Proton NMR studies of aliphatic ligand binding to human plasminogen kringle 4

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Abstract
A detailed 1H NMR analysis of ligand binding to the human plasminogen kringle 4 domain has been carried out at 300 MHz. The ligands that were investigated are N.alpha.-acetyl-L-lysine, L-lysine methyl ester, N.alpha.-acetyl-L-lysine methyl ester, L-lysine hydroxamic acid, trans-(aminomethyl)cyclohexanecarboxylic acid (AMCHA), and 4-(aminomethyl)bicyclo[2.2.2]octane-1-carboxylic acid (AMBOC). Specific ligand-binding effects were detected via two-dimensioanl COSY experiments. The side chains that are the most perturbed by ligand presence are those from Trp62, Phe64, and Trp72. Ligand-kringle saturation transfer (Overhauser) experiments show that the aromatic rings from these three residues, especially Trp72, are in direct contact with the ligand. These results add support to a previously reported model of the kringle 4 lysine-binding site [Ramesh, V., Petros. A. M., Llinas, M., Tulinsky, A., and Park, C. H. (1987) J. Mol. Biol. 198, 481-498] by which these aromatic groups are assigned a key role in establishing hydrophobic interactions with the ligand molecule. Equilibrium association constants (Ka) and kinetic rate constants (kon, koff) were determined for the binding of the various linear and cyclic ligands to kringle 4. We find that those ligands whose carboxylate function is blocked bind significantly weaker (Ka .gtorsim. 2 mM-1) than the corresponding analogues where the anionic center is present (Ka .gtorsim. 20 mM-1), which underscores the relevance of the polar gropu in stabilizing the interaction with the kringle 4 binding site. Furthermore, AMBOC (Ka .apprx. 48 mM-1) exhibits weaker binding than AMCHA (Ka .apprx. 159 mM-1), a fact that reflects the relative values of the dissociation rate constants (koff .apprx. 3.8 and 1.3 S-1, respectively) and that suggests a more constrained fit of the bulkier ligand at the binding site. Of all the investigated ligands, AMCHA is the one that exhibits the highest affinity for kringle 4. The numerical data are discussed in terms of optimal ligand structure and requirements for fibrin binding in vivo.

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