Ligand binding promotes the entropy-driven oligomerization of integrin αIIbβ3

Integrin α_IIbβ₃ clusters on the platelet surface after binding adhesive proteins in a process that regulates signal transduction. However, the intermolecular forces driving integrin self-association are poorly understood. This work provides new insights into integrin clustering mechanisms by demonstrating how temperature and ligand binding interact to affect the oligomeric state of α_IIbβ₃. The ligand-free receptor, solubilized in thermostable octyl glucoside micelles, exhibited a cooperative transition at ∼43°C, monitored by changes in intrinsic fluorescence and circular dichroism. Both signals changed in a direction opposite to that for global unfolding, and both were diminished upon binding the fibrinogen γ-chain ligand-mimetic peptide cHArGD. Free and bound receptors also exhibited differential sensitivity to temperature-enhanced oligomerization, as measured by dynamic light scattering, sedimentation velocity, and sedimentation equilibrium. Van't Hoff analyses of dimerization constants for α^IIbβ₃ complexed with cHArGD, cRGD, or eptifibatide yielded large, favorable entropy changes partly offset by unfavorable enthalpy changes. Transmission electron microscopy showed that ligand binding and 37°C incubation enhanced assembly of integrin dimers and larger oligomers linked by tail-to-tail contacts. Interpretation of these images was aided by threading models for α_IIbβ₃ protomers and dimers based on the ectodomain structure of α_vβ₃. We propose that entropy-favorable nonpolar interactions drive ligand-induced integrin clustering and outside-in signaling.