Membrane-coupled conformational switching of VISTA regulates CC′ loop accessibility and immune checkpoint signaling

V-domain Ig Suppressor of T cell activation (VISTA) has emerged as a critical target for anti-cancer immunotherapy. VISTA inhibits T cell activity, suppressing the anti-cancer immune responses. Here, we use atomistic molecular dynamics simulations to investigate the conformational behavior of membrane-bound human VISTA using both a glycan-free truncated model and a glycosylated, full-length model. Our simulations show that the extracellular and transmembrane domains remain structurally stable; notably, the unusually long CC′ loop is stabilized by extensive hydrogen-bonding interactions. Principal component analysis and conformational clustering reveal a dominant rotational motion of the extracellular domain relative to the membrane, giving rise to two recurrent conformational states: an “Up” state in which the CC′ loop is solvent-exposed and accessible for ligand engagement, and a “Down” state in which the loop transiently associates with the lipid bilayer. These transitions are allosterically coupled to proline-mediated bending of the transmembrane helix. We propose that this membrane-coupled Up/Down conformational switching regulates CC′ loop accessibility, favoring cis interactions with cognate protein partners on the same cell surface, consistent with recent experimental observations, while permitting context-dependent trans interactions. Together, our findings reveal a membrane-coupled conformational mechanism regulating VISTA immune checkpoint function.