Hirudin Binding Reveals Key Determinants of Thrombin Allostery

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 S3 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.The serine protease thrombin is a member of the large class of enzymes activated by monovalent cations (1) and requires Na ϩ for optimal catalytic activity. The effect of Na ϩ is allosteric (2) and shifts the conformation of the enzyme from a low activity slow (S) form to a high activity fast (F) form. The partitioning of thrombin between these two forms is of physiologic importance because the Na ϩ -bound F form accounts for the procoagulant (cleavage of fibrinogen) and prothrombotic/ signaling (cleavage of PAR1 and platelet activation) roles of the enzyme, whereas the Na ϩ -free S form is responsible for the anticoagulant (cleavage of protein C) role (3, 4). Ancillary procoagulant roles of thrombin, like activation of factor VIII, also require the enzyme to be in the F form (5). The Na ϩ -dependent allosteric regulation of catalytic activity is shared by other clotting proteases, such as activated protein C (6, 7) and factor Xa (8), that possess a Na ϩ binding site structurally similar (7, 9) to that found in thrombin (10). Na ϩ binding to thrombin increases both the rate of substrate diffusion into the active site and the rate of substrate acylation (2, 11). The result is that the F form interacts with chromogenic substrates, fibrinogen, and PAR1 with significantly higher k cat /K m compared with the S form (4). Recent crystal structures of the S and F forms have helped rationalize this difference (12). Na ϩ binding causes rearrangements of residues close to the Na ϩ site and located up to 15 Å away such as the catalytic Ser-195. The long-range communication of the Na ϩ effect is ensured by a network of water molecules that embed the Na ϩ site, the primary specificity pocket, and the active site. Asp-189 at the bottom of the primary specificity pocket is o...

Rapid Kinetics of Na+ Binding to Thrombin

“…Because Trp residues are distributed over the entire structure of thrombin, and their distance from the bound Na ϩ ranges from 13 (Trp-215) to 35 (Trp-51) Å, binding of Na ϩ to E likely elicits effects well beyond the immediate environment of the Na ϩ site and perturbs the structure of thrombin as a whole. This is in agreement with recent functional mapping of the Na ϩ -induced allosteric transition of thrombin (51). The amplitude of the fast phase increases significantly relative to wild type for the W29F, W51F, W60dF, and W96F mutants.…”

Thrombin Functions through Its RGD Sequence in a Non-canonical Conformation

“…This conformation would have little affinity toward hirudin, consistent with the results in Fig. 2, because of the massive disruption of the 220-loop hosting critical residues for hirudin recognition (26). The collapse of the primary specificity pocket would also compromise catalytic activity, which is, however, not required for RGD function (Figs.…”

Ligand Binding to Anion-binding Exosites Regulates Conformational Properties of Thrombin

“…Earlier fluorescence studies also documented Hirudin(54 -65) influences to the thrombin active site and to the autolysis loop (43). Mutagenesis studies revealed thrombin residues involved in establishing such networks (38).…”