Important Role of the Cys-191–Cys-220 Disulfide Bond in Thrombin Function and Allostery
| Autor: | Agustin O. Pineda, Leslie A. Bush-Pelc, Francesca Marino, Zhi-wei Chen, F. Scott Mathews, Enrico Di Cera |
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| Rok vydání: | 2007 |
| Předmět: |
biology
Protein Conformation Stereochemistry Chemistry Sodium Allosteric regulation Thrombin Active site Cell Biology Biochemistry Structure-Activity Relationship Protein structure Hydrolase Side chain biology.protein Cysteine Disulfides Binding site Oxyanion hole Crystallization Molecular Biology Allosteric Site |
| Zdroj: | Journal of Biological Chemistry. 282:27165-27170 |
| ISSN: | 0021-9258 |
| Popis: | Little is known on the role of disulfide bonds in the catalytic domain of serine proteases. The Cys-191-Cys-220 disulfide bond is located between the 190 strand leading to the oxyanion hole and the 220-loop that contributes to the architecture of the primary specificity pocket and the Na+ binding site in allosteric proteases. Removal of this bond in thrombin produces an approximately 100-fold loss of activity toward several chromogenic and natural substrates carrying Arg or Lys at P1. Na+ activation is compromised, and no fluorescence change can be detected in response to Na+ binding. A 1.54-A resolution structure of the C191A/C220A mutant in the free form reveals a conformation similar to the Na+-free slow form of wild type. The lack of disulfide bond exposes the side chain of Asp-189 to solvent, flips the backbone O atom of Gly-219, and generates disorder in portions of the 186 and 220 loops defining the Na+ site. This conformation, featuring perturbation of the Na+ site but with the active site accessible to substrate, offers a possible representation of the recently identified E* form of thrombin. Disorder in the 186 and 220 loops and the flip of Gly-219 are corrected by the active site inhibitor H-D-Phe-Pro-Arg-CH(2)Cl, as revealed by the 1.8-A resolution structure of the complex. We conclude that the Cys-191-Cys-220 disulfide bond confers stability to the primary specificity pocket by shielding Asp-189 from the solvent and orients the backbone O atom of Gly-219 for optimal substrate binding. In addition, the disulfide bond stabilizes the 186 and 220 loops that are critical for Na+ binding and activation. |
| Databáze: | OpenAIRE |
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