SECONDARY STRUCTURE OF FIBRONECTIN TYPE-1 AND EPIDERMAL GROWTH-FACTOR MODULES FROM TISSUE-TYPE PLASMINOGEN-ACTIVATOR BY NUCLEAR-MAGNETIC-RESONANCE

A Segment of human tissue-type plasminogen activator (t-PA) corresponding to the fibronectin type 1 (F1) and epidermal growth factor-like (G) pair of modules, residues 1-91, has been produced as a recombinant protein in Saccharomyces cerevisiae, with a single conservative Cys to Ser substitution. The sequence-specific assignment of the H-1 and N-15 nuclear magnetic resonances from the pair of modules has been completed using 2D H-1 nuclear magnetic resonance (NMR) spectra in conjunction with 3D, N-15- edited, H-1 and 2D N-15-H-1 NMR spectra. Slowly exchanging amide protons have been identified, and estimates of a number of backbone (3)J(NH-C alpha H) coupling constants were obtained by line-shape-fitting. The secondary structure of the F1 module in the pair closely matches that previously determined for the isolated F1 module from t-PA, and that of the G module conforms to the ''consensus'' G module structure determined previously from several isolated G modules. In the module pair, the residues linking the two modules appear to form an extended beta-strand, the carboxy-terminal end of which makes up a third strand of the major beta-sheet of the G module. The intermodule interface is defined by NOEs between residues in the ranges 22-24 in the F1 module and 65-72 in the G module. The NMR data indicate that there is little or no reorientation of the two modules with respect to one another but rather that they combine with a fixed hydrophobic contact dominated by the side chain of leucine-22.