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J. Biol. Chem., Vol. 282, Issue 37, 27165-27170, September 14, 2007

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Important Role of the Cys-191–Cys-220 Disulfide Bond in Thrombin Function and Allostery^*

From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110

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 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-Å 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-CH₂Cl, as revealed by the 1.8-Å 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.

Received for publication, April 16, 2006 , and in revised form, July 2, 2007.

The atomic coordinates and structure factors (code 2PGB and 2PGQ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by National Institutes of Health Research Grants HL49413, HL58141, and HL73813. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, St. Louis, MO. 63110. Tel.: 314-362-4185; Fax: 314-362-4311; E-mail: enrico{at}wustl.edu.

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