Rocker-Switch Mechanism of Choline Transmembrane Transport in FLVCR2: Dynamic Latch Residues and Cooperative Regulation of Transmembrane Helices

As a key member of the major facilitator superfamily, FLVCR2 plays a crucial role in choline transport. However, the kinetic mechanism and substrate recognition mode of its transmembrane transport remain elusive. Here, we integrate long-time-scale molecular dynamics simulations, potential of mean force, and functional validation of a key substrate-anchoring residue to elucidate the complete conformational cycle and energy regulation network of FLVCR2 at the atomic level, revealing its characteristic “rocker-switch” mechanism. We find that choline transmembrane transport is synergistically regulated by the electrostatic environment and hydrophobic surface of the extracellular tunnel, ensuring stable substrate migration within the channel lumen and efficient entry into the binding site. A network of negatively charged residues at the extracellular tunnel entrance drives the stepwise entry of choline through strong interactions, while W125 facilitates initial anchoring and avoids abnormal release. Subsequently, the rigid support of the transmembrane helix bundles and flexible hinge-like movements of the cytoplasmic loop drive the conformational transition of FLVCR2 from the outward-facing to the occluded state, accompanied by the closure of the extracellular tunnel and simultaneous opening of the intracellular tunnel. The latch residue pairs dynamically alternate closure, precisely recognizing the directionality of the conformational transitions. Prolonged simulation completely captures the coordinated sliding of transmembrane helices and dynamic latch regulation, revealing a continuous conformational transition from outward-facing to inward-facing. Periodic domain rocking correlates strongly with choline transport, while dynamic remodeling of the channel pore provides a physical pathway for substrate permeation. Intracellular choline release is coordinated through hydrophobic anchor disengagement, electrostatic gating regulation, and positively charged residue guidance. Electrostatic gate residues ensure directional substrate migration through a spatiotemporal balance of repulsion and attraction. Following choline release, FLVCR2 resets to its occluded state through reverse sliding of transmembrane helices and cytoplasmic latch tightening, maintaining structural stability to initiate new transport cycles.

作为主要促进因子超家族（major facilitator superfamily）的关键成员，FLVCR2在胆碱转运中发挥核心作用。然而，其跨膜转运的动力学机制与底物识别模式仍有待阐明。本研究整合长时间尺度分子动力学模拟、平均力势（potential of mean force）分析以及关键底物锚定残基的功能验证，在原子水平上揭示了FLVCR2的完整构象循环与能量调控网络，阐明了其特征性“摇杆开关（rocker-switch）”机制。研究发现，胆碱跨膜转运由胞外通道的静电环境与疏水表面协同调控，确保底物在通道内腔中稳定迁移并高效进入结合位点。胞外通道入口处的带负电残基网络通过强相互作用驱动胆碱逐步进入，而W125可助力初始锚定并避免异常释放。随后，跨膜螺旋束的刚性支撑与胞质环的柔性铰链样运动，驱动FLVCR2构象从外向开放态转变为闭塞构象，伴随胞外通道关闭与胞内通道同步开放。锁扣残基对动态交替闭合，精准识别构象转变的方向性。长时间模拟完整捕捉了跨膜螺旋的协同滑动与动态锁扣调控过程，揭示了从外向开放态到内向开放态的连续构象转变。结构域周期性摆动与胆碱转运显著相关，而通道孔道的动态重塑为底物渗透提供了物理通路。胞内胆碱释放通过疏水锚定脱离、静电门控调控以及带正电残基引导协同完成。静电门控残基通过斥力与吸引力的时空平衡确保底物定向迁移。胆碱释放完成后，FLVCR2通过跨膜螺旋的反向滑动与胞质锁扣收紧重置为闭塞构象，维持结构稳定性以启动新一轮转运循环。