2 NONCONTIGUOUS REGIONS CONTRIBUTE TO NIDOGEN BINDING TO A SINGLE EGF-LIKE MOTIF OF THE LAMININ-GAMMA-1 CHAIN

High affinity binding of nidogen to laminin is mediated by an EGF-like repeat gamma 1III4 of the mouse laminin gamma 1 chain and has now bee n restricted to two short noncontiguous regions of its 56 residue sequ ence by use of synthetic peptides and recombinant mutants. Disulfide l oop a,b of the repeat and a modified loop a,c could completely inhibit binding, with a 5000-fold or 300-fold reduced affinity respectively. Synthetic loops c and d lacked inhibitory activity, Some binding contr ibution of Tyr819 in loop c was, however, shown by mutation and side c hain modification. Together with studies of loop chimeras, this indica ted a distinct cooperativity between the two binding sites. The major binding site of loop a was localized to the heptapeptide NIDPNAV (posi tion 798-804). A change of Asp800 to Asn or Ala803 to Val caused a str ong reduction in binding activity, while only small effects were obser ved for the changes Pro801 to Gln and Ile799 to Val. The latter replac ement corresponds to the single substitution found in the same region of the Drosophila laminin gamma 1 chain. However, the changes Asn802 t o Ser or Val804 to Ser, both known to exist in the laminin gamma 2 cha in, were deleterious mutations. This demonstrated conservation of bind ing structures in laminins of distantly related species, but not betwe en homologous chains of laminin isoforms.