Stabilization of warfarin-binding pocket of VKORC1 and VKORL1 by a peripheral region determines their different sensitivity to warfarin inhibition

J Thromb Haemost. 2018 Jun;16(6):1164-1175. doi: 10.1111/jth.14127. Epub 2018 May 20.

Abstract

Essentials VKORL1 and VKORC1 have a similar overall structure and warfarin-binding pocket. A peripheral region stabilizing this pocket controls warfarin sensitivity of the VKOR paralogs. A human single nucleotide polymorphism in this region renders VKORL1 sensitive to warfarin. A group of warfarin-resistant mutations in VKORC1 acts by disrupting peripheral interactions.

Summary: Background The human genome encodes two paralogs of vitamin-K-epoxide reductase, VKORC1 and VKORL1, that support blood coagulation and other vitamin-K-dependent processes. Warfarin inhibits both enzymes, but VKORL1 is relatively resistant to warfarin. Objectives To understand the difference between VKORL1 and VKORC1, and the cause of warfarin-resistant (WR) mutations in VKORC1. Methods We performed systematic mutagenesis and analyzed warfarin responses with a cell-based activity assay. Mass spectrometry analyses were used to detect cellular redox state. Results VKORC1 and VKORL1 adopt a similar intracellular redox state with four-transmembrane-helix topology. Most WR mutations identified in VKORC1 also confer resistance in VKORL1, indicating that warfarin inhibits these paralogs at a common binding site. A group of WR mutations, distant from the warfarin-binding site, show significantly less resistance in VKORL1 than in VKORC1, implying that their different warfarin responses are determined by peripheral interactions. Remarkably, we identify a critical peripheral region in which single mutations, Glu37Lys or His46Tyr, drastically increase the warfarin sensitivity of VKORL1. In the background of these warfarin-sensitive VKORL1 mutants, WR mutations showing relative less resistance in wild-type VKORL1 become much more resistant, suggesting a structural conversion to resemble VKORC1. At this peripheral region, we also identified a human single nucleotide polymorphism that confers warfarin sensitivity of VKORL1. Conclusions Peripheral regions of VKORC1 and VKORL1 primarily maintain the stability of their common warfarin-binding pocket, and differences of such interactions determine their relative sensitivity to warfarin inhibition. This new model also explains most WR mutations located at the peripheral regions of VKORC1.

Keywords: blood coagulation; drug resistance; vitamin K; vitamin K epoxide reductases; warfarin.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anticoagulants / chemistry
  • Anticoagulants / metabolism
  • Anticoagulants / pharmacology*
  • Binding Sites
  • Drug Resistance* / genetics
  • HEK293 Cells
  • Humans
  • Models, Molecular
  • Mutation
  • Oxidation-Reduction
  • Polymorphism, Single Nucleotide
  • Protein Binding
  • Protein Conformation
  • Protein Stability
  • Structure-Activity Relationship
  • Vitamin K Epoxide Reductases / antagonists & inhibitors*
  • Vitamin K Epoxide Reductases / chemistry
  • Vitamin K Epoxide Reductases / genetics
  • Vitamin K Epoxide Reductases / metabolism
  • Warfarin / chemistry
  • Warfarin / metabolism
  • Warfarin / pharmacology*

Substances

  • Anticoagulants
  • Warfarin
  • VKORC1 protein, human
  • VKORC1L1 protein, human
  • Vitamin K Epoxide Reductases