LIGAND-BINDING TO THE TISSUE-TYPE PLASMINOGEN-ACTIVATOR KRINGLE-2 DOMAIN - STRUCTURAL CHARACTERIZATION BY H-1-NMR

Ligand binding to a recombinant human tissue-type plasminogen (tPA) kringle 2 domain has been characterized via H-1-NMR spectroscopy at 500 MHz. Seven omega-amino acid ligands were investigated: L-Lys, 6-aminohexanoic acid (6AHA), 7-aminoheptanoic acid (7AHA), trans-(aminomethyl)cyclohexanecarboxylic acid (AMCHA), p-(aminomethyl)benzoic acid (PAMBA), p-(aminoethyl)benzoic acid (PAEBA), and p-benzylaminesulfonic acid (BASA). The interactions with two peptides containing a C-terminal lysyl residue, Tyr-Leu-Leu-Lys (YLLK) and Ala-Phe-Gln-Tyr-His-Ser-Lys (AFQYHSK), were also studied, The sequence AFQYHSK is found within the plasminogen N-terminal activation peptide while the tetrapeptide YLLK corresponds the 119-122 segment of the fibrinogen B beta-chain. Spectral comparison of ligand-free and ligand-containing kringle 2 samples leads to the conclusion that all the small ligands as well as the peptides' C-terminal lysyl residues interact with a common binding site in kringle 2. Two-dimensional spectra show that besides the Tyr(36), Trp(62), His(64), Trp(72) and Tyr(74) aromatic rings, the Val(35) and Asp(55) aliphatic side chains also participate in ligand binding. Contact points with the ligands 6AHA and BASA were unambiguously identified from kringle 2-ligand nuclear Overhauser effects (NOEs), Overall, the ligand-induced chemical shifts and the intermolecular NOEs correlate remarkably well. Association constant (K-a) values for the kringle 2-ligand interactions were determined. Among the investigated ligands, BASA perturbs the kringle 2 spectrum the most and exhibits the highest affinity for kringle 2 (K-a similar to 233 mM(-1)). Of the two other aromatic ligands, PAEBA binds to kringle 2 less firmly (K-a = similar to 12 mM(-1)) than does the one-methylene group shorter analog PAMBA (K-a similar to 31 mM(-1)). By comparison, relative to 6AHA (K-a similar to 22 mM(-1)), the longer chain linear aliphatic ligand 7AHA interacts with kringle 2 with significantly higher affinity (K-a similar to 149 mM(-1)). By reference to the NMR-derived binding site structure, it is suggested that the higher affinity toward 7AHA may stem from (a) a relatively more favored ionic pairing between its carboxylate group and the Lys(34) + Arg(69) sidechain cationic centers and (b) an enhanced interaction between the ligand hydrocarbon moiety and the kringle hydrophobic pocket, in particular with the Leu(70) side chain. The latter is consistent with the relatively good affinity of kringle 2 for the cyclic hydrocarbon ligand AMCHA (K-a similar to 69 mM(-1)). When compared against L-Lys (K-a similar to 18 mM(-1)), the higher affinity exhibited by YLLK and AFQYHSK (K-a similar to 38 mM(-1)) indicates that the attached polypeptide chain segments contribute positively to their binding to kringle 2, Overall, the NMR ligand-binding data are in harmony with the binding site structure, solvent accessibility, and pH sensitivity of individual residues and confirm, as found for other kringles, that the ligand complexation event is not accompanied by any significant conformational change of the kringle fold.