Ligand interactions with the kringle 5 domain of plasminogen. A study by 1H NMR spectroscopy.
The binding of small molecules to the kringle 5 domain fragment of human plasminogen has been investigated by 1H NMR spectroscopy at 300 MHz. The compounds tested as potential ligands include L-arginine, L-lysine, and a number of aliphatic and aromatic analogs of similar size but different ionic charge configurations. Ligand/kringle 5 association constant (Ka) values were obtained from ligand titration experiments at 22 degrees C, pH 7.2. Neither L-arginine nor N alpha-acetyl-L-arginine and N alpha-acetyl-L-arginine methyl ester bind measurably to kringle 5 (Ka approximately less than 0.05 mM-1). In contrast, binding of hexylamine or epsilon-aminocaproic acid (epsilon ACA) is favored (Ka approximately 2.9 and 10.5 mM-1, respectively). Benzamidine and p-benzylaminesulfonic acid associate with kringle 5 with similar affinities (Ka approximately 3.4 and 2.2 mM-1, respectively) while benzylamine binds about twice as tightly (Ka approximately 6.3 mM-1). The higher affinities toward both benzylamine and epsilon ACA indicate that a free carboxylate group is not, by itself, a main determinant of ligand-binding to kringle 5. The experiments also reveal a definite affinity for L-arginine methyl ester, L-lysine, and N alpha-acetyl-L-lysine methyl ester. It is suggested that, although weak (0.1 approximately less than Ka approximately less than 0.6 mM-1), these interactions could be of physiological relevance in the context of plasminogen binding to the fibrin clot. Ligand-induced shifts of kringle 5 proton resonances indicate that the Trp25, His33, Tyr50, Trp62, and Tyr72 (kringle numbering convention) side chains form or neighbor the kringle 5-binding site. Benzamidine-kringle 5 magnetization transfer (Overhauser) experiments verify a close proximity of the bound ligand to these aromatic groups. A model of the binding site is proposed in which the above residues interact closely with each other and define a lipophilic surface which is accessible to the free ligand.
Thewes
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Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213-3890.
Constantine
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Byeon
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Llinás
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D000595:Amino Acid Sequence; D001120:Arginine / Q000031:analogs & derivatives; D001665:Binding Sites; D006801:Humans; D006859:Hydrogen; D007700:Kinetics; D008024:Ligands; D008239:Lysine; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D010958:Plasminogen / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid
D008024:Ligands; D006859:Hydrogen; D010958:Plasminogen; D001120:Arginine; D008239:Lysine