Amino acid sequences within the α subunit of integrin αMβ2 (Mac-1) critical for specific recognition of C3bi

Phagocytosis of opsonized particles by neutrophils and monocytes plays a central role in host defense mechanisms against foreign pathogens. This process depends on the interaction between C3bi, a degradation product derived from activation of the complement system, and the alpha(M)beta(2) (CD11/CD18, Mac-1) receptor, the major integrin on neutrophils. Previous studies had established a central role for the I domain, a stretch of similar to 200 amino acids within the alpha(M) subunit in the binding of C3bi, as well as many other alpha(M)beta(2) ligands. The present study was undertaken to establish the molecular basis of C3bi recognition by alpha(M)beta(2). The strategy employed the use of a series-of mutant receptors in which short segments of the I domain of alpha(M) were switched to the corresponding segments of alpha(L), which is structurally very similar but does not bind C3bi. We report three major findings: (1) The C3bi binding pocket is composed of three regions, P-147-R-152, P-201-K-217, and K-245-R-261 of alpha(M), which surround the cation binding site within the MIDAS motif of the I domain. (2) Within the latter segment, K-245 plays a critical role in mediating C3bi binding to alpha(M)beta(2). Mutation of K-245 to Ala significantly reduced C3bi binding but had no effect on binding of another alpha(M)beta(2) I domain ligand, NIF. (3) Blocking of C3bi binding to alpha(M)beta(2) by monoclonal antibodies is achieved through two different mechanisms: direct competition for the ligand binding site or induction of conformational changes. Overall, these studies support the hypothesis that many of the ligands of alpha(M)beta(2) bind to overlapping but not identical sites within the I domain. Although the same short structural segments within the I domain may be involved in binding, different amino acids within these segments may contact different ligands.