Ligand Preferences of Kringle 2 and Homologous Domains of Human Plasminogen:  Canvassing Weak, Intermediate, and High-Affinity Binding Sites by 1H-NMR

• Published in: Biochemistry (Easton) 1997-09, Vol.36 (39), p.11591-11604
• Main Authors: Marti, Daniel N, Hu, Chih-Kao, An, Seong Soo A, von Haller, Priska, Schaller, Johann, Llinás, Miguel
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Binding Sites; Humans; Kringles; Ligands; Magnetic Resonance Spectroscopy; Models, Chemical; Models, Molecular; Molecular Sequence Data; Plasminogen - chemistry; Plasminogen - metabolism; Protein Binding; Protein Conformation; Sequence Alignment
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi971316v

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 ω-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 α-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 1H-NMR ligand titration profiles. Among the aliphatic linear ligands, 5-APA (K a ∼ 3.4 mM-1) shows the strongest interaction with r-K2 followed by 6-AHA (K a ∼ 2.3 mM-1), 7-AHA (K a ∼ 0.45 mM-1), and 4-ABA (K a ∼ 0.22 mM-1). In contrast, r-K1, K4, and K5 exhibit a preference for 6-AHA (K a ∼ 74.2, 21.0, and 10.6 mM-1, respectively), a ligand ∼1.14 Å 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 ∼ 6.5, 3.9, 1.8, and 0.74 mM-1, respectively). We find that, while r-K2 definitely interacts with N α-acetyl-l-lysine and l-lysine (K a ∼ 0.96 and 0.68 mM-1, respectively), the affinity for analogs carrying a blocked carboxylate group is relatively weak (K a ∼ 0.1 mM-1). We also investigated the interaction of r-K2 with l-arginine (K a ∼ 0.31 mM-1) and its derivatives N α-acetyl-l-arginine (K a ∼ 0.55 mM-1), N α-acetyl-l-arginine methyl ester (K a ∼ 0.07 mM-1), and l-arginine methyl ester (K a ∼ 0.03 mM-1). Zwitterionic γ-guanidinobutyric acid, containing one less methylene group than arginine, exhibits a K a of ∼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 ∼ 7.3 mM-1) while the aromatic ligands BASA, benzylamine, and benzamidine exhibit K a values of ∼4.0, ∼0.04, and ∼0.03 mM-1, respectively. The rela