Electrostatic Steering and Ionic Tethering in the Formation of Thrombin−Hirudin Complexes: The Role of the Thrombin Anion-Binding Exosite-I

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Abstract
Electrostatic interactions between the thrombin anion-binding exosite-I (ABE-I) and the hirudin C-terminal tail play an important role in the formation of the thrombin−hirudin inhibitor complex and serves as a model for the interactions of thrombin with its many other ligands. The role of each solvent exposed basic residue in ABE-I (Arg35, Lys36, Arg67, Arg73, Arg75, Arg77a, Lys81, Lys109, Lys110, and Lys149e) in electrostatic steering and ionic tethering in the formation of thrombin−hirudin inhibitor complexes was explored by site directed mutagenesis. The contribution to the binding energy ( ) by each residue varied from 1.9 kJ mol-1 (Lys110) to 15.3 kJ mol-1 (Arg73) and were in general agreement to their observed interactions with hirudin residues in the thrombin−hirudin crystal structure [Rydel, T. J., Tulinsky, A., Bode, W., and Huber, R. (1991) J. Mol. Biol.221, 583−601]. Coupling energies ( ) were calculated for the major ion-pair interactions involved in ionic tethering using complementary hirudin mutants (h-D55N, h-E57Q, and h-E58Q). Cooperativity was seen for the h-Asp55/Arg73 ion pair ( 2.4 kJ mol-1); however, low coupling energies for h-Asp55/Lys149e ( 0.6 kJ mol-1) and h-Glu58/Arg77a ( 0.9 kJ mol-1) suggest these are not major interactions, as anticipated by the crystal structure. Interestingly, high coupling energies were seen for the intermolecular ion-pair h-Glu57/Arg75 ( 2.3 kJ mol-1) and for the solvent bridge h-Glu57/Arg77a ( 2.7 kJ mol-1) indicating that h-Glu57 interacts directly with both Arg75 and Arg77a in the thrombin−hirudin inhibitor complex. The remaining ABE-I residues that do not form major contacts in tethering the C-terminal tail of hirudin make small but collectively important contributions to the overall positive electrostatic field generated by ABE-I important in electrostatic steering.