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

Melissa A Chiasson,Marta Verby,Jason J Stephany,Katherine A Sitko,Douglas M Fowler,Frederick P Roth,Daniel Desloover,Kenneth A Matreyek,Nathan J Rollins,Song Sun,Allan E Rettie,Debora S Marks

doi:10.7554/elife.58026

Abstract

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 drug, warfarin. Despite VKOR's pivotal role in coagulation, its structure and active site remain poorly understood. In addition, VKOR variants can cause vitamin K-dependent clotting factor deficiency 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 functional data, along with an evolutionary coupling analysis, supports a four transmembrane domain topology, with variants in transmembrane domains exhibiting strongly deleterious effects on abundance and activity. Functionally constrained regions of the protein 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.

Highlights

The enzyme vitamin K epoxide reductase (VKOR) drives the vitamin K cycle, which activates blood coagulation factors
Having confirmed that human VKOR has four transmembrane domains, we explored the detailed pattern of mutational effects we observed in the context of a four transmembrane domain homology model
We evaluated a VKOR homology model in the context of the patterns of variant effects on abundance we measured and found that the homology model could explain these patterns

Summary

Introduction

The enzyme vitamin K epoxide reductase (VKOR) drives the vitamin K cycle, which activates blood coagulation factors. VKOR, an endoplasmic reticulum (ER) localized transmembrane protein encoded by the gene VKORC1, reduces vitamin K quinone and vitamin K epoxide to vitamin K hydroquinone (Li et al, 2004; Rost et al, 2004). Vitamin K hydroquinone is required to enable gamma-glutamyl carboxylase (GGCX) to carboxylate Gla domains on vitamin K-dependent blood clotting factors. VKOR is inhibited by the anticoagulant drug warfarin (Czogalla et al, 2017; Zimmermann and Matschiner, 1974), and VKORC1 polymorphisms contribute to an estimated ~25% of warfarin dosing variability (Owen et al, 2010).

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Conclusion