Title

The Structure of a Complex of Bovine &-Thrombin and Recombinant Hirudin at 2.8-A Resolution

Document Type

Article

Publication Date

9-5-1992

Publication Title

Journal of Biological Chemistry

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.

Comments

This work was supported in part by National Institutes of Health Grant GM 33192, the Wayne State University Center for Molecular Biology and Bristol-Myers Squibb Company (to B. F. P. E.), and by SRI Internal Research and Development Projects 870D32XJC and 391D32BKA and by funds provided by the Cigarette and Tobacco Surtax Fund of the State of California through the Tobacco-related Disease Research Program of the University of California (to P. H. cJ.).

Original Citation

Vitali, J.; Martin, P. D.; Malkowski, M. G.; Robertson, W. D.; Lazar, J. B.; Winant, R. C.; Johnson, P. H.; Edwards, B. F. The structure of a complex of bovine alpha-thrombin and recombinant hirudin at 2.8-A resolution. Journal of Biological Chemistry 1992, 267, 17670-17678.

Volume

267

Issue

25