1H 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 1H 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 1H-1H correlated two-dimensional spectra were identified via a natural abundance 1H-13C 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 2H2O yielded pKa* (pKa determined from acid/base titration in 2H2O, 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 2H2O affects mainly the His37, His40, His48a, and Tyr50 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.
Bokman
A M
AM
Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213-3890.
Jiménez-Barbero
J
J
Llinás
M
M
D000595:Amino Acid Sequence; D006801:Humans; D006859:Hydrogen; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008961:Models, Structural; D008969:Molecular Sequence Data; D010960:Plasminogen Activators / Q000737:chemistry; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry* / Q000378:metabolism
D006859:Hydrogen; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator