Hirudin binding reveals key determinants of thrombin allostery

doi:10.1074/jbc.M502678200

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Papers In Press, published online ahead of print May 27, 2005
J. Biol. Chem, 10.1074/jbc.M502678200
Submitted on March 10, 2005
Revised on May 26, 2005
Accepted on May 27, 2005

Hirudin binding reveals key determinants of thrombin allostery

Kristen E. Mengwasser, Leslie A. Bush, Peter Shih, Angelene M. Cantwell, and Enrico Di Cera

Dept. of Biochemistry & Molecular Biophysics, Washington Univ Med School, St Louis, 63110

Corresponding Author: enrico{at}wustl.edu

Thrombin exists in two allosteric forms, slow (S) and fast (F), that recognize natural substrates and inhibitors with significantly different affinities. Because under physiologic conditions the two forms are almost equally populated, investigation of thrombin function must address the contribution from the S and F forms and the molecular origin of their differential recognition of ligands. Using a panel of seventy-nine Ala mutants, we have mapped for the first time the epitopes of thrombin recognizing a macromolecular ligand, hirudin, in the S and F forms. Hirudin binding is a relevant model for the interaction of thrombin with fibrinogen and PAR1 and is likewise influenced by the allosteric SF transition. The epitopes are nearly identical and encompass two hot spots, one in exosite I and the other in the Na+ site at the opposite end of the protein. The higher affinity of the F form is due to the preferential interaction of hirudin with Lys-36, Leu-65, Thr-74, Arg-75 in exosite I, Gly-193 in the oxyanion hole, and Asp-221 and Asp-222 in the Na+ site. Remarkably, no correlation is found between the energetic and structural involvements of thrombin residues in hirudin recognition, which invites caution in the analysis of protein-protein interactions in general.

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