Missense VKOR mutants exhibit severe warfarin resistance but lack VKCFD via shifting to an aberrantly reduced state.

Missense VKOR mutations in patients cause their resistance to warfarin treatment, but not abnormal bleeding due to defective VKOR activity. The underlying mechanism of these phenotypes remains unknown. Here we show that the redox state of these mutants is essential to their activity and warfarin resistance. Using a mass-spectrometry-based footprinting method, we found that severe warfarin-resistant mutations change VKOR active site to an aberrantly reduced state in cells. Molecular dynamics simulation based on our recent crystal structures of VKOR reveals that these mutations induce an artificial opening of the protein conformation that increases access of small molecules to reduce the active site, generating constitutive activity uninhibited by warfarin. Increased activity also compensates for the weakened substrate binding caused by these mutations, thereby maintaining normal VKOR function. The uninhibitable nature of severe resistance mutations suggests that patients showing such signs should be genotyped and treated by alternative anticoagulation strategies.