Triple-helical ligands selectively targeting the closed αI domain of integrin α2β1

Integrins α1β1, α2β1, α10β1, and α11β1 serve as primary collagen receptors, recognizing the GxxGEx motif of the collagen triple helix via their αI domains. The Glu residue of the motif coordinates with a divalent metal cation at the metal ion-dependent adhesion site of the αI domain. Owing to this conserved recognition mechanism, previously identified binding sequences show low subtype selectivity. In this study, we identified triple-helical peptide ligands capable of selectively binding to the α2I domain in a manner independent of both Glu and a divalent metal cation. By employing yeast-two hybrid screening using a randomized triple-helical peptide library, we identified sequences in which canonical Glu was substituted by aliphatic residues such as Met. X-ray crystallographic analysis of the complex formed by the α2I domain and a triple-helical peptide containing GFOGMR (O: 4-hydroxyproline)—a Met-substituted variant of the canonical integrin-binding sequence GFOGER—revealed a novel binding mode. In this mode, the peptide was recognized by the closed form of the α2I domain in a metal cation-independent manner. Furthermore, this interaction mode enabled Met-containing peptides to specifically bind to the integrin α2I domain. These findings deepen our understanding of collagen recognition by collagen-binding integrins and provide insight for the discovery of ligands capable of selectively targeting collagen receptor-type integrins.