Two non-contiguous 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 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 gamma 1 chain. However, the changes Asn802 to Ser or Val804 to Ser, both known to exist in the laminin gamma 2 chain, were deleterious mutations. This demonstrated conservation of binding structures in laminins of distantly related species, but not between homologous chains of laminin isoforms.
Pöschl
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Max-Planck Connective Tissue Clinical Research Group for Rheumatology, Erlangen, Germany.
Fox
J W
JW
Block
D
D
Mayer
U
U
Timpl
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001485:Basement Membrane / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D007797:Laminin / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011869:Radioligand Assay; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D007797:Laminin; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C089141:laminin gamma 1; C029967:nidogen