TY - JOUR
T1 - Characterization of Warfarin Inhibition Kinetics Requires Stabilization of Intramembrane Vitamin K Epoxide Reductases
AU - Li, Shuang
AU - Liu, Shixuan
AU - Yang, Yihu
AU - Li, Weikai
N1 - Funding Information:
W.L. is supported by NHLBI ( R01 HL121718 ), W. M. Keck Foundation (Basic Science @ the Forefront of Science), Children's Discovery Institute ( MC-II-2020-854 ), NEI ( R21 EY028705 ), and NIGMS ( R01 GM131008 ).
Publisher Copyright:
© 2020 Elsevier Ltd
PY - 2020/8/21
Y1 - 2020/8/21
N2 - 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 inhibitor-binding conformation enables the characterization of enzyme kinetics in difficult cases.
AB - 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 inhibitor-binding conformation enables the characterization of enzyme kinetics in difficult cases.
KW - VKOR
KW - intramembrane enzyme
KW - protein stability
KW - tight-binding inhibition
KW - warfarin
UR - http://www.scopus.com/inward/record.url?scp=85086508433&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2020.05.009
DO - 10.1016/j.jmb.2020.05.009
M3 - Article
C2 - 32445640
AN - SCOPUS:85086508433
VL - 432
SP - 5197
EP - 5208
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 18
ER -