Critical amino acid residues for ligand binding are clustered in a predicted beta‐turn of the third N‐terminal repeat in the integrin alpha 4 and alpha 5 subunits.

Integrin alpha 4 beta 1 is a receptor for vascular cell adhesion molecule (VCAM)‐1 and fibronectin (CS‐1). The alpha 4 beta 1‐ligand interaction is involved in the pathogenesis of diseases and is, therefore, a therapeutic target. Here, we identified critical residues of alpha 4 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 alpha 4 beta 1. These critical residues are clustered in a predicted beta‐turn structure (residues 181–190) of the third N‐terminal repeat in alpha 4. The repeat does not contain divalent cation binding motifs. Notably, the mutations within the corresponding region of alpha 5 significantly reduced fibronectin‐alpha 5 beta 1 interaction. These findings suggest that the predicted beta‐turn structure could be ubiquitously involved in ligand binding of non‐I domain integrins.