠-质子反转运蛋白，负责通过将两个质子交换为一个钠离子来维持细胞离子和体积稳态。尽管进行了二十年的研究，但对NhaA的转运机制仍知之甚少。最近的晶体结构和计算研究表明，Lys300是第二个质子结合位点。然而，几个K300突变体的功能测量证明了电转运，从而使人们对Lys300的作用产生怀疑。为了解决这一争议，我们对NhaA突变体K300A，K300R，K300Q / D163N和K300Q / D163N / D133A进行了最新的连续恒定pH分子动力学模拟。模拟表明，K300突变体通过利用替代质子结合残基Asp133来维持电原性运输。出奇，Asp133独自负责结合K300R中的第二个质子，Asp133和Asp163共同结合K300A中的第二个质子，Asp133和Asp164共同结合K300Q / D163N中的两个质子。有趣的是，Asp133与Asp163或Asp164之间的耦合是通过质子耦合的氢键网络在两个破裂的螺旋的灵活交点处实现的。这些数据解决了争议，并突出了NhaA突变体的补偿性转运机制的复杂性。替代的质子结合位点和远距离天冬氨酸之间的质子共享可能代表了次级活性转运子中质子偶联转运的重要一般机制。Asp133与Asp163或Asp164之间的耦合是通过质子耦合氢键网络在两个破裂的螺旋的灵活交点处实现的。这些数据解决了争议，并突出了NhaA突变体的补偿性转运机制的复杂性。替代的质子结合位点和远距离天冬氨酸之间的质子共享可能代表了次级活性转运子中质子偶联转运的重要一般机制。Asp133与Asp163或Asp164之间的耦合是通过质子耦合氢键网络在两个破裂的螺旋的灵活交点处实现的。这些数据解决了争议，并突出了NhaA突变体的补偿性转运机制的复杂性。替代的质子结合位点和远距离天冬氨酸之间的质子共享可能代表了次级活性转运子中质子偶联转运的重要一般机制。