Redox state determines the warfarin sensitivity of wild-type VKOR and the resistance level and catalytic activity of WR mutants. (A) Structural locations of the WR mutations (spheres) identified from patients. The mutations are either at the warfarin (War) binding pocket or at peripheral regions, stabilizing this pocket. The VKOR structure in the O state (PDB 6WV3) is shown. (B) VKOR structures in the PO state with warfarin (PDB 6WV4; top) and KO (PDB 6WV5; bottom). WR mutations (side chains) interfering with warfarin binding should also affect KO binding. The Cys51-Cys132 disulfide bond stabilizes the cap region despite the disorder of a preceding region (dashed line). (C) The warfarin inhibition curves of wild-type (WT) VKOR in cells and with GSH and DTT. Because DTT has a much lower redox potential than GSH, the overly reduced VKOR is not in the stable O or PO state (panels A-B) and is prohibited from binding warfarin. (D) Relative activities of WR mutants (normalized to WT). Activities of WR mutants in GSH are close to those observed in cells. In contrast, DTT reduction interferes with substrate binding that is further weakened by WR mutations, resulting in lower relative activities. (E) Resistance levels of WR mutants in GSH correspond well with their cellular resistance levels. With DTT, however, most WR mutants do not show resistance.