title: 1H NMR characterization of the urokinase kringle module. Structural, but not functional, relatedness to homologous domains.
abstract: 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.
authors.affiliation: Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213-3890.
authors.forename: A M
authors.initials: AM
authors.lastname: Bokman
authors.forename: J
authors.initials: J
authors.lastname: Jiménez-Barbero
authors.forename: M
authors.initials: M
authors.lastname: Llinás
mesh_terms: 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
chemical_list: D006859:Hydrogen; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator