Complete assignment of the aromatic proton magnetic resonance spectrum of the kringle 1 domain from human plasminogen: structure of the ligand-binding site

The kringle 1 domain of human plasminogen has been investigated by 1H NMR spectroscopy at 300 and 600 MHz on the basis of a fragment obtained via controlled proteolysis of the zymogen with staphylococcus aureus V8 protease. The aromatic spectrum has been fully analyzed and all resonances assigned. The Tyr ring signals were identified by reference to the recently reported spectra of the plasminogen kringle 4 homologues from human, bovine, and porcine origin. Ligand binding was investigated by monitoring the effects of the antifibrinolytic drugs epsilon-aminohexanoic acid and p-benzylaminesulfonic acid (BASA) on the 1H NMR spectrum of kringle 1. Proton Overhauser experiments centered on aromatic transitions from these residues reveal efficient cross-relaxation with BASA, which indicates direct contacts between the hydrophobic side chain rings and the ligand hydrocarbon moiety at the binding site. Excellent overall agreement is found between the NMR data and the molecular folding of the prothrombin kringle I determined crystallographically. A structure is proposed here for the kringle 1 lysine-binding site which is based upon the NMR results, the x-ray structure, and computer graphics modeling. It is concluded that although features of the lysine-binding site are common to plasminogen kringles 1 and 4, in kringle 1 the binding site extends beyond the kringle inner loop as it encompasses residues Arg34 and Phe36 as well. Furthermore, it appears that in kringle 1 Arg34 and Asp55 are likely to play a direct role in the ligand-kringle 1 interaction by reinforcing the polarity of the cationic and anionic centers of the side chains of Arg71 and Asp57, which have been implicated to provide the electrostatic charges in kringle 4 that balance those of the ligand dipole at the binding site