600 MHz 1H nuclear magnetic resonance studies of the kringle 4 fragment of human plasminogen

Abstract

Kringle 4, a ~10,000-Da domain in the heavy chain of human plasminogen, has been isolated intact and studied by 1H NMR spectroscopy at 600 MHz. The spectroscopic data indicates that kringle 4 possesses a globular and flexible structure which exhibits relatively fast amide-hydrogen exchange. About 17 NH groups show retarded exchange, with half-lives of ~7 h in 2H 2O at pH ∗ 6.45, 25 °C, which indicates that regions of the kringle are buried and shielded from direct interaction with the solvent. Analysis of the methyl region spectrum accounts for all singlets and doublets in terms of the amino acid composition; resonances from the C- and N-termini residues could be identified from the magnitude of their 3 J couplings and their response to pH titration. It is shown that elastase digestion of plasminogen generates two species of kringle 4, one that terminates with Ala 85 and another that extends to Val 87. The heterogeneity can be resolved by chromatography on CM-Sephadex. The interaction of kringle 4 with BASA ( p-benzylaminesulfonic acid), an antifibrinolytic drug presumed to bind to the plasminogen lysine-binding sites, has been investigated through the effects of added ligand on the kringle spectrum. The kringle lysine-binding site would appear to be integrated by a cluster of interacting His and aromatic residues since many of these resonances follow a definite saturation curve pattern upon BASA titration. In contrast, only minor changes are detected in the aliphatic methyl spectra. The association constant for the BASA-kringle 4 interaction is estimated to be K a ~ 74 m m −1, which should be compared with K a ~ 145 m m −1 previously measured for kringle 1 under identical conditions. It is proposed that residues in the proximity of the Cys 80-Cys 1 disulfide bridge are proximal to, or form part of, the lysine-binding site.