Journal article

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

Barbara 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 Jose Barbosa Pereira



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-termin..

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University of Melbourne Researchers


Awarded by Fundacao para a Ciencia e a Tecnologia

Awarded by project Programa PESSOA/PHC PESSOA 2016


Awarded by National Health and Medical Research Council

Awarded by FRISBI within the Grenoble Partnership for Structural Biology

Awarded by CALIPSOplus

Awarded by GRAL within the Grenoble Partnership for Structural Biology

Funding Acknowledgements

This work was funded in part by Fundacao para a Ciencia e a Tecnologia through contracts DL 57/2016/CP1355/CT0011 (to J.R.-R.) and IF/00092/2014/CP1255/CT0004 (to N.V.), project Programa PESSOA 441.00 (to P.J.B.P.)/PHC PESSOA 2016 (35797UH) (to D.d.S.), and grant UIDB/QUI/50006/2020 (LAQV-REQUIMTE), and by the National Health and Medical Research Council (Project Grant APP1120941 to R.J.P. and P.J.B.P. and Investigator Grant APP1174941 to R.J.P.). We acknowledge access to the platforms of the Grenoble Instruct Centre (ISBG; UMS 3518 CNRS-CEAUJF-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology. We also acknowledge support from the John A. Lamberton Scholarship (to L.J.D. and C.F.). We thank Luca Signor for assistance with and access to the mass spectrometry facility, the ESRF (Grenoble, France) for provision of synchrotron radiation facilities, and their staff for help with data collection. Some of these experiments were performed at beamline BL13-XALOC of the ALBA Synchrotron (Cerdanyola del Valle's, Spain), with the collaboration of ALBA staff and CALIPSOplus (grant 730872) funding. The support of the X-Ray Crystallography and BioSciences Screening platforms of i3S (Porto, Portugal) is also acknowledged.