Molecular basis of ligand recognition by integrin α₅β₁. II. Specificity of Arg-Gly-Asp binding is determined by Trp¹⁵⁷ of the α subunit

Different β₁ integrins bind Arg. Gly-Asp (RGD) peptides with differing specificities, suggesting a role for residues in the α subunit in determining ligand specificity. Integrin α₅β₁ has been shown to bind with high affinity to peptides containing an Arg-Gly-Asp-Gly-Trp (RGDGW) sequence but with relatively low affinity to other RGD peptides. The residues within the ligand-binding pocket that determine this specificity are currently unknown. A cyclic peptide containing the RGDGW sequence was found to strongly perturb the binding of the anti-α₅ monoclonal antibody (mAb) 16 to α₅β₁. In contrast, RGD peptides lacking the tryptophan residue acted as weak inhibitors of mAb 16 binding. The epitope of mAb 16 has previously been localized to a region of the α₅ subunit that contains Ser¹⁵⁶-Trp¹⁵⁷. Mutation of Trp¹⁵⁷ (but not of Ser¹⁵⁶ or surrounding residues) to alanine blocked recognition of mAb 16 and perturbed the high affinity binding of RGDGW-containing peptides to α₅β₁. The same mutation also abrogated recognition of the α₅β₁-specific ligand peptide Arg-Arg-Glu-Thr-Ala-Trp- Ala (RRETAWA). Based on these findings, we propose that Trp¹⁵⁷ of α₅ participates in a hydrophobic interaction with the tryptophan residue in RGDGW, and that this interaction determines the specificity of α₅β₁ for RGDGW-containing peptides. Since the RGD sequence is recognized predominantly by amino acid residues on the β₁ subunit, our results suggest that Trp¹⁵⁷ of α₅ must lie very close to these residues. Our findings therefore provide new insights into the structure of the ligand-binding pocket of α₅β₁.