Identification of the Principal Binding Site for RGD-Containing Ligands in the α_Vβ₃ Integrin:  A Photoaffinity Cross-Linking Study

Abstract

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.

A novel αvβ3-blocking disintegrin containing the RGD motive, DisBa-01, inhibits bFGF-induced angiogenesis and melanoma metastasis

“…Various studies have indicated that the aspartic acid present at the RGD sequence should be involved in interactions with a metal coordinated at metal-ion-dependent adhesion site (MIDAS) of b subunits [24][25][26]. Thus, we superposed the RGD motif of DisBa-01 to the same sequence found in a peptide complexed to a v b 3 integrin (PDB: 1L5G [25]), removed the peptide and replaced the original manganese atoms by calcium atoms.…”

The Disintegrin Echistatin Stabilizes Integrin αIIbβ3's Open Conformation and Promotes Its Oligomerization

“…38 In addition, echistatin has a putative auxiliary binding site at its carboxy terminus. 39,40 Thus, we examined the interactions of full-length (rEch (1-49) M28L) and truncated (rEch (1-40) M28L) recombinant echistatin variants 41 with the purified aIIbb3 integrin, isolated in a functional conformation in octyl glucoside micelles. 10,11,13 We assessed the ability of each recombinant echistatin variant to inhibit platelet aggregation and platelet adhesion to fibrin, two activities that are dependent on the aIIbb3 integrin.…”

“…Tirofiban's smaller size and higher affinity for the aIIbb3 integrin 32 could explain why its effects on aIIbb3 conformation were smaller than echistatin's. We may speculate that echistatin operates by an induced fit mechanism, 58 perturbing the receptor's conformation to maximize electrostatic contacts with its target integrin 15,39,40,59 at the expense of binding energy.…”

Enhanced tumor MR imaging with gadolinium‐loaded polychelating polymer‐containing tumor‐targeted liposomes

“…Previous attempts to target contrast liposomes to tumors for enhanced imaging through the use of targeting moieties include antibodies against the transferrin receptor (13) or RGD peptides (14). Although promising data have been obtained, these approaches are restricted only to those tumors that overexpress the transferrin receptor (15), or only to tumor endothelium since the RGD peptide specifically binds endothelial integrins (16). In contrast to the studies described above, a universal tumor-imaging agent could be prepared if an antibody possessing broad tumor specificity is used as a targeting moiety.…”