SOLVENT-ACCESSIBLE RESIDUES ON THE METAL ION-DEPENDENT ADHESION SITE FACE OF INTEGRIN CR3 MEDIATE ITS BINDING TO THE NEUTROPHIL INHIBITORY FACTOR

Neutrophil adhesion dependent functions such as chemotaxis, spreading, and phagocytosis are inhibited by neutrophil inhibitory factor (NIF), a glycoprotein produced by the hookworm Ancylostoma caninum. The NIF binding site has been localized to the A-domain of integrin CR3 (CD11b /CD18) and shown to be metal-dependent, The recently solved crystal st ructure of the A-domain from CD11b revealed a putative metal ion-depen dent adhesion site (MIDAS) on the top of the structure. To determine i f NIF binds to the A-domain at its MIDAS face, amino acid substitution s involving 24 residues present in surface loops and adjacent helices in the structure were created. The expressed CD11b A-domain and CR3 he terodimers were then tested in a blinded manner for their ability to b ind to biotinylated NIF. The solvent-exposed Gly(143), Asp(149), Glu(1 78)-Glu(179), and Arg(208), all located on the MIDAS face, in close pr oximity to the metal ion, were involved in CR3-NIF interaction. These data show that the natural integrin antagonist, NIF, binds to CR3 thro ugh the MIDAS region and identify putative contact residues in this re gion that could be targeted therapeutically.