Characterization of αX I-Domain Binding to Receptors for Advanced Glycation End Products (RAGE)

The beta 2 integrins are cell surface transmembrane proteins regulating leukocyte functions, such as adhesion and migration. Two members of beta 2 integrin, alpha M beta 2 and alpha X beta 2, share the leukocyte distribution profile and integrin alpha X beta 2 is involved in antigen presentation in dendritic cells and transendothelial migration of monocytes and macrophages to atherosclerotic lesions. Receptor for advanced glycation end products (RAGE), a member of cell adhesion molecules, plays an important role in chronic inflammation and atherosclerosis. Although RAGE and alpha X beta 2 play an important role in inflammatory response and the pathogenesis of atherosclerosis, the nature of their interaction and structure involved in the binding remain poorly defined. In this study, using I-domain as a ligand binding motif of alpha X beta 2, we characterize the binding nature and the interacting moieties of alpha X I-domain and RAGE. Their binding requires divalent cations (Mg2+ and Mn2+) and shows an affinity on the sub-micro molar level: the dissociation constant of alpha X I-domains binding to RAGE being 0.49 alpha M. Furthermore, the alpha X I-domains recognize the V-domain, but not the C1 and C2-domains of RAGE. The acidic amino acid substitutions on the ligand binding site of alpha X I-domain significantly reduce the I-domain binding activity to soluble RAGE and the alanine substitutions of basic amino acids on the flat surface of the V-domain prevent the V-domain binding to alpha X I-domain. In conclusion, the main mechanism of alpha X I-domain binding to RAGE is a charge interaction, in which the acidic moieties of alpha X I-domains, including E244, and D249, recognize the basic residues on the RAGE V-domain encompassing K39, K43, K44, R104, and K107.