Hirudin Binding Reveals Key Determinants of Thrombin Allostery

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

doi:10.1074/jbc.m502678200

Kristen E Mengwasser, Leslie A Bush + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m502678200

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Abstract

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 79 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 S-->F 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, and 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.

Highlights

The serine protease thrombin is a member of the large class of enzymes activated by monovalent cations [1] and requires Naϩ for optimal catalytic activity
The value of Ki for hirudin binding was determined in the S and F forms (Table I and Fig. 1 using Eq 2 and the values of Kd for Naϩ binding reported elsewhere [12]
Energetic mapping of the epitopes of thrombin recognizing hirudin in the S and F forms is relevant to understand how the enzyme fulfills its procoagulant, prothrombotic, and signaling roles and to identify the residues of thrombin responsible for the higher affinity of the F form

Summary

Introduction

The serine protease thrombin is a member of the large class of enzymes activated by monovalent cations [1] and requires Naϩ for optimal catalytic activity. Ser-195 is within H-bonding distance of its catalytic partner His-57 in the F form, but not in the S form, which ensures optimal substrate acylation Whether such changes around Asp-189 and Ser-195 are responsible for the higher specificity of the F form toward fibrinogen and/or PAR1 remains to be demonstrated. The compact N-terminal domain of the inhibitor occludes the active site of the enzyme and contacts the 60-loop, the aryl binding site, and the Naϩ site [22, 23] These are the same regions probed by fibrinogen and PAR1 upon binding to thrombin [24, 25]. Hirudin mimics important physiologic substrates in their interaction with thrombin, and, at the same time, it is sensitive to structural changes between the F and S forms that are at the basis of the procoagulant, prothrombotic, and signaling functions of the enzyme.

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