There is ongoing debate regarding the mechanism through which cation/proton antiporters (CPAs), like Thermus thermophilus NapA (TtNapA) and Escherichia coli NapA (EcNhaA), alternate between their outward- and inward-facing conformations in the membrane. CPAs comprise two domains, and it is unclear whether the transition is driven by their rocking-bundle or elevator motion with respect to each other. Here we address this question using metadynamics simulations of TtNapA, where we bias conformational sampling along two axes characterizing the two proposed mechanisms: angular and translational motions, respectively. By applying the bias potential for the two axes simultaneously, as well as to the angular, but not the translational, axis alone, we manage to reproduce each of the two known states of TtNapA when starting from the opposite state, in support of the rocking-bundle mechanism as the driver of conformational change. Next, starting from the inward-facing conformation of EcNhaA, we sample what could be its long-sought-after outward-facing conformation and verify it using cross-linking experiments.

关于阳离子/质子反转运蛋白 (CPA)（如嗜热栖热菌NapA (TtNapA) 和大肠杆菌 NapA (EcNhaA)）在膜中的向外和向内构象之间交替的机制，一直存在争论。 CPA 包含两个域，目前尚不清楚这种转变是由它们之间的摇动束还是电梯运动驱动的。在这里，我们使用 TtNapA 的元动力学模拟来解决这个问题，其中我们沿着两个轴偏置构象采样，这两个轴分别表征了两种提出的机制：角运动和平移运动。通过同时对两个轴以及角度轴而非平移轴施加偏置电势，我们设法在从相反状态开始时再现 TtNapA 的两个已知状态中的每一个，以支持摇摆-束机制作为构象变化的驱动力。接下来，从 EcNhaA 的内向构象开始，我们对可能是其长期寻找的外向构象进行采样，并通过交联实验对其进行验证。