Kringle‐2 domain of the tissue‐type plasminogen activator

A recombinant 90-residue polypeptide fragment containing the three-loop kringle-2 domain of human tissue-type plasminogen activator (t-PA) has been studied by two-dimensional 1H-NMR spectroscopy at 500 MHz. Complete sequence-specific resonance assignments were derived. Overall, the kringle exhibits a compact, folded conformation with more than 50% of the residues in irregular structures. Elements of secondary structure were identified from sequential, medium- and long-range dipolar (Overhauser) interproton interactions. These identifications were corroborated by analysis of spin-spin scalar 3J alpha N splittings and identification of backbone amide NH protons exhibiting retarded 1H/2H exchange in 2H2O. Three antiparallel beta-sheets and six tight turns were located. In addition, one short alpha-helical region was found in the Ser43-Ala44-Gln44a-Ala44b-Leu44c-Gly45+ ++ segment; this region contains three-residue insertions unique to the t-PA and urokinase kringles. Although the secondary structure of the t-PA kringle 2 in solution is in overall agreement with that observed in the crystallographic structure of the prothrombin kringle 1 [Tulinsky, A., Park, C.H. & Skrzypczak-Jankun, E. (1988) J. Mol. Biol. 202, 885-901], the alpha-helical segment and other details of the secondary structure differ somewhat from the prothrombin homolog.