The structure of a complex of bovine alpha-thrombin and recombinant hirudin at 2.8-A resolution.

Crystalsof the complex of bovine a-thrombin with recombinant hirudin variant 1 have space group C22Z1 with cel! 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 21F0J -lFel 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-Sh, Ser-19h, and Asn-ZOh, 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 A,-,, 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.a-Thrombin (EC 3.4.21.5) is a serine protease with a high specificity for arginine bonds that plays a central role in thrombosis and hemostasis.It is the product of prothrombin cleavage by factor Xa in the final step of the blood clotting cascade (reviewed by Mann, 1987; Davie et al., 1991).During clotting, a-thrombin converts fibrinogen into fibrin by removing fibrinopeptide A from the Aa-chain and fibrinopeptide B from the BP-chains of fibrinogen.Thrombin also functions as a major regulator of hemostasis (Fenton, 1986;Davie et al.,