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 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.
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Protein Engineering Section, National Research Council Canada, Montréal, Québec.
DiMaio
J
J
Szewczuk
Z
Z
Purisima
E O
EO
Ni
F
F
Konishi
Y
Y
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
D006629:Hirudins; D010446:Peptide Fragments; D013917:Thrombin