Structural disruption of VKORC1 by resistance mutations reveals a conserved hydrophobic pattern essential for...

Vitamin K epoxide reductase (VKORC1) catalyzes the reduction of vitamin K epoxide to quinone and hydroquinone, a cofactor essential for the activation of clotting factors. Mutations in VKORC1 are known to confer resistance to vitamin K antagonists (VKAs) such as warfarin in both humans and rodents, yet the structural mechanisms underlying these resistances remain poorly understood. Using AlphaFold3-predicted structures, molecular docking and molecular dynamics simulations, we investigated the effects of four major resistance-causing mutations - W59G and L128S in Mus musculus, L120Q and Y139C in Rattus norvegicus - on VKORC1 structure and warfarin binding. Three mutations (W59G, L120Q and L128S) disrupted a hydrophobic cluster linking the ER luminal loop to transmembrane helices TM3-TM4, inducing and ER loop drift and altering helices orientation. These structural changes reduced warfarin affinity compared to the wild-type enzyme. In contrast, the Y139C mutation caused minimal perturbation and preserved wild-type binding energy, suggesting that it may confer resistance through a different mechanism. Collectively, six hydrophobic residues (V54, W59, L120, I123, L124, L128) were identified as a conserved structural pattern critical for VKORC1 stability and inhibitor sensitivity. This integrative structural study provides a mechanistic framework for understanding VKA resistance in VKORC1. It highlights a hydrophobic core essential for enzyme integrity and drug binding, offering a molecular basis for monitoring emerging mutations and for designing next-generation anticoagulants active against resistant VKORC1 variants.