Identification of the Binding Site for Fibrinogen Recognition Peptide γ383-395 within the α_MI-Domain of Integrin α _Mβ₂

The leukocyte integrin α_Mβ₂ (Mac-1, CD11b/CD18) is a cell surface adhesion receptor for fibrinogen. The interaction between fibrinogen and α_Mβ₂ mediates a range of adhesive reactions during the immune-inflammatory response. The sequence γ³⁸³TMKIIPFNRLTIG³⁹⁵, P2-C, within the γ-module of the D-domain of fibrinogen, is a recognition site for α_Mβ₂ and α_Xβ ₂. We have now identified the complementary sequences within the α_MI-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 α _Mβ₂, α_Xβ₂, and α_Lβ₂, i.e. the α_MI- and α_XI-domains bind P2-C, and the α_LI-domain did not bind this ligand. The Lys²⁴⁵-Arg²⁶¹ sequence, which forms a loop βD-α5 and an adjacent helix α5 in the three-dimensional structure of the α_MI-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 α_MI-domain segments ²⁴⁵KFG and Glu²⁵³-Arg²⁶¹ were switched to the homologous α_LI-domain segments failed to support adhesion to P2-C; 2) synthetic peptides duplicating the Lys²⁴⁵-Tyr²⁵² and Glu²⁵³-Arg²⁶¹ sequences directly bound the D fragment and P2-C derivative, γ384-402, and this interaction was blocked efficiently by the P2-C peptide; 3) mutation of three amino acid residues within the Lys²⁴⁵-Arg²⁶¹ segment, Phe²⁴⁶, Asp²⁵⁴, and Pro²⁵⁷, resulted in the loss of the binding function of the recombinant α_MI-domains; and 4) grafting the α_M(Lys²⁴⁵-Arg²⁶¹) segment into the α_LI-domain converted it to a P2-C-binding protein. These results demonstrate that the α_M(Lys²⁴⁵-Arg ²⁶¹) segment, a site of the major sequence and structure difference among α_MI-, α_XI-, α_XI-, and α_LI-domains, is responsible for recognition of a small segment of fibrinogen, γThr³⁸³-Gly³⁹⁵, by serving as ligand binding site.