Madanin-1 and chimadanin are two small cysteine-free thrombin inhibitors that facilitate blood feeding in the tick Haemaphysalis longicornis. Here, we report a post-translational modification—tyrosine sulfation—of these two proteins that is critical for potent anti-thrombotic and anticoagulant activity. Inhibitors produced in baculovirus-infected insect cells displayed heterogeneous sulfation of two tyrosine residues within each of the proteins. One-pot ligation–desulfurization chemistry enabled access to homogeneous samples of all possible sulfated variants of the proteins. Tyrosine sulfation of madanin-1 and chimadanin proved crucial for thrombin inhibitory activity, with the doubly sulfated variants three orders of magnitude more potent than the unmodified inhibitors. The three-dimensional structure of madanin-1 in complex with thrombin revealed a unique mode of inhibition, with the sulfated tyrosine residues binding to the basic exosite II of the protease. The importance of tyrosine sulfation within this family of thrombin inhibitors, together with their unique binding mode, paves the way for the development of anti-thrombotic drug leads based on these privileged scaffolds.

中文翻译：

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酪氨酸硫酸化调节tick信号凝血酶抑制剂的活性

Madanin-1和chimadanin是两种小的无半胱氨酸的凝血酶抑制剂，可促进the血中的血供。在这里，我们报道了这两种蛋白质的翻译后修饰（酪氨酸硫酸化），对有效的抗血栓形成和抗凝活性至关重要。在杆状病毒感染的昆虫细胞中产生的抑制剂表现出每种蛋白质中两个酪氨酸残基的异质硫酸化。一锅式连接-脱硫化学试剂可以获取蛋白质所有可能的硫酸化变体的均质样品。证明madanin-1和chimadanin的酪氨酸硫酸化对凝血酶抑制活性至关重要，双硫酸化变体的效力比未修饰的抑制剂高三个数量级。madanin-1与凝血酶复合的三维结构显示出独特的抑制模式，硫酸化的酪氨酸残基与蛋白酶的碱性外位II结合。