ESRF - The European Synchrotron, Structural Biology Group, 38000 Grenoble, France; ALBA Synchrotron, 08290 Cerdanyola del Vallès, Spain.
IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal; Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal.
Cell Chem Biol. 2021 Jan 21;28(1):26-33.e8. doi: 10.1016/j.chembiol.2020.10.002. Epub 2020 Oct 22.
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.
尽管仅含有 32 个残基，但采采蝇凝血酶抑制剂 (TTI) 是目前为止描述的最有效的抗凝剂之一，对凝血酶的抑制活性达到皮摩尔级。出人意料的是，从采采蝇中分离得到的 TTI 比合成和重组变体的活性高 2000 倍，分子量也重 180Da。我们预测 TTI 存在酪氨酸 O-硫酸化的翻译后修饰，这促使我们研究该修饰对抗凝活性的影响。我们采用化学合成和功能测定相结合的方法发现，硫酸化显著提高了 TTI 对凝血酶的抑制活性。通过 X 射线晶体学，我们表明 TTI 的 N 端硫酸化片段与凝血酶的基本外显子 II 结合，建立了与生理底物相似的相互作用，而 C 端片段则使凝血酶的催化活性丧失。这种非典型的抑制模式，加上其强大的活性和较小的分子量，使 TTI 成为设计新型抗血栓药物的有吸引力的支架。
