Identification of the binding site for fibrinogen recognition peptide γ383-395 within the αMI-domain of integrin αMβ2

The leukocyte integrin alpha (M)beta (2) (Mac-1, CD11b/CD18) is a cell surface adhesion receptor for fibrinogen. The interaction between fibrinogen and alpha (M)beta (2) mediates a range of adhesive reactions during the immune-inflammatory response. The sequence gamma (383)TMKIIPFNRLTIG(395), P2-C, within the gamma -module of the D-domain of fibrinogen, is a recognition site for alpha (M)beta (2) and alpha (x)beta (2). We have now identified the complementary sequences within the alpha I-M-domain of the receptor responsible for recognition of P2-C, The strategy to localize the binding site for P2-C was based on distinct P2-C binding properties of the three structurally similar I-domains of alpha (M)beta (2), alpha (X)beta (2), and alpha (L)beta (2) i.e. the alpha I-M-. and alpha I-X-domains bind P2-C, and the alpha I-L-domain did not bind this ligand. The Lys(245)-Arg(261) sequence, which forms a loop betaD-alpha5 and an adjacent helix alpha5 in the three-dimensional structure of the alpha I-M-domain, was identified as the binding site for P2-C. This conclusion is supported by the following data: 1) mutant cell lines in which the alpha I-M-domain segments (245)KFG and Glu(253)-Arg(261) were switched to the homologous alpha I-L-domain segments failed to support adhesion to P2-C; 2) synthetic peptides duplicating the Lys(245)-Tyr(252) and Glu(253)-Arg(261) sequences directly bound the D fragment and P2-C derivative, gamma 384-402, and this interaction was blocked efficiently by the P2-C peptide; 3) mutation of three amino acid residues within the Lys245-Arg261 segment, Phe(246), Asp(254), and Pro(257), resulted in the loss of the binding function of the recombinant alpha I-M-domains; and 4) grafting the alpha (M) (Lys(245)-Arg(261)) Segment into the alpha I-L-domain converted it to a P2-C-binding protein. These results demonstrate that the alpha (M)(Lys(245)-Arg(261)) segment, a site of the major sequence and structure difference among alpha I-M-, alpha I-X-, and alpha I-L-domains, is responsible for recognition of a small segment of fibrinogen, gamma Thr(383)-Gly(395), by serving as ligand binding site.