# Structural and Functional Basis of the Vitamin K Cycle

> **NIH NIH R01** · WASHINGTON UNIVERSITY · 2021 · $393,750

## Abstract

The vitamin K cycle supports blood coagulation, bone mineralization, and vascular calcium homeostasis. A key
enzyme in this cycle, vitamin K epoxide reductase (VKOR), is the target of vitamin K antagonists (VKAs). Despite
their extensive clinical use, the dose of VKAs (e.g., warfarin) is hard to regulate and overdose can lead to fatal
bleeding. Improving the dose regulation requires understanding how VKAs inhibit VKOR, which is a membrane-
embedded enzyme that is difficult to characterize with structural and biochemical studies. Our long-term goal is
to elucidate the physiological process of the entire vitamin K cycle and its interaction with VKAs. This cycle
begins with γ-carboxylation, a modification required for the activity of vitamin-K-dependent proteins, including
several coagulation factors. The carboxylase activity requires the epoxidation of vitamin K hydroquinone. VKOR
regenerates this cofactor by reducing the epoxide, and this reductase activity is maintained by electron-transfer
pathways. VKOR also has a paralog, VKORL, which has the same activity but is relatively insensitive to warfarin
inhibition. Owing to differences in tissue distribution, VKOR primarily supports blood coagulation and VKORL
likely supports non-coagulation processes. The objective of this application is to elucidate the mechanisms of
VKOR and VKORL catalysis and vitamin K antagonism using our expertise in membrane structure biology. Our
hypotheses are: (1) the narrow therapeutic window of warfarin is in part because it is a tight-binding inhibitor
whose dose range is limited by VKOR levels; (2) the cellular activity of VKOR is maintained by alternative
electron-transfer pathways; and (3) a common structural mechanism governs the warfarin insensitivity of VKORL
and warfarin-resistant mutations in VKOR. To support these hypotheses, we have achieved a long-standing goal
of determining the crystal structures of human VKOR with several VKAs and with the substrate, vitamin K epoxide,
and have determined the structures of a VKORL homolog in its warfarin-bound and ligand-free states. We found
distinct groups of warfarin-resistant mutations in VKOR, and identified key residues that control the warfarin
sensitivity of VKORL. We also showed that warfarin is a tight-binding inhibitor in vitro. We will test our hypotheses
with three specific aims: (1) we will show the tight binding of warfarin in a cellular environment, understand its
correlation with VKOR's redox status, and test whether reducing VKOR can release bound warfarin; (2) we will
determine how VKAs inhibit VKOR catalysis, elucidate the reduction steps and reaction intermediate of VKOR,
and characterize the electron-transfer pathways that maintain VKOR activity; and 3) we will define the structural
basis of warfarin resistance and investigate whether VKORL variations lead to osteoporosis. Armed with our
recently developed structural tools, we will demonstrate innovative concepts about the inhibition range of VKAs,
t...

## Key facts

- **NIH application ID:** 10163694
- **Project number:** 5R01HL121718-08
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** Weikai Li
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $393,750
- **Award type:** 5
- **Project period:** 2014-05-15 → 2023-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10163694

## Citation

> US National Institutes of Health, RePORTER application 10163694, Structural and Functional Basis of the Vitamin K Cycle (5R01HL121718-08). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10163694. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*