School of Chemistry, The University of Sydney, Sydney, New South Wales 2006, Australia.
IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal.
Nat Chem. 2017 Sep;9(9):909-917. doi: 10.1038/nchem.2744. Epub 2017 Mar 20.
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.
Madanin-1 和 chimadanin 是两种不含半胱氨酸的凝血酶抑制剂，可促进蜱虫长角血蜱的吸血。在这里，我们报告了这两种蛋白质的一种翻译后修饰——酪氨酸硫酸化，这对于强大的抗血栓和抗凝活性至关重要。杆状病毒感染的昆虫细胞中产生的抑制剂显示出两种蛋白质中每个蛋白质的两个酪氨酸残基的不均一硫酸化。一锅连接-脱硫化学方法可获得所有可能的蛋白质硫酸化变体的同质样品。Madanin-1 和 chimadanin 的酪氨酸硫酸化被证明对凝血酶抑制活性至关重要，双硫酸化变体比未修饰的抑制剂强三个数量级。Madanin-1 与凝血酶复合物的三维结构揭示了一种独特的抑制模式，硫酸化的酪氨酸残基与蛋白酶的碱性外切酶 II 结合。该家族凝血酶抑制剂中的酪氨酸硫酸化及其独特的结合模式，为基于这些特权支架开发抗血栓药物先导化合物铺平了道路。
