Several distinct regions of the integrin αIIb subunit have been implicated in ligand binding. To localize the ligand binding sites in αIIb, we swapped all 27 predicted loops with the corresponding sequences of α4 or α5. 19 of the 27 swapping mutations had no effect on binding to both fibrinogen and ligand-mimetic antibodies (e.g. LJ-CP3), suggesting that these regions do not contain major ligand binding sites. In contrast, swapping the remaining 8 predicted loops completely blocked ligand binding. Ala scanning mutagenesis of these critical predicted loops identified more than 30 discontinuous residues in repeats 2-4 and at the boundary between repeats 4 and 5 as critical for ligand binding. Interestingly, these residues are clustered in the predicted β-propeller model, consistent with this model. Most of the critical residues are located at the edge of the upper face of the propeller, and several critical residues are located on the side of the propeller domain. None of the predicted loops in repeats 1, 6, and 7, and none of the four putative Ca2+-binding predicted loops on the lower surface of the β-propeller were important for ligand binding. The results map an important ligand binding interface at the edge of the top and on the side of the β-propeller toroid, centering on repeat 3.
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