1H NMR studies of aliphatic ligand binding to human plasminogen kringle 4.
A detailed 1H NMR analysis of ligand binding to the human plasminogen kringle 4 domain has been carried out at 300 MHz. The ligands that were investigated are N alpha-acetyl-L-lysine, L-lysine methyl ester, N alpha-acetyl-L-lysine methyl ester, L-lysine hydroxamic acid, trans-(aminomethyl)cyclohexanecarboxylic acid (AMCHA), and 4-(aminomethyl)bicyclo[2.2.2]octane-1-carboxylic acid (AMBOC). Specific ligand-binding effects were detected via two-dimensional COSY experiments. The side chains that are the most perturbed by ligand presence are those from Trp62, Phe64, and Trp72. Ligand-kringle saturation transfer (Overhauser) experiments show that the aromatic rings from these three residues, especially Trp72, are in direct contact with the ligand. These results add support to a previously reported model of the kringle 4 lysine-binding site [Ramesh, V., Petros, A. M., Llinás, M., Tulinsky, A., & Park, C. H. (1987) J. Mol. Biol. 198, 481-498] by which these aromatic groups are assigned a key role in establishing hydrophobic interactions with the ligand molecule. Equilibrium association constants (Ka) and kinetic rate constants (kon, koff) were determined for the binding of the various linear and cyclic ligands to kringle 4. We find that those ligands whose carboxylate function is blocked bind significantly weaker (Ka approximately less than 2 mM-1) than the corresponding analogues where the anionic center is present (Ka approximately greater than 20 mM-1), which underscores the relevance of the polar group in stabilizing the interaction with the kringle 4 binding site.(ABSTRACT TRUNCATED AT 250 WORDS)
Petros
A M
AM
Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.
Ramesh
V
V
Llinás
M
M
D006639:Histidine; D006801:Humans; D006859:Hydrogen; D007700:Kinetics; D008024:Ligands; D008239:Lysine / Q000031:analogs & derivatives / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D010649:Phenylalanine; D010958:Plasminogen / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D014364:Tryptophan; D014443:Tyrosine
D008024:Ligands; D014443:Tyrosine; D010649:Phenylalanine; D006639:Histidine; D006859:Hydrogen; D014364:Tryptophan; D010958:Plasminogen; D008239:Lysine