Multiplexed measurement of variant abundance and activity reveals VKOR topology, active site and human variant impact

Vitamin K epoxide reductase (VKOR) drives the vitamin K cycle, activating vitamin K-dependent blood clotting factors. VKOR is also the target of the widely used anticoagulant, warfarin. Despite VKOR9s pivotal role in coagulation, its structure and active site remain poorly understood. In addition, VKOR variants can cause disease or alter warfarin response. Here, we used multiplexed, sequencing-based assays to measure the effects of 2,695 VKOR missense variants on abundance and 697 variants on activity in cultured human cells. The large-scale data, along with an evolutionary coupling analysis, supports a four transmembrane domain topology, with variants in transmembrane domains exhibiting deleterious effects on abundance and activity. Functionally constrained regions of VKOR define the active site, and we find that, of four conserved cysteines putatively critical for function, only three are absolutely required. Finally, 25% of human VKOR missense variants show reduced abundance or activity, possibly conferring warfarin sensitivity or causing disease.