Structural basis of antagonizing the vitamin K catalytic cycle for anticoagulation

Anticoagulants take over In its fully reduced form, vitamin K helps to catalyze an oxidation reaction essential for blood coagulations, but it must be regenerated in each reaction through reduction. Liu et al. determined the structures of the enzyme responsible, vitamin K epoxide reductase, bound to an oxidized vitamin K substrate or with anticoagulants, including the widely prescribed drug warfarin. Understanding how these molecules bind and inhibit explains some variations in the therapeutic response as well as resistance for anticoagulants used in pest control. Science, this issue p. 43 X-ray crystal structures reveal how anticoagulants inhibit a vitamin K regenerating enzyme. INTRODUCTION Vitamin K antagonists (VKAs), such as warfarin, are oral anticoagulants commonly used to treat and prevent thromboembolic diseases, including stroke and heart attack. Vitamin K supplementation, on the other hand, has saved the lives of numerous newborns with deficient hemostasis. Central to these therapeutic processes is vitamin K epoxide reductase (VKOR), an endoplasmic membrane enzyme that generates the active form of vitamin K to support blood coagulation. VKAs inhibit VKOR catalysis, which is carried out by two cysteine pairs, one directly reducing substrates and another mediating electron transfers. VKOR homologs and paralogs (VKOR-like) constitute a large family of integral membrane thiol oxidoreductases, but understanding their catalytic and inhibitory mechanisms has been challenging in the absence of high-resolution structures. RATIONALE Overdose of VKAs often causes major or fatal bleeding, accounting for one-third of hospitalizations for all adverse drug reactions in older adults. Despite decades of clinical difficulties, mechanistic insights are lacking for the antagonism of VKAs and for their target enzyme VKOR, which manages to transfer electrons across the water-membrane interface to support its distinct activity of epoxide reduction. Here, we present 11 crystal structures of human VKOR and a VKOR-like paralog with various substrates and antagonists in different functional states, revealing nearly the entire catalytic cycle of VKOR enzymes and the action of VKAs. RESULTS Structures with four representative VKAs show that their hydrogen bonding with Asn80 and Tyr139 provides the recognition specificity in a largely hydrophobic pocket. This VKA-binding pocket, also serving as the active site, is surrounded by a four-transmembrane-helix bundle and covered by a cap domain. Mutations that destabilize the cap domain or directly disrupt the warfarin-binding interactions result in warfarin resistance. Metabolic inactivation of warfarin is through a hydroxyl modification that is energetically unfavorable in the hydrophobic pocket. High-potency inhibition by “superwarfarins” is afforded by their large side groups that bind to a tunnel designated for the isoprenyl chain of vitamin K. Structural comparison with a ligand-free state suggests that local binding interactions of warfarin lead to a global change from open to closed protein conformation. Substrate-bound structures reveal that a reduced active-site cysteine is required to form a charge-transfer complex or covalent complex. These substrate adducts form hydrogen bonds with Asn80 and Tyr139 to facilitate the catalytic chemistry, and their stable binding interactions induce the closed protein conformation. This conformational change brings together the two pairs of cysteines, triggering electron transfer that enables the reduction of substrates. CONCLUSION The structures reveal an activation mechanism in which stably bound substrate adducts trigger restructuring of VKOR to promote electron transfer. The high potency of VKAs results from mimicking the conformational change at the global level and the substrate hydrogen-bonding interactions at the local level. These mechanistic insights provide the basis to design new therapeutic strategies of anticoagulation. The catalytic cycle and inhibition of VKOR. Partially oxidized VKOR forms a cysteine adduct with substrates, vitamin K epoxide, or quinone, whose binding induces a closed conformation, juxtaposing all cysteines (S–S or SH) for unimpeded electron transfer. VKOR becomes fully oxidized with an open conformation that releases reaction products, vitamin K quinone, or hydroquinone. Warfarin locks VKOR in both redox states into the closed conformation. The luminal and transmembrane domains of VKOR are shown as a pink hemisphere and gray cylinder, respectively. Y139, Tyr139; N80, Asn80. Vitamin K antagonists are widely used anticoagulants that target vitamin K epoxide reductases (VKOR), a family of integral membrane enzymes. To elucidate their catalytic cycle and inhibitory mechanism, we report 11 x-ray crystal structures of human VKOR and pufferfish VKOR-like, with substrates and antagonists in different redox states. Substrates entering the active site in a partially oxidized state form cysteine adducts that induce an open-to-closed conformational change, triggering reduction. Binding and catalysis are facilitated by hydrogen-bonding interactions in a hydrophobic pocket. The antagonists bind specifically to the same hydrogen-bonding residues and induce a similar closed conformation. Thus, vitamin K antagonists act through mimicking the key interactions and conformational changes required for the VKOR catalytic cycle.