Amino acid residues of the kringle-4 and kringle-5 domains of human plasminogen that stabilize their interactions with omega-amino acid ligands.

Regions of the human plasminogen (Pg) cDNA containing its kringle 4 (K4) and K5 domains have been expressed in Escherichia coli, and binding constants of omega-amino acid ligands for recombinant (r)-K4Pg and r-K5Pg have been obtained. In each case, the results showed that of a series of aliphatic alpha, omega-amino acid analogues, 6-aminohexanoic acid showed maximal affinity for these modules, and all ligands interacted more strongly with r-K4Pg than with r-K5Pg. Site-directed mutagenesis investigations demonstrated that the major amino acid side chain contributors to ligand binding were similar for each of these kringles. Ligand binding was stabilized by charged groups at Asp56 of r-K4Pg and Asp57 of r-K5Pg as well as by Arg69 of both r-K4Pg and r-K5Pg. Some hydrophobic amino acids that contributed significantly to the binding strength of the ligands were identified. These were provided by homologous residues in each of the domains, viz. Trp60 and Trp70 of r-K4Pg and Trp62 and Tyr72 of r-K5Pg. Tyr74 of r-K5Pg also substantially contributed to its ligand binding energy.