Identification of a contact domain between echistatin and the integrin α_vβ₃ by photoaffinity cross-linking

The integrin α_vβ₃ is the major receptor mediating the attachment of osteoclasts to the extracellular matrix in bone and plays a critical role in bone resorption and bone remodeling. Most of the ligands interacting with the α_vβ₃ receptor contain an Arg-Gly-Asp (RGD) motif. Recently, we have identified two small RGD peptides, containing a benzophenone moiety at either the carboxyl or amino terminus, that photo-cross-linked within the β₃[99-118] [Bitan, G., et al. (1999) Biochemistry 38, 3414-3420] or the β₃[167-171] [Bitan, G., et al. (2000) Biochemistry 39, 11014-11023] sequence, respectively, of the α_vβ₃ receptor in a selective fashion. Here, we report the synthesis of a photoreactive analogue of echistatin (a 49-amino acid peptide), a potent RGD-containing antagonist of the α_vβ₃ receptor both in vitro and in vivo. This bioactive analogue is substituted at position 45 with a p-benzoyl moiety (pBz₂), located within the flexible C-terminal domain and removed 20 amino acid residues from the R²⁴GD²⁶ triad. This C-terminal domain was reported to contribute to receptor binding affinity by acting as an auxiliary binding site. The radiolabeled ¹²⁵I-[Arg³⁵, Lys⁴⁵(N_ε-pBz₂)]-echistatin photo-cross-links effectively to a site within the β₃[209-220] sequence. Residues in this domain have been reported to be part of the metal ion-dependent adhesion site (MIDAS). Receptor fragments overlapping this domain were reported to bind to fibrinogen and block fibrinogen binding to α_IIbβ₃, the platelet integrin receptor. Taken together, position 45 in echistatin, located within an auxiliary binding site in echistatin, cross-links to a site distinct from the two previously reported sites, β₃[99-118] and β₃[167- 171], which cross-link to photophores flanking the RGD triad. These cross-linking data support the hypothesis that the ligand-bound conformation of the integrin β₃ subunit differs from the known conformation of I domains.