Crystal Structure of Anticoagulant Thrombin Variant E217K Provides Insights into Thrombin Allostery
| Autor: | Timothy Myles, Wendy J. Carter, Lawrence L.K. Leung, Craig S. Gibbs, James A. Huntington |
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| Rok vydání: | 2004 |
| Předmět: |
Models
Molecular Protein Conformation Allosteric regulation Crystallography X-Ray Fibrinogen Biochemistry Catalysis Fibrin Thrombin medicine Humans Binding site Saturated mutagenesis Molecular Biology Binding Sites biology Chemistry Sodium Active site Hydrogen Bonding Cell Biology Mutagenesis Site-Directed biology.protein Biophysics Oxyanion hole Allosteric Site Protein Binding circulatory and respiratory physiology medicine.drug |
| Zdroj: | Journal of Biological Chemistry. 279:26387-26394 |
| ISSN: | 0021-9258 |
| Popis: | Thrombin is the ultimate protease of the blood clotting cascade and plays a major role in its own regulation. The ability of thrombin to exhibit both pro- and anti-coagulant properties has spawned efforts to turn thrombin into an anticoagulant for therapeutic purposes. This quest culminated in the identification of the E217K variant through scanning and saturation mutagenesis. The antithrombotic properties of E217K thrombin are derived from its inability to convert fibrinogen to a fibrin clot while maintaining its thrombomodulin-dependent ability to activate the anticoagulant protein C pathway. Here we describe the 2.5-A crystal structure of human E217K thrombin, which displays a dramatic restructuring of the geometry of the active site. Of particular interest is the repositioning of Glu-192, which hydrogen bonds to the catalytic Ser-195 and which results in the complete occlusion of the active site and the destruction of the oxyanion hole. Substrate binding pockets are further blocked by residues previously implicated in thrombin allostery. We have concluded that the E217K mutation causes the allosteric inactivation of thrombin by destabilizing the Na+ binding site and that the structure thus may represent the Na+-free, catalytically inert “slow” form. |
| Databáze: | OpenAIRE |
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