Heparin Binds Lamprey Angiotensinogen and Promotes Thrombin Inhibition through a Template Mechanism
| Autor: | Ying Zheng, Hermann Ragg, Yunjie Wang, Haiyan Cai, Zhenquan Wei, Ruoxi Zhang, Jiawei Wu, Aiwu Zhou, Xin Huang, Lina Ma, Fei Zhang, Hudie Wei, Lingling Feng |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Fish Proteins Conformational change Angiotensins Peptide Serpin Biochemistry Protein Structure Secondary 03 medical and health sciences Thrombin Protein Domains medicine Animals Binding site Molecular Biology Reactive center chemistry.chemical_classification 030102 biochemistry & molecular biology Chemistry Heparin Lampreys Cell Biology Dissociation constant 030104 developmental biology Mutation Protein Structure and Folding medicine.drug |
| Zdroj: | The Journal of biological chemistry. 291(48) |
| ISSN: | 1083-351X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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