2PGB

Inhibitor-free human thrombin mutant C191A-C220A

Summary for 2PGB

• Entry DOI: 10.2210/pdb2pgb/pdb
• Related: 1SHH 2PGQ
• Descriptor: Prothrombin, 2-acetamido-2-deoxy-beta-D-glucopyranose, SULFATE ION, ... (5 entities in total)
• Functional Keywords: serine protease, hydrolase
• Biological source: Homo sapiens (human); More
• Total number of polymer chains: 2
• Total formula weight: 34225.96

Authors

Bush-Pelc, L.A.,Marino, F.,Chen, Z.,Pineda, A.O.,Mathews, F.S.,Di Cera, E. (deposition date: 2007-04-09, release date: 2007-07-17, Last modification date: 2024-10-30)

Primary citation

Bush-Pelc, L.A.,Marino, F.,Chen, Z.,Pineda, A.O.,Mathews, F.S.,Di Cera, E.
Important role of the cys-191 cys-220 disulfide bond in thrombin function and allostery
J.Biol.Chem., 282:27165-27170, 2007

PubMed Abstract: 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.

PubMed: 17636263
DOI: 10.1074/jbc.M703202200

Experimental method

X-RAY DIFFRACTION (1.54 Å)