Molecular basis of ligand recognition by integrin alpha(5)beta(1) - II. Specificity of Arg-Gly-Asp binding is determined by Trp(157) of the alpha subunit

Different beta(1) integrins bind Arg-Gly-Asp (RGD) peptides with differing specificities, suggesting a role for residues in the a subunit in determini ng ligand specificity, Integrin alpha(5)beta(1) has been shown to bind with high affinity to peptides containing an Arg-Gly-Asp-Gly-Trp (RGDGW) sequen ce but with relatively low affinity to other RGD peptides, The residues wit hin the ligand-binding pocket that determine this specificity are currently unknown, A cyclic peptide containing the RGDGW sequence was found to stron gly perturb the binding of the anti-a, monoclonal antibody (mAb) 16 to alph a(5)beta(1) 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 alpha(5) subunit that contains Ser(156)-T rp(157). Mutation of Trp(157) (but not of Ser(156) or surrounding residues) to alanine blocked recognition of mAb 16 and perturbed the high affinity b inding of RGDGW-containing peptides to alpha(5)beta(1). The same mutation a lso abrogated recognition of the alpha(5)beta(1)-specific ligand peptide Ar g-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA). Based on these findings, we propose th at Trp(167) of alpha(5) participates in a hydrophobic interaction with the tryptophan residue in RGDGW, and that this interaction determines the speci ficity of alpha(5)beta(1) for RGDGW-containing peptides, Since the RGD sequ ence is recognized predominantly by amino acid residues on the beta(1) subu nit, our results suggest that Trp167 Of alpha(5) must lie very close to the se residues. Our findings therefore provide new insights into the structure of the ligand-binding pocket of alpha(5)beta(1).