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 h as been investigated by H-1-NMR spectroscopy at 500 MHz. The results a re compared against ligand-binding properties of the homologous, lysin e-binding HPg kringle 1 (K1), kringle 4 (K4), and kringle 5 (K5). The investigated ligands include the omega-aminocarboxylic acids 4-aminobu tyric acid (4-ABA), 5-aminopentanoic acid (5-APA), 6-aminohexanoic aci d (6-AHA), 7-aminoheptanoic acid (7-AHA), lysine and arginine derivati ves 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-benzylaminesulfon ic acid (BASA), and the nonzwitterions benzylamine and benzamidine. Eq uilibrium association constant (K-a) values were determined from H-1-N MR ligand titration profiles. Among the aliphatic linear ligands, 5-AP A (K-a similar to 3.4 mM(-1)) shows the strongest interaction with r-K 2 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, 2 1.0, 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 a nd 0.68 mM(-1), respectively), the affinity for analogs carrying a blo cked 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 sim ilar to 0.31 mM(-1)) and its derivatives Na-acetyl L-arginine (K-a sim ilar to 0.55 mM(-1)), N-alpha-acetyl-L-arginine methyl ester (K-a simi lar 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, benz ylamine, 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 stabi lity of these interactions indicates a strict requirement for both cat ionic 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 Ove rhauser effect (NOESY) experiments in the presence of 6-AHA reveal dir ect involvement of residues Tyr(36), Trp(62), Phe(64), and Trp(72) (kr ingle residue numbering convention) in ligand binding. Starting from t he 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 electros tatic pairing between the K2 Arg(56) and Glu(57) side chains. Competit ion between these two conformations in equilibrium may account for the relatively lesser affinity of the K2 domain for zwitterionic lysine-t ype ligands.