Identification of the principal binding site for RGD-containing ligands in the alpha(V)beta(3) integrin: a photoaffinity cross-linking study.

By superimposing data obtained by photo-cross-linking RGD-containing ligands to the human alpha(V)beta(3) integrin onto the crystal structure of the ectopic domain of this receptor (Xiong et al. (2001) Science 294, 339-345), we have identified the binding site for the RGD triad within this integrin. We synthesized three novel analogues of the 49-amino acid disintegrin, echistatin: [Bpa(21),Leu(28)]-, [Bpa(23),Leu(28)]-, and [Bpa(28)]echistatin. Each contains a photoreactive p-benzoyl-phenylalanyl (Bpa) residue in close proximity to the RGD motif which spans positions 24-26; together, the photoreactive positions flank the RGD motif. The analogues bind with high affinity to the purified recombinant alpha(V)beta(3) integrin, but very poorly to the closely related human alpha(IIb)beta(3) platelet integrin. While echistatin analogues containing Bpa in either position 23 or 28 cross-link specifically and almost exclusively to the beta(3) subunit of alpha(V)beta(3), [Bpa(21),Leu(28)]echistatin cross-links to both the alpha(V) and the beta(3) subunits, with cross-linking to the former favored. [Bpa(23),Leu(28)]echistatin cross-links 10-30 times more effectively than the other two analogues. We identified beta(3)[109-118] as the domain that encompasses the contact site for [Bpa(28)]echistatin. This domain is included in beta(3)[99-118] (Bitan et al. (2000) Biochemistry 39, 11014-11023), a previously identified contact domain for a cyclic RGD-containing heptapeptide with a benzophenone moiety in a position that is similar to the placement of the benzophenone in [Bpa(28)]echistatin relative to the RGD triad. Recently, we identified beta(3)[209-220] as the contact site for an echistatin analogue with a photoreactive group in position 45, near the C-terminus of echistatin (Scheibler et al. (2001) Biochemistry 40, 15117-14126). Taken together, these results support the hypothesis that the very high binding affinity of echistatin to alpha(V)beta(3) results from two distinct epitopes in the ligand, a site including the RGD triad and an auxiliary epitope at the C-terminus of echistatin. Combining our results from photoaffinity cross-linking studies with data now available from the recently published crystal structure of the ectopic domain of alpha(V)beta(3), we characterize the binding site for the RGD motif in this receptor.