H-1-NMR CHARACTERIZATION OF THE UROKINASE KRINGLE MODULE - STRUCTURAL, BUT NOT FUNCTIONAL, RELATEDNESS TO HOMOLOGOUS DOMAINS

The human urokinase (uPA) kringle (K) domain has been characterized via high resolution NMR spectroscopy. The H-1 spectrum is analogous to that of the K2 domain of tissue-type plasminogen activator (tPA) and other homologous domains from the plasminogen (Pgn) heavy chain. This indicates a similar folding for the uPA/K. Comparisons of the high-field methyl and aromatic regions of the uPA/K and tPA/K2 spectra against those from the Pgn/K1 and K4 homologues afford the immediate assignment of signals stemming from conserved residues, such as the characteristic high-field shifted Leu46 delta,delta'-methyl doublets, and the aromatic side chains at the hydrophobic core, in particular those from Trp25, His48a, Tyr50, and Trp62. Resonances unresolved due to spectral overlaps in the H-1-H-1 correlated two-dimensional spectra were identified via a natural abundance H-1-C-13 single/multiple quantum correlated experiment. Spin systems unique to the uPA/K, such as His7, His37, His40, and His78, were assigned from Overhauser experiments and sequence information. Acid/base titrations of His imidazole signals in (H2O)-H-2 yielded pK(a)* (pK(a) determined from acid/base titration in (H2O)-H-2, uncorrected for deuterium isotope effects) values of 6.2 for His7, 6.3 for His37, 6.4 for His40, 4.1 for His48a, and 6.2 for His78, which suggests a significant structural protection for His48a, consistent with a buried location within the hydrophobic core. Binding of low molecular weight heparin to the uPA/K in (H2O)-H-2 affects mainly the His37, His40, His48a, and Tyr 50 resonances, in a concerted and saturable fashion (association constant approximately 58 mM-1). The absence of perturbation of the His7 and His78 side chains indicates that segment 37-50 is selectively sensitive to heparin binding. Thus, the kringle outer B-loop is likely to be proximal to the basic residues responsible for the interaction with the polyanion ligand.