title: Characterization of the interactions of a bifunctional inhibitor with alpha-thrombin by molecular modelling and peptide synthesis.
abstract: 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 linker portion hirudin 49-54, was designed based on the hirudin sequence [DiMaio et al. (1990) J. Biol. Chem., 265, 21698-21798]. A three-dimensional model of the complex between the B-chain of human thrombin and the inhibitor [D-Phe45, Arg47] hirudin 45-65 was constructed using molecular modelling starting from the X-ray C alpha coordinates of the thrombin-hirudin complex and the NMR-derived structure of the thrombin-bound hirudin 55-65. The contribution of the H49-54 fragment to the thrombin-inhibitor interaction was deduced by examining a series of analogs containing single glycine substitution and analogs with reduced number of residues within the linker. The results were consistent with the molecular modelling observations i.e. the H49-54 fragment serves the role of a spacer in the binding interaction and could be replaced by four glycine residues. The studies on the interaction of the exosite-directed portion of the inhibitor with thrombin using a series of synthetic H55-65 analogs demonstrated that residues AspH55 to ProH60 play a major role in binding to human thrombin where the side chains of PheH56, IleH59 and GluH57 showed critical contributions. Molecular modelling suggested that these side chains may contribute to inter- and intramolecular hydrophobic and electrostatic interactions, respectively.
authors.affiliation: Protein Engineering Section, National Research Council Canada, Montréal, Québec.
authors.forename: S Y
authors.initials: SY
authors.lastname: Yue
authors.forename: J
authors.initials: J
authors.lastname: DiMaio
authors.forename: Z
authors.initials: Z
authors.lastname: Szewczuk
authors.forename: E O
authors.initials: EO
authors.lastname: Purisima
authors.forename: F
authors.initials: F
authors.lastname: Ni
authors.forename: Y
authors.initials: Y
authors.lastname: Konishi
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D006629:Hirudins / Q000031:analogs & derivatives / Q000494:pharmacology*; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology*; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D006629:Hirudins; D010446:Peptide Fragments; D013917:Thrombin