Structural Investigation of the Vitamin K Epoxide Reductase (VKORC1) Binding Site with Vitamin K

Abstract

The vitamin K epoxide reductase (VKORC1) enzyme is of primary importance in many physiological processes, i.e., blood coagulation, energy metabolism, and arterial calcification prevention, due to its role in the vitamin K cycle. Indeed, VKORC1 catalyzes reduction of vitamin K epoxide to quinone and then to hydroquinone. However, the three-dimensional VKORC1 structure remains experimentally undetermined, because of the endoplasmic reticulum membrane location of this enzyme. Here we present a molecular modeling investigation of the VKORC1 enzymatic site structure and function, supported by in vitro enzymatic assays. Four VKORC1 mutants were designed in silico (F55G, F55Y, N80G, and F83G) based on a previous study that identified residues F55, N80, and F83 as being crucial for vitamin K epoxide binding. F55G, N80G, and F83G nonconservative mutants were all predicted to be inactive by molecular modeling analyses. However, the F55Y conservative mutant was expected to be active compared to wild-type VKORC1. In vitro enzymatic assays performed on recombinant proteins assessed our molecular modeling hypotheses and led us to describe the role of accurate VKORC1 active site residues with respect to VKORC1. Residues F55, N80, and F83 appeared to act in a concerted manner to keep vitamin K epoxide close to the C135 catalytic residue. Residues F55 and N80 prevent naphthoquinone head rotation away from the active site, assisted by residue F83 that prevents vitamin K from sliding outside the enzymatic pocket, through hydrophobic tail stabilization. Our results thus highlighted the specific functions of VKORC1 catalytic pocket residues and evidenced the ability of our structural model to predict biological effects of VKORC1 mutations.

Potential impact on coagulopathy of gene variants of coagulation related proteins that interact with SARS-CoV-2

“…VKORC1 and ORF7a were confirmed to have strong binding affinity. Interactions are generally limited to transmembrane helices as opposed to intervening loops where warfarin is known to bind [ 60 ]. The top scoring complexes are shown in Fig 1 .…”

“…Tetherin inhibits 3 virion dispersal [105], and several viruses, including HIV, have auxiliary proteins to counter this 3 effect. The structures of tetherin and VKORC1 are noticeably similar, sharing a coiled-coil 3 architecture: VKORC1 has four alpha helices bound together [60], while tetherin exists as a 3…”

“…Tetherin inhibits virion dispersal [ 105 ], and several viruses, including HIV, have auxiliary proteins to counter this effect. The structures of tetherin and VKORC1 are noticeably similar, sharing a coiled-coil architecture: VKORC1 has four alpha helices bound together [ 60 ], while tetherin exists as a homomer of four alpha helices [ 106 ] [ 107 ]. Reduced VKORC1 expression or binding to ORF7a, as it may occur with some variants, may increase the availability of ORF7a to bind and inhibit tetherin increasing the severity of SARS-CoV-2 infection.…”

“…VKORC1 2 and ORF7a were confirmed to have strong binding affinity. Interactions are generally limited to 2 transmembrane helices as opposed to intervening loops where warfarin is known to bind [60]. PABPC4, due to the lack of structural data from similar proteins, sections of the models for 2 PABPC4 and SERPING1 were of low quality.…”

Computational ligands to VKORC1s and CYPs. Could they predict new anticoagulant rodenticides?

“…The modeled 3D structure of rat VKORC1 contains 5 short α-helices and 6 Cysteins (C), 4 of which are conserved among species. Cysteins C51-C132 form a disulphide bridge in humans 13 . Helices are highly hydrophobic at their carboxy-terminal segment ( Figure 1D), containing the most abundant mutations correlating with rat anticoagulant resistance ( Figure 1,B,D).…”