V. Nienaber, L. Mersinger, C. Kettner
Jul 30, 1996
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Biochemistry
Abstract
Kinetic study of a series of compounds containing the thrombin-directed peptide D-Phe-ProboroArg-OH had indicated that the structure of the N-terminal blocking group may be correlated with binding [Kettner, C., Mersinger, L., & Knabb, R. (1990) J. Biol. Chem. 265, 18289-18297]. In order to further study this phenomenon, a second series of compounds that contains a C-terminal methyl ester in place of the boronic acid was synthesized, binding measured, and the three-dimensional structure in complex with human thrombin determined by X-ray crystallography. Incubation of Ac-D-Phe-Pro-Arg-OMe, Boc-D-Phe-Pro-Arg-OMe, and H-D-Phe-Pro-Arg-OMe resulted in the formation of thrombin-product complexes within the crystal. Ki values for the corresponding products (free carboxylic acids) were 60 +/- 12 microM, 7.8 +/- 0.1 microM, 0.58 +/- 0.02 microM, respectively, indicating that the nature of the N-terminal blocking group has a significant effect on affinity. Examination of the crystal structures indicated that the higher affinity of the H-D-Phe peptide is due to rearrangement of one residue comprising the S3 site (Glu192) in order to maximize electrostatic interactions with the "NH3(+)-" of H-D-Phe. The relative affinity of Boc-D-Phe-Pro-Arg-OH is due to favorable hydrophobic interactions between thrombin and the bulky butyl group. However, this results in less favorable binding of Arg-P1 in the oxyanion hole as shown by long hydrogen-bonding distances. This work gave rise to some general observations applicable to structure-based drug design: (1) altering the structure of an inhibitor at one site can affect binding at an unchanged distal site; (2) minor alteration of inhibitor structure can lead to small, but significant reorganization of neighboring protein structure; (3) these unexpected reorganizations can define alternate binding motifs.