Stabilization of warfarin‐binding pocket of VKORC1 and VKORL1 by a peripheral region determines their different sensitivity to warfarin inhibition

Warfarin is an oral anticoagulant taken by ~1% of the US population to treat and prevent deep vein thrombosis, pulmonary embolism, stroke, and myocardial infarction. Warfarin targets vitamin K epoxide reductase in the vitamin K cycle [1–3], which supports the activity of vitamin-K-dependent proteins including several coagulation factors. The cycle begins with the γ-carboxylation of selected glutamic acids in these proteins, a post-translational modification that allows their membrane association and activation. The γ-carboxylase activity is driven by the epoxidation of the vitamin K hydroquinone, which is regenerated by vitamin K epoxide reductases to complete the vitamin K cycle. The epoxide reductase activity has been identified for two paralogs found in the human genome, VKORC1 and VKORL1, which share ~74% similarity (48% identity) in their protein sequences [3–7] (Fig. 1). VKORC1 is highly expressed in liver where coagulation factors are produced, whereas VKORL1 is more abundant in extrahepatic tissues where other vitamin-K-dependent proteins, such as osteocalcin and matrix Gla protein, are made [8,9]. Compared to VKORC1, VKORL1 shows 30–50-fold more resistance to warfarin in an in vitro assay [8]. Thus, at a warfarin dose administered to inhibit VKORC1-supported blood coagulation, the VKORL1 activity is largely retained, possibly explaining the small effects that short-term warfarin administration usually has against the bone mineralization and the inhibition of vascular calcification [8,9]. The mechanism underlying the relative resistance of VKORL1 to warfarin inhibition, however, is unclear. Open in a separate window Figure 1. Mapping of WR mutations in VKORC1 with sequence alignment of VKORC1 (C1) and VKORL1 (L1). Identical residues (48%) are shadowed in dark grey, and similar residues (26%) in grey. Prediction of secondary structures (top) are based on the crystal structure of a bacterial VKOR homolog [22]. Regions I–IV are identified (bottom) based on the distribution of WR mutations in VKORC1. The underlying panels show the resistant level of WRs in VKORC1 [15], with a Y-axis break at NRwar = 5. Residues with NRwar > 5 mutations are shown in bold letters in the sequence alignment. The orange-colored WR mutations in VKORC1 are selected for mutagenesis analysis of the corresponding residues in VKORL1 (Fig. 3A–C). Matching mutations (Fig. 4B) and the human SNP (Fig. 6) that increase the warfarin sensitivity of VKORL1 are indicated by green and red dots, respectively.