Characterization of the interactions of a bifunctional inhibitor with {alpha}-thrombin by molecular modelling and peptide synthesis

A potent thrombin inhibitor, [D-Phe⁴⁵, Arg⁴⁷] hirudin 45-65,that contains an active site-directed sequence D-Phe-Pro-Arg-Pro,an exosite specific fragment hirudin 55-65 (H55-65) and a linkerportion hirudin 49-54, was designed based on the hirudin sequence[DiMaio et al. (1990) J. Biol. Chem., 265, 21698-21798]. A three-dimensionalmodel of the complex between the B-chain of human thrombin andthe inhibitor [D-Phe45, Arg47⁴⁵, Arg⁴⁷] hirudin 45-65 was constructedusing molecular modelling starting from the X-ray Ca coordinatesof the thrombin-hirudin complex and the NMR-derived structureof the thrombin-bound hirudin 55 - 65. The contribution of theH49 -54 fragment to the thrombin - inhibitor interaction wasdeduced by examining a series of analogs containing single glycinesubstitution and analogs with reduced number of residues withinthe linker. The results were consistent with the molecular modellingobservations i.e. the H49-54 fragment serves the role of a spacerin the binding interaction and could be replaced by four glycineresidues. The studies on the interaction of the exosite-directedportion of the inhibitor with thrombin using a series of syntheticH55 -65 analogs demonstrated that residues AspH55 to ProH60play a major role in binding to human thrombin where the sidechains of PheH56, IleH59 and GluH57 showed critical contributions.Molecular modelling suggested that these side chains may contributeto inter- and intramolecular hydrophobic and electrostatic interactions,respectively.