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

Biochemistry. 1998 Mar 10;37(10):3258-71. doi: 10.1021/bi972284e.

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Binding Sites / genetics
  • Cattle
  • Crystallography, X-Ray
  • DNA Primers / genetics
  • Drug Design
  • Humans
  • Hydrogen Bonding
  • In Vitro Techniques
  • Kringles / genetics
  • Ligands
  • Lysine / metabolism
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Plasminogen / chemistry*
  • Plasminogen / genetics
  • Plasminogen / metabolism
  • Protein Conformation
  • Protein Engineering
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Thermodynamics

Substances

  • DNA Primers
  • Ligands
  • Recombinant Fusion Proteins
  • Plasminogen
  • Lysine