Identification of the binding site for fibrinogen recognition peptide gamma 383-395 within the alpha(M)I-domain of integrin alpha(M)beta2.
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(M)I-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(M)I- and alpha(X)I-domains bind P2-C, and the alpha(L)I-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(M)I-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(M)I-domain segments (245)KFG and Glu(253)-Arg(261) were switched to the homologous alpha(L)I-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, gamma384-402, and this interaction was blocked efficiently by the P2-C peptide; 3) mutation of three amino acid residues within the Lys(245)-Arg(261) segment, Phe(246), Asp(254), and Pro(257), resulted in the loss of the binding function of the recombinant alpha(M)I-domains; and 4) grafting the alpha(M)(Lys(245)-Arg(261)) segment into the alpha(L)I-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(M)I-, alpha(X)I-, and alpha(L)I-domains, is responsible for recognition of a small segment of fibrinogen, gammaThr(383)-Gly(395), by serving as ligand binding site.
Joseph J. Jacobs Center for Thrombosis and Vascular Biology and the Department of Molecular Cardiology, Lerner Research Institute, Cleveland, Ohio 44195, USA.
V P
VP
Yakubenko
D A
DA
Solovjov
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L
Zhang
V C
VC
Yee
E F
EF
Plow
T P
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Ugarova
D000595:Amino Acid Sequence; D001665:Binding Sites; D002448:Cell Adhesion; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005340:Fibrinogen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D005998:Glycine / Q000737:chemistry; D006801:Humans; D008024:Ligands; D016177:Macrophage-1 Antigen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013912:Threonine / Q000737:chemistry
D008024:Ligands; D016177:Macrophage-1 Antigen; C427756:P2-C protein, fibrinogen; D010446:Peptide Fragments; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013912:Threonine; D005340:Fibrinogen; D005998:Glycine