NUCLEAR-MAGNETIC-RESONANCE SOLUTION STRUCTURE OF THE PLASMINOGEN-ACTIVATOR PROTEIN STAPHYLOKINASE

Staphylokinase, a 15.5 kDa protein from Staphylococcus aureus, is a pl asminogen activator which is currently undergoing clinical trials for the therapy of myocardial infarction and peripheral thrombosis. The th ree-dimensional (3D) NMR solution structure has been determined by mul tidimensional heteronuclear NMR spectroscopy on uniformly N-15- and N- 15, C-13-labeled samples of staphylokinase. Structural constraints wer e obtained from 82 (3)J(HNH alpha) as well as 22 (3)J(NH beta) scalar coupling constants and 2345 NOE cross-peaks, derived from N-15-edited and C-13-edited 3D NOE spectra. NOE cross-peak assignments were confir med by analysis of {N-15, C-13}-edited and {C-13, C-13}-edited 4D NOE spectra. The structure is presented as a family of 20 conformers which show an average rmsd of 1.02 +/- 0.15 Angstrom from the mean structur e for the backbone atoms. The tertiary structure of staphylokinase sho ws a well-defined global structure consisting of a central 13-residue alpha-helix flanked by a two-stranded beta-sheet, both of which are lo cated above a five-stranded beta-sheet. Two of the connecting loops ex hibit a higher conformational heterogeneity. Overall, staphylokinase s hows a strong asymmetry of hydrophilic and hydrophobic surfaces. The N -terminal sequence, including Lys10 which is the site of the initial p roteolytic cleavage during activation of plasminogen, folds back onto the protein core, thereby shielding amino acids with functional import ance in the plasminogen activation process. From a comparison of the s tructure with mutational studies, a binding region for plasminogen is proposed.