Although warfarin is the most widely used anticoagulant worldwide, the mechanism by which warfarin inhibits its target, human vitamin K epoxide reductase (hVKOR), remains unclear. Here we show that warfarin blocks a dynamic electron-transfer process in hVKOR. A major fraction of cellular hVKOR is in an intermediate redox state containing a Cys51-Cys132 disulfide, a characteristic accommodated by a four-transmembrane-helix structure of hVKOR. Warfarin selectively inhibits this major cellular form of hVKOR, whereas disruption of the Cys51-Cys132 disulfide impairs warfarin binding and causes warfarin resistance. Relying on binding interactions identified by cysteine alkylation footprinting and mass spectrometry coupled with mutagenesis analysis, we conducted structure simulations, which revealed a closed warfarin-binding pocket stabilized by the Cys51-Cys132 linkage. Understanding the selective warfarin inhibition of a specific redox state of hVKOR should enable the rational design of drugs that exploit the redox chemistry and associated conformational changes in hVKOR.
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- Supplementary Figure 1: Cys51-Cys132 is an intramolecular disulfide bond. (464 KB)
(a) The wild-type hVKOR protein shows a monomeric band on a non-reducing SDS-PAGE gel. To prevent disulfide shuffling, the cells were treated with NEM to block reactive free cysteines. Uncropped blot/gel images are shown in Supplementary Data Set 2. (b-c) Cys51-Cys132 linked peptides of various lengths (in addition to Fig. 2c,d) have been identified by mass measurements before and after reduction of the disulfide bond (left), and by MS/MS (right). (d) Cys51-Cys132 is preserved in a C43A C135A double mutant, which eliminates alternative ways of forming disulfide bonds between the four conserved cysteines, so that Cys51-Cys132 becomes the only possible form (see inset). Both Cys51 and Cys132 are oxidized in this double mutant, and both can be reduced by DTT (as wild-type hVKOR in Fig. 2b). Error bars, same analysis as in Fig. 2b. MS and MS/MS spectra can be found in Supplementary Data Set 1. (e) A Cys51-Cys132 linked peptide is identified in this double mutant.
- Supplementary Figure 2: The electron-transfer pathway and associated conformational changes. (175 KB)
(a) Scheme of an electron transfer pathway proposed to regenerate hVKOR activity. The observed oxidation level of each conserved cysteine (Figs. 2b, 3, and 4) reflects a combination of different redox states in this pathway. State I: a pair of free cysteines (green) from a redox partner reduces (blue arrow) the Cys43-Cys51 disulfide (red) in fully oxidized hVKOR. State II to III: electron transfer (blue arrows) generates alternative disulfide bonds (red bars) and Cys51-Cys132 is formed as an intermediate state (III). State IV to V: all cysteines are reduced and hVKOR is catalytically active to reduce KO. State V to I: Reduction of KO (KO to K and K to hydroquinone KH2) generates the Cys132-Cys135 disulfide, and the Cys43-Cys51 disulfide (as in Fig. 4b-e), thereby regenerating State I. (b) Electron transfer is associated with conformational changes in the crystal structures of a bacterial VKOR homolog. Left, A C56A mutant (corresponding to C51A in hVKOR; in parentheses) generates a structure (PDB code: 3KP9) with the Cys130-Cys133 disulfide (Cys132-Cys135 in hVKOR). Right, A C50A mutant (C43A in hVKOR) generates a structure (PDB code: 4NV2 and 4NV5) with the Cys56-Cys130 disulfide (Cys51-Cys132 in hVKOR). Comparison of these two structures shows that the formation of different disulfide bonds (during electron transfer as in a) is associated with conformational changes in HL1-2 (red). In particular, the N-terminal part of the short helix alternates between extended (left) and helical (right) conformations, which directly change the VKOR active site.
- Supplementary Figure 3: Cysteine mutations result in a predictable pattern of redox changes in hVKOR. (161 KB)
(a) Redox changes after cysteine mutations (for comparison, Fig. 3c shows only the four conserved cysteines). Error bars, same analysis as in Fig. 2b. MS and MS/MS spectra can be found in Supplementary Data Set 1. (b) Predictions (left) based on a redistribution mechanism (Supplementary Note) fit well with the observed redox changes (right) after cysteine mutations. The P values from two-tailed Student t test (right) are calculated by comparing the changes between mutants and wild-type (observed in a); the values are shown in green for cysteines that become more reduced, in red for those that become more oxidized, and in black for insignificant changes.
- Supplementary Figure 4: Cysteine mutations block substrate-induced oxidation through electron transfer. (537 KB)
The KO treatment is 5 h and these mutants are analyzed in the same way as for the wild-type hVKOR in Fig. 4. The red arrows indicate the direction of oxidation and the cross signs indicate mutations. All subsequent oxidation in the pathway are blocked by the mutation of conserved cysteines (right). In contrast, mutation of non-conserved cysteines (left) has no effect. Error bars, same analysis as in Fig. 2b. MS and MS/MS spectra can be found in Supplementary Data Set 1.
- Supplementary Figure 5: MS analysis of cysteine protection induced by warfarin binding. (232 KB)
(a) The untreated and warfarin-treated wild-type hVKOR were alkylated with NEM and compared by quantitative MS. Warfarin inhibits the alkylation of four cysteines (**P < 0.001, ***P < 0.0001) in hVKOR. Error bars, same analysis as in Fig. 2b. (b) Cysteine mutations change the pattern of warfarin-induced protection. Wild-type hVKOR (data from a) and each cysteine mutant was monitored for the change in NEM alkylation of individual cysteines induced by warfarin binding. Each bar shows the difference (as in Fig. 6c) between apparent oxidation levels for warfarin-treated samples and untreated samples. Mutations of C51A, C132A, and C135A block warfarin-induced changes (**P < 0.001, ***P < 0.0001; compared with wild type), thereby confirming the gel shift analyses (Fig. 5c) indicating that these mutations interfere with warfarin binding. In contrast, C43A, C16A, C85A, and C96A mutations have insignificant effects. Error bars, same analysis as in Fig. 2b but with error propagation calculated for the subtraction. MS and MS/MS spectra can be found in Supplementary Data Set 1.
- Supplementary Figure 6: The Cys51-Cys132 disulfide is important for warfarin binding. (77 KB)
(a) Cysteine mutations were tested for warfarin-induced gel shift as control experiments for Fig. 5c. Uncropped blot/gel images are shown in Supplementary Data Set 2. (b) The activity of cysteine mutants. C132A and C135A are inactive mutants because they delete active site residues. The activity of C43A is also low and does not allow reliable analysis for warfarin resistance in Fig. 5d. Error bars defined in Online Methods. Source data for basal activity are available in Supplementary Data Set 4.
- Supplementary Figure 7: Confirming the MD-predicted warfarin-binding pocket by clustering of WRs. (222 KB)
(a) Novel WRs identified by alanine scanning mutagenesis (orange) and naturally occurring WRs (blue) together indicate that the warfarin binding region is located at the luminal portion of the hVKOR protein according to the four-TM model (bars below). These data are shown as a heat map in Fig. 7a. Error bars defined in Online Methods. Source data for resistance and basal activity are available in Supplementary Data Set 3, 4. (b) Strong WR residues are highly conserved in eukaryotic VKOR homolog proteins. The sequences of these proteins were generated from the Psi-Blast search of the human VKOR against eukaryotic proteins in a non-redundant NCBI database. Top 500 sequences from the search were used to plot the sequence logo.
- Video 2: Electron transfer is associated with conformational changes at the VKOR active site. (3.14 MB, Download)
- Four structures (PDB code: 3KP9, 4NV2, 4NV5, and 4NV6) of the bacterial VKOR homolog are combined and a trajectory of conformational changes is modeled. These structures show a short helix (part of HL1-2; red) at the active site change conformations when alternative disulfide bonds are formed during electron transfer. The sequence of this helical region is highly conserved between the bacterial homolog and hVKOR. Therefore, similar conformational changes may occur in hVKOR, and warfarin may selectively bind to a conformational state stabilized by the Cys132-Cys135 disulfide.
- Supplementary Text and Figures (1,677 KB)
Supplementary Figures 1–7 and Supplementary Note
- Supplementary Data Set 1 (4,598 KB)
MS and product-ion (MS/MS) spectra of all the cysteine-containing peptides.
- Supplementary Data Set 2 (14,231 KB)
Uncropped western blots.
- Supplementary Data Set 6 (113 KB)
PDB validation report for the hVKOR model.
- Supplementary Data Set 3 (25 KB)
Inhibition and resistance.
- Supplementary Data Set 4 (14 KB)
- Supplementary Data Set 5 (3,206 KB)
Coordinates of a representative structure from an ensemble generated via molecular dynamics simulation of hVKOR with warfarin.