Multiple discontinuous ligand-mimetic antibody binding sites define a ligand binding pocket in integrin alpha(IIb)beta(3).
Integrin alpha(IIb)beta(3), a platelet fibrinogen receptor, is critically involved in thrombosis and hemostasis. However, how ligands interact with alpha(IIb)beta(3) has been controversial. Ligand-mimetic anti-alpha(IIb)beta(3) antibodies (PAC-1, LJ-CP3, and OP-G2) contain the RGD-like RYD sequence in their CDR3 in the heavy chain and have structural and functional similarities to native ligands. We have located binding sites for ligand-mimetic antibodies in alpha(IIb) and beta(3) using human-to-mouse chimeras, which we expect to maintain functional integrity of alpha(IIb)beta(3). Here we report that these antibodies recognize several discontinuous binding sites in both the alpha(IIb) and beta(3) subunits; these binding sites are located in residues 156-162 and 229-230 of alpha(IIb) and residues 179-183 of beta(3). In contrast, several nonligand-mimetic antibodies (e.g. 7E3) recognize single epitopes in either subunit. Thus, binding to several discontinuous sites in both subunits is unique to ligand-mimetic antibodies. Interestingly, these binding sites overlap with several (but not all) of the sequences that have been reported to be critical for fibrinogen binding (e.g. N-terminal repeats 2-3 but not repeats 4-7, of alpha(IIb)). These results suggest that ligand-mimetic antibodies and probably native ligands may make direct contact with these discontinuous binding sites in both subunits, which may constitute a ligand-binding pocket.
Puzon-McLaughlin
W
W
Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Kamata
T
T
Takada
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D005340:Fibrinogen / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D009154:Mutation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D011485:Protein Binding; D020449:Repetitive Sequences, Amino Acid; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D008024:Ligands; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D005340:Fibrinogen