Kringle‐2 domain of the tissue‐type plasminogen activator

Abstract

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_αN splittings and identification of backbone amide NH protons exhibiting retarded ¹H/²H exchange in ²H₂O. Three antiparallel β‐sheets and six tight turns were located. In addition, one short α‐helical region was found in the Ser⁴³‐Ala⁴⁴ ‐Gln^44a‐Ala^44b‐Leu^44c‐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 α‐helical segment and other details of the secondary structure differ somewhat from the prothrombin homolog.