Two non-contiguous regions contribute to nidogen binding to a single EGF-like motif of the laminin gamma 1 chain.

Abstract

High affinity binding of nidogen to laminin is mediated by an EGF-like repeat γ1III4 of the mouse laminin γ1 chain and has now been restricted to two short noncontiguous regions of its 56 residue sequence by use of synthetic peptides and recombinant mutants. Disulfide loop 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 contribution of Tyr819 in loop c was, however, shown by mutation and side chain modification. Together with studies of loop chimeras, this indicated a distinct cooperativity between the two binding sites. The major binding site of loop a was localized to the heptapeptide NIDPNAV (position 798-804). A change of Asp800 to Asn or Ala803 to Val caused a strong reduction in binding activity, while only small effects were observed for the changes Pro801 to Gln and Ile799 to Val. The latter replacement corresponds to the single substitution found in the same region of the Drosophila laminin γ1 chain. However, the changes Asn802 to Ser or Val804 to Ser, both known to exist in the laminin γ2 chain, were deleterious mutations. This demonstrated conservation of binding structures in laminins of distantly related species, but not between homologous chains of laminin isoforms.