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 structure of the A-domain from CD11b revealed a putative metal ion-dependent adhesion site (MIDAS) on the top of the structure. To determine if NIF binds to the A-domain at its MIDAS face, amino acid substitutions involving 24 residues present in surface loops and adjacent helices in the structure were created. The expressed CD11b A-domain and CR3 heterodimers were then tested in a blinded manner for their ability to bind to biotinylated NIF. The solvent-exposed Gly143, Asp149, Glu178-Glu179, and Arg208, all located on the MIDAS face, in close proximity to the metal ion, were involved in CR3-NIF interaction. These data show that the natural integrin antagonist, NIF, binds to CR3 through the MIDAS region and identify putative contact residues in this region that could be targeted therapeutically.
Rieu
P
P
Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts 02129, USA.
Sugimori
T
T
Griffith
D L
DL
Arnaout
M A
MA
D000595:Amino Acid Sequence; D000722:Ancylostoma; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001665:Binding Sites; D018821:CD18 Antigens / Q000096:biosynthesis / Q000378:metabolism; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D015801:Helminth Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D016177:Macrophage-1 Antigen / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism*; D008565:Membrane Proteins; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012997:Solvents; D014162:Transfection
D000911:Antibodies, Monoclonal; D018821:CD18 Antigens; D017931:DNA Primers; D006023:Glycoproteins; D015801:Helminth Proteins; D016177:Macrophage-1 Antigen; D008565:Membrane Proteins; D011994:Recombinant Proteins; D012997:Solvents; C086581:NIF protein, Ancylostoma caninum; D008345:Manganese