Department of Biochemistry and Molecular Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel.
Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
Proc Natl Acad Sci U S A. 2020 Dec 15;117(50):31850-31860. doi: 10.1073/pnas.2002710117. Epub 2020 Nov 30.
There is ongoing debate regarding the mechanism through which cation/proton antiporters (CPAs), like 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/质子反向转运体（CPAs），如 NapA（TtNapA）和大肠杆菌 NapA（EcNhaA），在膜中如何在外向和内向构象之间交替，目前仍存在争议。CPAs 由两个结构域组成，目前尚不清楚这种转变是由它们彼此之间的摇摆束还是提升运动驱动的。在这里，我们使用 TtNapA 的元动力学模拟来解决这个问题，我们沿着两个轴对两种拟议的机制进行构象采样：分别是角度和平移运动。通过同时应用两个轴的偏压，以及仅对角度而不对平移轴施加偏压，我们成功地从相反的状态重现了 TtNapA 的两种已知状态，支持了摇摆束机制是构象变化的驱动力。接下来，从 EcNhaA 的内向构象开始，我们采样了可能是其长期以来寻求的外向构象，并使用交联实验对其进行了验证。
