Important role of the cys-191 cys-220 disulfide bond in thrombin function and allostery

J Biol Chem. 2007 Sep 14;282(37):27165-27170. doi: 10.1074/jbc.M703202200. Epub 2007 Jul 18.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Allosteric Site
  • Crystallization
  • Cysteine
  • Disulfides / chemistry*
  • Protein Conformation
  • Sodium / metabolism
  • Structure-Activity Relationship
  • Thrombin / chemistry*
  • Thrombin / physiology*


  • Disulfides
  • Sodium
  • Thrombin
  • Cysteine

Associated data

  • PDB/2PGB
  • PDB/2PGQ