Experimental support for a β-propeller domain in integrin α-subunits and a calcium binding site on its lower surface

Integrins are large, heterodimeric surface molecules of wide importance in cell adhesion. The N-terminal half of all integrin α-subunits contains seven weak sequence repeats of ≈60 amino acids that are important in ligand binding and have been predicted to fold cooperatively into a single β-propeller domain with seven β-sheets. We provide evidence supporting this model with a mouse mAb to human Mac-1 (αMβ2, CD11b/CD18). This antibody, CBRM1/20, binds to amino acid residues that are in different repeats and are 94 residues apart in the primary structure in the loop between strands 1 and 2 of β-sheet 5 and in the loop between strands 3 and 4 of β-sheet 6. The 1–2 loops of β-sheets 5–7 in integrins have EF hand-like Ca(2+)-binding motifs. CBRM1/20 binds to Mac-1 in the presence of Ca(2+) or Sr(2+) with an EC(50) of 0.2 mM. Mg(2+) or Mn(2+) cannot substitute. Antibodies to other epitopes on the Mac-1 β-propeller domain bind in the absence of calcium. mAb CBRM1/20 does not block ligand binding. Thus, the region on the lower surface of the β-propeller domain to which mAb CBRM1/20 binds does not bind ligand and, furthermore, cannot bind other integrin domains, such as those of the β-subunit.