Molecular dynamics simulations coupled with functional analyses of the major yeast phosphatidylinositol/phosphatidylcholine transfer protein Sec14p identify structural elements involved in regulating the ability of Sec14p to execute phospholipid exchange. The molecular dynamics simulations suggest large rigid body motions within the Sec14p molecule accompany closing and opening of an A10/T4/A11 helical gate, and that "state-of-closure" of this helical gate determines access to the Sec14p phospholipid binding cavity. The data also project that conformational dynamics of the helical gate are controlled by a hinge unit (residues F 212, Y213, K239, I240, and I 242) that links to the N- and C-terminal ends of the helical gate, and by a novel gating module (composed of the B1LB2 and A12LT5 substructures) through which conformational information is transduced to the hinge. The 114TDKDGR119 motif of B1LB2 plays an important role in that transduction process. These simulations offer new mechanistic possibilities for an important half-reaction of the Sec14p phospholipid exchange cycle that occurs on membrane surfaces after Sec14p has ejected bound ligand, and is reloading with another phospholipid molecule. These conformational transitions further suggest structural rationales for known disease missense mutations that functionally compromise mammalian members of the Sec14-protein superfamily. © 2007 by The American Society for Cell Biology.