Unconstrained MD simulation of warfarin binding to VKOR, Y139A, and A26T mutants. (A) The backbone RMSD profiles of VKOR (red) and the Y139A mutant (blue) during the 200-nanosecond unconstrained MD simulation. The green curve shows the backbone RMSD profile of wild-type VKOR when warfarin is removed at the 80-nanosecond time point and the structure is reequilibrated before additional simulation. (B) Warfarin binding to wild-type VKOR at the last snapshot of the 200-nanosecond unconstrained MD simulation. A T-shaped π-π stacking interaction between warfarin and Y139 is shown (nearest carbon-carbon distance of 3.36 Å). (C) Warfarin binding to the Y139A mutant at the last snapshot of the 200-nanosecond unconstrained MD simulation. Warfarin drifts away from its initial site. (D) Configuration of Y25, Y139, and warfarin at the last snapshot of the 448-picosecond steered MD simulation. Y25 stabilizes Y139 by forming a hydrogen bond network via water molecules from the environment. (E) Warfarin-binding pocket at the 24-nanosecond snapshot of the unconstrained conventional MD simulation. Y139 was stabilized by forming a new hydrogen bond network via water molecules with the backbone of A26. (F) Configuration of the warfarin-binding pocket of the A26T mutant at the 200-nanosecond snapshot of the unconstrained conventional MD simulation. Y139 was stabilized by directly forming a hydrogen bond with A26T.