Changes in interactions in complexes of hirudin derivatives and human α‐thrombin due to different crystal forms

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Abstract
The three-dimensional structures of d-Phe-Pro-Arg-chloromethyl ketone-inhibited thrombin in complex with Tyr-63-sulfated hirudin55–65 (ternary complex) and of thrombin in complex with the bifunctional inhibitor d-Phe-Pro-Arg-Pro-(Gly)4-hirudin54–65 (CGP 50, 856, binary complex) have been determined by X-ray crystallography in crystal forms different from those described by Skrzypczak-Jankun et al. (Skrzypczak-Jankun, E., Carperos, V.E., Ravichandran, K.G., & Tulinsky, A., 1991, J. Mol. Biol. 221, 1379–1393). In both complexes, the interactions of the C-terminal hirudin segments of the inhibitors binding to the fibrinogen-binding exosite of thrombin are clearly established, including residues 60–64, which are disordered in the earlier crystal form. The interactions of the sulfate group of Tyr-63 in the ternary complex structure explain why natural sulfated hirudin binds with a 10-fold lower Ki than the desulfated recombinant material. In this new crystal form, the autolysis loop of thrombin (residues 146–150), which is disordered in the earlier crystal form, is ordered due to crystal contacts. Interactions between the C-terminal fragment of hirudin and thrombin are not influenced by crystal contacts in this new crystal form, in contrast to the earlier form. In the bifunctional inhibitor–thrombin complex, the peptide bond between Arg-Pro (P1-P1′) seems to be cleaved.