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.