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  • Sulfotyrosine-Mediated Recognition of Human Thrombin by a Tsetse Fly Anticoagulant Mimics Physiological Substrates.

Sulfotyrosine-Mediated Recognition of Human Thrombin by a Tsetse Fly Anticoagulant Mimics Physiological Substrates.

Cell chemical biology (2020-10-24)
Bárbara M Calisto, Jorge Ripoll-Rozada, Luke J Dowman, Charlotte Franck, Stijn M Agten, Benjamin L Parker, Rita Carvalho Veloso, Nuno Vale, Paula Gomes, Daniele de Sanctis, Richard J Payne, Pedro José Barbosa Pereira
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Poly(ethylene glycol), BioXtra, average mol wt 3,350, powder
Sigma-Aldrich
Magnesium sulfate hydrate, puriss., dried, 99.0-101.0% MgSO4 basis (in dried substance)
Sigma-Aldrich
Sodium cacodylate trihydrate, ≥98%