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) kr ingle 2 domain has been characterized via H-1-NMR spectroscopy at 500 MHz. Seven omega-amino acid ligands were investigated: L-Lys, 6-aminoh exanoic 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 lys yl residue, Tyr-Leu-Leu-Lys (YLLK) and Ala-Phe-Gln-Tyr-His-Ser-Lys (AF QYHSK), were also studied, The sequence AFQYHSK is found within the pl asminogen N-terminal activation peptide while the tetrapeptide YLLK co rresponds the 119-122 segment of the fibrinogen B beta-chain. Spectral comparison of ligand-free and ligand-containing kringle 2 samples lea ds to the conclusion that all the small ligands as well as the peptide s' C-terminal lysyl residues interact with a common binding site in kr ingle 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 po ints with the ligands 6AHA and BASA were unambiguously identified from kringle 2-ligand nuclear Overhauser effects (NOEs), Overall, the liga nd-induced chemical shifts and the intermolecular NOEs correlate remar kably well. Association constant (K-a) values for the kringle 2-ligand interactions were determined. Among the investigated ligands, BASA pe rturbs the kringle 2 spectrum the most and exhibits the highest affini ty for kringle 2 (K-a similar to 233 mM(-1)). Of the two other aromati c ligands, PAEBA binds to kringle 2 less firmly (K-a = similar to 12 m M(-1)) than does the one-methylene group shorter analog PAMBA (K-a sim ilar 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 sug gested that the higher affinity toward 7AHA may stem from (a) a relati vely more favored ionic pairing between its carboxylate group and the Lys(34) + Arg(69) sidechain cationic centers and (b) an enhanced inter action between the ligand hydrocarbon moiety and the kringle hydrophob ic pocket, in particular with the Leu(70) side chain. The latter is co nsistent with the relatively good affinity of kringle 2 for the cyclic hydrocarbon ligand AMCHA (K-a similar to 69 mM(-1)). When compared ag ainst 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 atta ched polypeptide chain segments contribute positively to their binding to kringle 2, Overall, the NMR ligand-binding data are in harmony wit h the binding site structure, solvent accessibility, and pH sensitivit y of individual residues and confirm, as found for other kringles, tha t the ligand complexation event is not accompanied by any significant conformational change of the kringle fold.