Critical amino acid residues for ligand binding are clustered in a predicted β-turn of the third N-terminal repeat in the integrin α4 and α5 subunits

Integrin α4β1 is a receptor for vascular cell adhesion molecule (VCAM)-1 and fibronectin (CS-1). The α4β1-ligand interaction is involved in the pathogenesis of diseases and is, therefore, a therapeutic target. Here, we identified critical residues of α4 for ligand binding using alanine-scanning mutagenesis of the previously localized putative ligand binding sites (residues 108-268). Among 43 mutations tested, mutations of Tyr187, Trp188 and Gly190 significantly inhibited cell adhesion to both VCAM-1 and CS-1. This inhibition was not due to any gross structural changes of α4β1. These critical residues are clustered in a predicted β-turn structure (residues 181-190) of the third N-terminal repeat in α4. The repeat does not contain divalent cation binding motifs. Notably, the mutations within the corresponding region of α5 significantly reduced fibronectin-α5β1 interaction. These findings suggest that the predicted β-turn structure could be ubiquitously involved in ligand binding of non-I domain integrins.