High integrin αβ affinity reached by hybrid domain deletion slows ligand-binding on-rate.

The role of the hybrid domain in integrin affinity regulation is unknown, as is whether the kinetics of ligand binding is modulated by integrin affinity state. Here, we compare cell surface and soluble integrin αβ truncation mutants for ligand-binding affinity, kinetics, and thermodynamics. Removal of the integrin transmembrane/cytoplasmic domains or lower legs has little effect on αβ affinity, in contrast to β integrins. In integrin opening, rearrangement at the interface between the βI and hybrid domains is linked to remodeling at the ligand-binding site at the opposite end of the βI domain, which greatly increases in affinity in the open conformation. The larger size of the βI-hybrid interface in the closed state suggests that the hybrid domain stabilizes closing. In agreement, deletion of the hybrid domain raised affinity by 50-fold. Surface plasmon resonance and isothermal titration calorimetry gave similar results and the latter revealed tradeoffs between enthalpy and entropy not apparent from affinity. At extremely high affinity reached in Mn with hybrid domain truncation, αβ on-rate for both pro-TGF-β1 and fibronectin declined. The results suggest that the open conformation of αβ has lower on-rate than the closed conformation, correlate with constriction of the ligand-binding pocket in open αβ structures, and suggest that the extended-closed conformation is kinetically selected for ligand binding. Subsequent transition to the extended-open conformation is stabilized by its much higher affinity for ligand and would also be stabilized by force exerted across ligand-bound integrins by the actin cytoskeleton.