Cell Chemical Biology
Volume 28, Issue 1, 21 January 2021, Pages 26-33.e8
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Article
Sulfotyrosine-Mediated Recognition of Human Thrombin by a Tsetse Fly Anticoagulant Mimics Physiological Substrates

https://doi.org/10.1016/j.chembiol.2020.10.002Get rights and content
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Highlights

  • The diminutive TTI is one of the most potent anticoagulants reported to date

  • Post-translational tyrosine O-sulfation enhances TTI activity over 1000-fold

  • TTI is a bivalent inhibitor that targets thrombin's exosite II and active site

  • The binding mode of TTI resembles that of physiologic thrombin substrates

Summary

Despite possessing only 32 residues, the tsetse thrombin inhibitor (TTI) is among the most potent anticoagulants described, with sub-picomolar inhibitory activity against thrombin. Unexpectedly, TTI isolated from the fly is 2000-fold more active and 180 Da heavier than synthetic and recombinant variants. We predicted the presence of a tyrosine O-sulfate post-translational modification of TTI, prompting us to investigate the effect of the modification on anticoagulant activity. A combination of chemical synthesis and functional assays was used to reveal that sulfation significantly improved the inhibitory activity of TTI against thrombin. Using X-ray crystallography, we show that the N-terminal sulfated segment of TTI binds the basic exosite II of thrombin, establishing interactions similar to those of physiologic substrates, while the C-terminal segment abolishes the catalytic activity of thrombin. This non-canonical mode of inhibition, coupled with its potency and small size, makes TTI an attractive scaffold for the design of novel antithrombotics.

Keywords

coagulation inhibitor
macromolecular recognition
post-translational modification
tyrosine sulfation
protein-protein interaction
anticoagulant
thrombin inhibitor
three-dimensional structure
X-ray crystallography
Glossina morsitans

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These authors contributed equally

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