We have employed echistatin, a 5.4 kDa snake venom disintegrin, as a model protein to investigate the paradox that small ligand-mimetics can bind to the resting αIIbβ3 integrin while adhesive macromolecules cannot. We characterized the interactions between purified human αIIbβ3 and two recombinant echistatin variants: rEch (1-49) M28L, chosen for its selectivity toward β3-integrins, and rEch (1-40) M28L, a carboxy-terminal truncation mutant. While both contain an RGD integrin targeting sequence, only rEch (1-49) M28L was an effective inhibitor of αIIbβ3 function. Electron microscopy of rotary shadowed specimens yielded a variety of αIIbβ3 conformers ranging from compact, spherical particles (maximum dimension 22 nm) to the classical "head with two tails" forms (32 nm). The population of larger particles (42-56 nm) increased from 17% to 28% in the presence of rEch (1-49) M28L, indicative of ligand-induced oligomerization. Sedimentation velocity measurements demonstrated that both full length and truncated echistatin perturbed αIIbβ3's solution structure, yielding slower-sedimenting open conformers. Dynamic light scattering showed that rEch (1-49) M28L protected αIIbβ3 from thermal aggregation, raising its transition mid-point from 46°C to 69°C; a smaller shift resulted with rEch (1-40) M28L. Sedimentation equilibrium demonstrated that both echistatin ligands induced substantial αIIbβ3 dimerization. van't Hoff analysis revealed a pattern of entropy/enthalpy compensation similar to tirofiban, a small RGD ligand-mimetic that binds tightly to αIIbβ3, but yields smaller conformational perturbations than echistatin. We propose that echistatin may serve as a paradigm for understanding multidomain adhesive macromolecules because its ability to modulate αIIbβ3's structure resides on an RGD loop, while full disintegrin activity requires an auxiliary site that includes the carboxy-terminal nine amino acid residues.
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