Ligand preferences of kringle 2 and homologous domains of human plasminogen: Canvassing weak, intermediate, and high-affinity binding sites by H-1-NMR

The interaction of various small aliphatic and aromatic ionic ligands with the human plasminogen (HPg) recombinant kringle 2 (r-K2) domain has been investigated by H-1-NMR spectroscopy at 500 MHz. The results are compared against ligand-binding properties of the homologous, lysine-binding HPg kringle 1 (K1), kringle 4 (K4), and kringle 5 (K5). The investigated ligands include the omega-aminocarboxylic acids 4-aminobutyric acid (4-ABA), 5-aminopentanoic acid (5-APA), 6-aminohexanoic acid (6-AHA), 7-aminoheptanoic acid (7-AHA), lysine and arginine derivatives with free and blocked alpha-amino and/or carboxylate groups, and a number of cyclic analogs, zwitterions of similar size such as trans-(aminomethyl)cyclohexanecarboxylic acid (AMCHA) and p-benzylaminesulfonic acid (BASA), and the nonzwitterions benzylamine and benzamidine. Equilibrium association constant (K-a) values were determined from H-1-NMR ligand titration profiles. Among the aliphatic linear ligands, 5-APA (K-a similar to 3.4 mM(-1)) shows the strongest interaction with r-K2 followed by B-AHA (K-a similar to 2.3 mM(-1)), 7-AHA (K-a similar to 0.45 mM(-1)), and 4-ABA (K-a similar to 0.22 mM(-1)). In contrast, r-K1, K4, and 1(5 exhibit a preference for 6-AHA (K-a similar to 74.2, 21.0, and 10.6 mM(-1), respectively), a ligand similar to 1.14 Angstrom longer than 5-APA. Mutations R220G and E221D increase the affinity of r-K2 for these ligands but leave the selectivity profile essentially unaffected: 5-APA > 6-AHA > 7-AHA > 4-ABA (K-a similar to 6.5, 3.9, 1.8, and 0.74 mM(-1), respectively). We find that, while r-K2 definitely interacts with Na-acetyl-L-lysine and L-lysine (K-a similar to 0.96 and 0.68 mM(-1), respectively), the affinity for analogs carrying a blocked carboxylate group is relatively weak (K-a similar to 0.1 mM(-1)). We also investigated the interaction of r-K2 with L-arginine (K-a similar to 0.31 mM(-1)) and its derivatives Na-acetyl L-arginine (K-a similar to 0.55 mM(-1)), N-alpha-acetyl-L-arginine methyl ester (K-a similar to 0.07 mM(-1)), and L-arginine methyl ester (K-a similar to 0.03 mM(-1)). Zwitterionic gamma-guanidinobutyric acid, containing one less methylene group than arginine, exhibits a K-a of similar to 0.28 mM(-1). The affinity of r-K2 for lysine and arginine derivatives suggests that K2 could play a role in intermolecular as well as intramolecular interactions of HPg. As is the case for the HPg K1, K4, and K5, among the tested ligands, AMCHA is the one which interacts most firmly with r-K2 (K-a similar to 7.3 mM(-1)) while the aromatic ligands BASA, benzylamine, and benzamidine exhibit K-a values of similar to 4.0, similar to 0.04, and similar to 0.03 mM(-1), respectively. The relative stability of these interactions indicates a strict requirement for both cationic and anionic polar groups in the ligand, whereas the presence of a lipophilic aromatic group seems to be of lesser consequence. Ligand-induced shifts of r-K2 H-1-NMR signals and two-dimensional nuclear Overhauser effect (NOESY) experiments in the presence of 6-AHA reveal direct involvement of residues Tyr(36), Trp(62), Phe(64), and Trp(72) (kringle residue numbering convention) in ligand binding. Starting from the X-ray crystallographic structure of HPg K4 and the intermolecular H-1-NMR NOE data, two models of the K2 lysine binding site complexed to 6-AHA have been derived which differ mainly in the extent of electrostatic pairing between the K2 Arg(56) and Glu(57) side chains. Competition between these two conformations in equilibrium may account for the relatively lesser affinity of the K2 domain for zwitterionic lysine-type ligands.