Hudie Wei, Haiyan Cai, Jiawei Wu, Zhenquan Wei, Fei Zhang, Xin Huang, Lina Ma, Lingling Feng, Ruoxi Zhang, Yunjie Wang, Hermann Ragg, Ying Zheng, Aiwu Zhou
The Journal of biological chemistry 2016 Nov 25Lamprey 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 Å 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 ∼10 nm involving ∼8 monosaccharides and ∼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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
Hudie Wei, Haiyan Cai, Jiawei Wu, Zhenquan Wei, Fei Zhang, Xin Huang, Lina Ma, Lingling Feng, Ruoxi Zhang, Yunjie Wang, Hermann Ragg, Ying Zheng, Aiwu Zhou. Heparin Binds Lamprey Angiotensinogen and Promotes Thrombin Inhibition through a Template Mechanism. The Journal of biological chemistry. 2016 Nov 25;291(48):24900-24911
PMID: 27681598
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