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 1H-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 (Ka) values were determined from 1H-NMR ligand titration profiles. Among the aliphatic linear ligands, 5-APA (Ka approximately 3.4 mM-1) shows the strongest interaction with r-K2 followed by 6-AHA (Ka approximately 2.3 mM-1), 7-AHA (Ka approximately 0.45 mM-1), and 4-ABA (Ka approximately 0.22 mM-1). In contrast, r-K1, K4, and K5 exhibit a preference for 6-AHA (Ka approximately 74.2, 21.0, and 10.6 mM-1, respectively), a ligand approximately 1.14 A 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 (Ka approximately 6.5, 3.9, 1.8, and 0.74 mM-1, respectively). We find that, while r-K2 definitely interacts with Nalpha-acetyl-L-lysine and L-lysine (Ka approximately 0.96 and 0.68 mM-1, respectively), the affinity for analogs carrying a blocked carboxylate group is relatively weak (Ka approximately 0.1 mM-1). We also investigated the interaction of r-K2 with L-arginine (Ka approximately 0.31 mM-1) and its derivatives Nalpha-acetyl-L-arginine (Ka approximately 0.55 mM-1), Nalpha-acetyl-L-arginine methyl ester (Ka approximately 0.07 mM-1), and L-arginine methyl ester (Ka approximately 0.03 mM-1). Zwitterionic gamma-guanidinobutyric acid, containing one less methylene group than arginine, exhibits a Ka of approximately 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 (Ka approximately 7.3 mM-1) while the aromatic ligands BASA, benzylamine, and benzamidine exhibit Ka values of approximately 4.0, approximately 0.04, and approximately 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 (1)H-NMR signals and two-dimensional nuclear Overhauser effect (NOESY) experiments in the presence of 6-AHA reveal direct involvement of residues Tyr36, Trp62, Phe64, and Trp72 (kringle residue numbering convention) in ligand binding. Starting from the X-ray crystallographic structure of HPg K4 and the intermolecular 1H-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 Arg56 and Glu57 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.