title: Structure and ligand binding determinants of the recombinant kringle 5 domain of human plasminogen.
abstract: The X-ray crystal structure of the recombinant (r) kringle 5 domain of human plasminogen (K5HPg) has been solved by molecular replacement methods using K1HPg as a model and refined at 1.7 A resolution to an R factor of 16.6%. The asymmetric unit of K5HPg 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 His33-Thr37, Pro54-Val58, Pro61-Tyr64, and Leu71-Tyr74 and is occupied in the apo-form by water molecules. A unique feature of the LBS of apo-K5HPg is the substitution by Leu71 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-K5HPg interacted weakly with these types of ligands, replacement by site-directed mutagenesis of Leu71 by arginine led to substantially increased affinity of the ligands for the LBS of K5HPg. As a result, binding of omega-amino acids to this mutant kringle (r-K5HPg[L71R]) was restored to levels displayed by the companion much stronger affinity HPg kringles, K1HPg and K4HPg. Correspondingly, alkylamine binding to r-K5HPg[L71R] was considerably attenuated from that shown by wtr-K5HPg. Thus, employing a rational design strategy based on the crystal structure of K5HPg, 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 K1HPg and K4HPg.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Chang
authors.forename: I
authors.initials: I
authors.lastname: Mochalkin
authors.forename: S G
authors.initials: SG
authors.lastname: McCance
authors.forename: B
authors.initials: B
authors.lastname: Cheng
authors.forename: A
authors.initials: A
authors.lastname: Tulinsky
authors.forename: F J
authors.initials: FJ
authors.lastname: Castellino
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D018360:Crystallography, X-Ray; D017931:DNA Primers / Q000235:genetics; D015195:Drug Design; D006801:Humans; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D018082:Kringles / Q000235:genetics; D008024:Ligands; D008239:Lysine / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010958:Plasminogen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D017931:DNA Primers; D008024:Ligands; D011993:Recombinant Fusion Proteins; D010958:Plasminogen; D008239:Lysine