Heparin Binds Lamprey Angiotensinogen and Promotes Thrombin Inhibition through a Template Mechanism
Heparin Binds Lamprey Angiotensinogen and Promotes Thrombin Inhibition through a Template Mechanism
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肝素结合七鳃鳗血管紧张素原并通过模板机制促进凝血酶抑制。
DOI：
10.1074/jbc.m116.725895
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发表时间：
2016-11-25
影响因子：
4.8
通讯作者：
Zhou, Aiwu
中科院分区：
文献类型：
--
作者：
Wei, Hudie;Cai, Haiyan;Zhou, Aiwu
七鳃鳗血管紧张素原（l-ANT）是调节血压的激素载体，同时也是七鳃鳗凝血系统中肝素依赖性凝血酶抑制剂。 l-ANT 如何携带血管紧张素以及肝素如何结合 l-ANT 并介导凝血酶抑制的详细机制尚不清楚。在这里，我们以 2.7 埃的分辨率解析了裂解的 l-ANT 的晶体结构，并表征了其肝素结合和蛋白酶抑制的特性。该结构表明，l-ANT 具有保守的丝氨酸蛋白酶抑制剂折叠和不稳定的 N 端血管紧张素肽，并且当反应中心环被裂解时，会经历典型的应激至松弛构象变化。肝素以类似于 10 nM 的解离常数紧密结合 l-ANT，涉及类似于 8 个单糖和类似于 6 个离子相互作用。肝素结合位点位于螺旋 D 周围广泛的带正电荷的表面区域，涉及残基 Lys-148、Lys-151、Arg-155 和 Arg-380。尽管l-ANT本身是一种较差的凝血酶抑制剂，其二阶速率常数为500 M-1 s(-1)，但其与凝血酶的相互作用被高分子量肝素加速90倍，遵循钟形剂量依赖性曲线。 6-20 个单糖单元的短肝素链不足以促进凝血酶抑制。此外，P1 Ile 突变为 Arg 的 l-ANT 突变体抑制凝血酶的速度比野生型快近 1500 倍，高分子量肝素进一步加速抑制凝血酶的速度。综上所述，这些结果表明肝素在螺旋 D 周围的保守肝素结合位点结合 l-ANT，并通过脊椎动物中保守的模板机制促进 l-ANT 和凝血酶之间的相互作用。
Lamprey angiotensinogen (l-ANT) is a hormone carrier in the regulation of blood pressure, but it is also a heparin-dependent thrombin inhibitor in lamprey blood coagulation system. The detailed mechanisms on how angiotensin is carried by l-ANT and how heparin binds l-ANT and mediates thrombin inhibition are unclear. Here we have solved the crystal structure of cleaved l-ANT at 2.7 angstrom resolution and characterized its properties in heparin binding and protease inhibition. The structure reveals that l-ANT has a conserved serpin fold with a labile N-terminal angiotensin peptide and undergoes a typical stressed-to-relaxed conformational change when the reactive center loop is cleaved. Heparin binds l-ANT tightly with a dissociation constant of similar to 10 nM involving similar to 8 monosaccharides and similar to 6 ionic interactions. The heparin binding site is located in an extensive positively charged surface area around helix D involving residues Lys-148, Lys-151, Arg-155, and Arg-380. Although l-ANT by itself is a poor thrombin inhibitor with a second order rate constant of 500 M-1 s(-1), its interaction with thrombin is accelerated 90-fold by high molecular weight heparin following a bell-shaped dose-dependent curve. Short heparin chains of 6-20 monosaccharide units are insufficient to promote thrombin inhibition. Furthermore, an l-ANT mutant with the P1 Ile mutated to Arg inhibits thrombin nearly 1500-fold faster than the wild type, which is further accelerated by high molecular weight heparin. Taken together, these results suggest that heparin binds l-ANT at a conserved heparin binding site around helix D and promotes the interaction between l-ANT and thrombin through a template mechanism conserved in vertebrates.