title: The structure of a complex of bovine alpha-thrombin and recombinant hirudin at 2.8-A resolution.
abstract: Crystals of the complex of bovine alpha-thrombin with recombinant hirudin variant 1 have space group C222(1) with cell constants a = 59.11, b = 102.62, and c = 143.26 A. The orientation and position of the thrombin component was determined by molecular replacement and the hirudin molecule was fit in 2 magnitude of Fo - magnitude of Fc electron density maps. The structure was refined by restrained least squares and simulated annealing to R = 0.161 at 2.8-A resolution. The binding of hirudin to thrombin is generally similar to that observed in the crystals of human thrombin-hirudin. Several differences in the interactions of the COOH-terminal polypeptide of hirudin, specifically of residues Asp-55h, Phe-56h, Glu-57h, and Glu-58h, and a few differences in the interactions of the hirudin core, specifically of residues Asp-5h, Ser-19h, and Asn-20h, with thrombin from human thrombin-hirudin suggest that there is some flexibility in the binding of these 2 molecules. Most of the residues in the 9 subsites that bind fibrinopeptide A7-16 to thrombin also interact with the NH2-terminal domain of hirudin. The S1 subsite is a notable exception in that only 1 of its 6 residues, namely Ser-214, interacts with hirudin. The only difference between human and bovine thrombins that appears to influence the binding of hirudin is the replacement of Lys-149E by an acidic glutamate in the bovine enzyme.
authors.affiliation: Department of Biochemistry, Wayne State University, Detroit, Michigan 48201.
authors.forename: J
authors.initials: J
authors.lastname: Vitali
authors.forename: P D
authors.initials: PD
authors.lastname: Martin
authors.forename: M G
authors.initials: MG
authors.lastname: Malkowski
authors.forename: W D
authors.initials: WD
authors.lastname: Robertson
authors.forename: J B
authors.initials: JB
authors.lastname: Lazar
authors.forename: R C
authors.initials: RC
authors.lastname: Winant
authors.forename: P H
authors.initials: PH
authors.lastname: Johnson
authors.forename: B F
authors.initials: BF
authors.lastname: Edwards
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003460:Crystallization; D006629:Hirudins / Q000737:chemistry* / Q000378:metabolism; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism; D014961:X-Ray Diffraction / Q000379:methods
chemical_list: D006629:Hirudins; D011994:Recombinant Proteins; D013917:Thrombin