title: Interactions of hirudin-based inhibitor with thrombin: critical role of the IleH59 side chain of the inhibitor.
abstract: Hirudin is the most potent and specific thrombin inhibitor from medicinal leech with a Ki value of 2.2 x 10(-14) M. It consists of an active site inhibitor segment, hirudin1-48, a fibrinogen-recognition exosite inhibitor segment, hirudin55-65, and a linker, hirudin49-54, connecting these inhibitor segments. The role of the side chain of the hirudin 59th residue, Ile, is studied by using a series of synthetic bivalent thrombin inhibitors, which mimic the binding mode of hirudin. The synthetic inhibitors based on the hirudin sequence have a general sequence of Ac-(D-Phe)-Pro-Arg-Pro-(4-aminobutyric acid)-(7-amino-heptanoic acid)-Asp-Phe-Glu-Glu-Xaa-Pro-Glu-Glu-Tyr-Leu-Gln-OH, in which the 59th residue, Xaa, is substituted by various natural and unnatural L-amino acids. For example, substitution of IleH59 by Val, which is equivalent to removing the delta-methyl group of IleH59, reduces the affinity of the inhibitor 5.7-fold (delta delta G0 = 1.0 kcal/mol) to a Ki value of 4.7 nM compared to that (Ki = 0.82 nM) of the corresponding inhibitor with IleH59. Removal of the entire side chain of IleH59, i.e., a substitution of IleH59 by Gly, reduces the affinity of the inhibitor 6300-fold, revealing the critical role of the IleH59 side chain in the inhibitor binding. Theoretical free energy calculation successfully reproduces the binding free energy of most of the analogs. It suggests that intra- and intermolecular van der Waals interactions of delta-CH3, gamma-CH3, and gamma-CH2 of IleH59 play the major role in the binding affinity.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Biotechnology Research Institute, National Research Council of Canada, Quebec.
authors.forename: Y
authors.initials: Y
authors.lastname: Tsuda
authors.forename: Z
authors.initials: Z
authors.lastname: Szewczuk
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.forename: S Y
authors.initials: SY
authors.lastname: Yue
authors.forename: E
authors.initials: E
authors.lastname: Purisima
authors.forename: Y
authors.initials: Y
authors.lastname: Konishi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D006629:Hirudins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D007532:Isoleucine / Q000737:chemistry*; D008969:Molecular Sequence Data; D011485:Protein Binding; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D006629:Hirudins; D007532:Isoleucine; D013917:Thrombin