Identification of a Contact Domain between Echistatin and the Integrin αvβ3by Photoaffinity Cross-Linking

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Abstract
The integrin αvβ3 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 αvβ3 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) Biochemistry38, 3414−3420] or the β3[167−171] [Bitan, G., et al. (2000) Biochemistry39, 11014−11023] sequence, respectively, of the αvβ3 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 αvβ3 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(Nε-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 αIIbβ3, 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.