Identification of the principal binding site for RGD-containing ligands in the α_vβ₃ integrin: A photoaffinity cross-linking study

By superimposing data obtained by photo-cross-linking RGD-containing ligands to the human α_vβ₃ 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: [Bpa²¹,Leu²⁸]-, [Bpa²³, Leu²⁸]-, and [Bpa²⁸]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 α_vβ₃ integrin, but very poorly to the closely related human α_IIbβ₃ platelet integrin. While echistatin analogues containing Bpa in either position 23 or 28 cross-link specifically and almost exclusively to the β₃ subunit of α_vβ₃, [Bpa²¹,Leu²⁸]echistatin cross-links to both the α_v and the β₃ subunits, with cross-linking to the former favored. [Bpa²³,Leu²⁸]echistatin cross-links 10-30 times more effectively than the other two analogues. We identified β₃[109-118] as the domain that encompasses the contact site for [Bpa²⁸]echistatin. This domain is included in β₃[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 [Bpa²⁸]echistatin relative to the RGD triad. Recently, we identified β₃[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 α_vβ₃ 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 α_vβ₃, we characterize the binding site for the RGD motif in this receptor.