Abstract:
: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.
journal_name
J Mol Bioljournal_title
Journal of molecular biologyauthors
Li S,Liu S,Yang Y,Li Wdoi
10.1016/j.jmb.2020.05.009subject
Has Abstractpub_date
2020-08-21 00:00:00pages
5197-5208issue
18eissn
0022-2836issn
1089-8638pii
S0022-2836(20)30350-8journal_volume
432pub_type
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