Structure-Based Understanding of Ligand Affinity Using Human Thrombin as a Model System

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Abstract
Kinetic study of a series of compounds containing the thrombin-directed peptide d-Phe-Pro-boroArg-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 μM, 7.8 ± 0.1 μM, 0.58 ± 0.02 μM, 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.