NMR resonance assignments of thrombin reveal the conformational and dynamic effects of ligation
- 21 July 2010
- journal article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences of the United States of America
- Vol. 107 (32), 14087-14092
- https://doi.org/10.1073/pnas.1005255107
Abstract
The serine protease thrombin is generated from its zymogen prothrombin at the end of the coagulation cascade. Thrombin functions as the effector enzyme of blood clotting by cleaving several procoagulant targets, but also plays a key role in attenuating the hemostatic response by activating protein C. These activities all depend on the engagement of exosites on thrombin, either through direct interaction with a substrate, as with fibrinogen, or by binding to cofactors such as thrombomodulin. How thrombin specificity is controlled is of central importance to understanding normal hemostasis and how dysregulation causes bleeding or thrombosis. The binding of ligands to thrombin via exosite I and the coordination of Na(+) have been associated with changes in thrombin conformation and activity. This phenomenon has become known as thrombin allostery, although direct evidence of conformational change, identification of the regions involved, and the functional consequences remain unclear. Here we investigate the conformational and dynamic effects of thrombin ligation at the active site, exosite I and the Na(+)-binding site in solution, using modern multidimensional NMR techniques. We obtained full resonance assignments for thrombin in seven differently liganded states, including fully unliganded apo thrombin, and have created a detailed map of residues that change environment, conformation, or dynamic state in response to each relevant single or multiple ligation event. These studies reveal that apo thrombin exists in a highly dynamic zymogen-like state, and relies on ligation to achieve a fully active conformation. Conformational plasticity confers upon thrombin the ability to be at once selective and promiscuous.Keywords
This publication has 37 references indexed in Scilit:
- Molecular basis of thrombomodulin activation of slow thrombinJournal of Thrombosis and Haemostasis, 2009
- Slow thrombin is zymogen-likeJournal of Thrombosis and Haemostasis, 2009
- How Na+ activates thrombin – a review of the functional and structural dataBiological Chemistry, 2008
- ThrombinMolecular Aspects of Medicine, 2008
- Mechanism of Na+ binding to thrombin resolved by ultra-rapid kineticsBiophysical Chemistry, 2007
- Expression of Allosteric Linkage between the Sodium Ion Binding Site and Exosite I of Thrombin during Prothrombin ActivationPublished by Elsevier BV ,2007
- Solvent accessibility of the thrombin-thrombomodulin interfaceJournal of Molecular Biology, 2001
- Molecular Recognition by Thrombin. Role of the Slows→Fast Transition, Site-specific Ion Binding Energetics and Thermodynamic Mapping of Structural ComponentsJournal of Molecular Biology, 1994
- Protein crystals and their stabilityJournal of Crystal Growth, 1992
- On the size of the active site in proteases. I. PapainBiochemical and Biophysical Research Communications, 1967