Structure and ligand binding determinants of the recombinant kringle 5 domain of human plasminogen

The X-ray crystal structure of the recombinant (r) kringle 5 domain of human plasminogen (K5(HPg)) has been solved by molecular replacement methods using K1(HPg) as a model and refined at 1.7 Angstrom resolution to an R factor of 16.6%. The asymmetric unit of K5(HPg) is composed of two molecules related by a noncrystallographic 2-fold rotation axis approximately parallel to the z-direction. The lysine binding site (LBS) is defined by the regions His(33)-Thr(37), Pro(54)-Val(58), Pro(61)-Tyr(64), and Leu(71)-Tyr(74) and is occupied in the ape-form by water molecules. A unique feature of the LBS of apo-K5(HPg) is the substitution by Leu(71) for the basic amino acid, arginine, that in other kringle polypeptides forms the donor cationic center for the carboxylate group of omega-amino acid ligands. While wild-type (wt) r-K5(HPg) interacted weakly with these types of ligands, replacement by site-directed mutagenesis of Leu(71) by arginine led to substantially increased affinity of the ligands for the LBS of K5(HPg) AS a result, binding of omega-amino acids to this mutant kringle (r-K5(HPg)[(LR)-R-71]) was restored to levels displayed by the companion much stronger affinity HPg kringles, K1(HPg) and K4(HPg). Correspondingly, alkylamine binding to r-K5(HPg)[(LR)-R-71] was considerably attenuated from that shown by wtr-K5(HPg) Thus, employing a rational design strategy based on the crystal structure of K5(HPg), successful remodeling of the LBS has been accomplished, and has resulted in the conversion of a weak ligand binding kringle to one that possesses an affinity for omega-amino acids that is similar to K1(HPg) and K4(HPg).