Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 432, Issue 18, Pages 5197-5208Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2020.05.009
Keywords
VKOR; warfarin; intramembrane enzyme; protein stability; tight-binding inhibition
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Funding
- NHLBI [R01 HL121718]
- W. M. Keck Foundation (Basic Science @ the Forefront of Science), Children's Discovery Institute [MC-II-2020-854]
- NEI [R21 EY028705]
- NIGMS [R01 GM131008]
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Intramembrane enzymes are often difficult for biochemical characterization. Human vitamin K epoxide reductase (VKOR) is the target of warfarin. However, this intramembrane enzyme becomes insensitive to warfarin inhibition in vitro, preventing the characterization of inhibition kinetics for decades. Here we employ structural biology methods to identify stable VKOR and VKOR-like proteins and purify them to near homogeneity. We find that the key to maintain their warfarin sensitivity is to stabilize their native protein conformation in vitro. Reduced glutathione drastically increases the warfarin sensitivity of a VKOR-like protein from Takifugu rubripes, presumably through maintaining a disulfide-bonded conformation. Effective inhibition of human VKOR-like requires also the use of LMNG, a mild detergent developed for crystallography to increase membrane protein stability. Human VKOR needs to be preserved in ER-enriched microsomes to exhibit warfarin sensitivity, whereas human VKOR purified in LMNG is stable only with pre-bound warfarin. Under these optimal conditions, warfarin inhibits with tight-binding kinetics. Overall, our studies show that structural biology methods are ideal for stabilizing intramembrane enzymes. Optimizing toward their inhibitorbinding conformation enables the characterization of enzyme kinetics in difficult cases. (C) 2020 Elsevier Ltd. All rights reserved.
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