Identification of amino acid residues that form part of the ligand- binding pocket of integrin α5β1

Abstract

Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA) is a novel ligand peptide for integrin α5β1, which blocks α5β1-mediated cell adhesion to fibronectin (Koivunen, E., Wang, B., and Ruoslahti, E. (1994) J. Cell Biol. 124, 373-380). Here we have localized the binding site for RRETAWA on α5β1 using inhibitory monoclonal antibodies (mAbs) and site-directed mutagenesis. A cyclic peptide containing this sequence (*CRRETAWAC*) had little effect on the binding of most anti-α5 and anti-β1 mAbs to α5β1 but completely blocked binding of the anti-α5 mAb 16 in a directly competitive manner. Hence, the binding site of RRETAWA appears to closely overlap with the epitope of mAb 16. *CRRETAWAC* also acted as a direct competitive inhibitor of the binding of Arg-Gly-Asp (RGD)-containing fibronectin fragments to α5β1, suggesting that the binding site for RRETAWA is also closely overlapping with that for RGD. However, differences between the binding sites of RRETAWA and RGD were apparent in that (i) RGD peptides allosterically inhibited the binding of mAb 16 to α5β1, and (ii) several mAbs that perturbed binding of α5β1 to RGD had little effect on binding of α5β1 to RRETAWA. A double mutation in α5 (S156G/W157S) blocked the interaction of both RRETAWA and mAb 16 with α5β1 but had no effect on fibronectin binding or on the binding of other anti-α5 mAbs. Ser156-Trp157 is located near the apex of a putative loop region on the upper surface of a predicted β-propeller structure formed by the NH2- terminal repeats of α5. Our findings suggest that this sequence forms part of the ligand-binding pocket of α5β1. Furthermore, as Ser156-Trp157 is unique to the α5 subunit, it may be responsible for the specific recognition of RRETAWA by α5β1.