Low affinity integrin states have faster binding kinetics than the high affinity state

Integrin conformational ensembles contain two low-affinity states, bent-closed and extended-closed, and an active, high-affinity, extended-open state. It is widely thought that integrins must be activated before they bind ligand; however, one model holds that activation follows ligand binding. As ligand-binding kinetics are not only rate limiting for cell adhesion but also have important implications for the mechanism of activation, we measure them here for integrins α4β1 and α5β1 and show that the low-affinity states bind substantially faster than the high-affinity state. On and off-rate measurements are similar for integrins on cell surfaces and ectodomain fragments. Although the extended-open conformation’s on-rate is ∼20-fold slower, its off-rate is ∼25,000-fold slower, resulting in a large affinity increase. The tighter ligand-binding pocket in the open state may slow its on-rate. These kinetic measurements, together with previous equilibrium measurements of integrin conformational state affinity and relative free energy on intact cells, are key to a definitive understanding of the mechanism of integrin activation.