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.