Entropy Drives Integrin αIIbβ3:Echistatin Binding—Evidence from Surface Plasmon Resonance Spectroscopy^†

This investigation examined the molecular mechanisms that enable the alpha IIb beta 3 integrin to bind efficiently, tightly, and selectively to echistatin, an RGD disintegrin. We used surface plasmon resonance spectroscopy to measure the rate, extent, and stability of complexes formed between micellar alpha IIb beta 3 and recombinant echistatin (rEch) mutants, immobilized on the surface of a biosensor chip. alpha IIb beta 3 bound readily and tightly to wild-type RGD-rEch and RGDF-rEch but not to RGA-rEch or AGD-rEch, demonstrating that both of those charged moieties contribute to integrin recognition. van't Hoff analysis of the temperature dependence of the RGD-rEch K-d data yielded an unfavorable enthalpy change, Delta H degrees = 14 +/- 3 kcal/mol, offset by a favorable entropy term, T Delta S degrees = 23 +/- 3 kcal/mol. Eyring analysis of the temperature dependence of the kinetic parameters yielded Delta H-a degrees(double dagger) = 9 2 kcal/mol and T Delta S-a degrees(double dagger) = -4 +/- 2 kcal/mol, indicating that a substantial activation enthalpy barrier and a smaller activation entropy hinder assembly of the encounter complex. Thus, equilibrium thermodynamic data demonstrate that entropy is the dominant factor stabilizing integrin:echistatin binding, while transition-state thermodynamic parameters indicate that the rate of complex formation is enthalpy-limited. When electrostatic contacts are the major source of receptor:ligand stability, theory and experiment indicate that entropy-favorable ion-pair desolvation often provides the driving force for molecular recognition.