Kinetics of calmodulin binding to calcineurin

Calcineurin (CaN) binds Ca2+-saturated calmodulin (CaM) with relatively high affinity; however, an accurate steady-state Kd value has not been determined. In this report, we describe, using steady-state and stopped-flow fluorescence techniques, the rates of association and dissociation of Ca2+-saturated CaM from CaN heterodimer (CaNA/CaNB) and CaNA only. The rate of Ca2+/CaM association was determined to be 4.6 × 107 M−1 s−1. The rate of Ca2+/CaM dissociation from CaN was slower than previously reported and was approximately 0.0012 s−1. In preparations of CaNA alone (no regulatory CaNB subunit), the dissociation rate was slowed further to 0.00026 s−1. From these data we calculate a Kd for binding of Ca2+-saturated CaM to CaN of 28 pM. This Kd is significantly lower than previously reported estimates of ∼1 nM and indicates that CaN is one of the highest affinity CaM-binding proteins identified to date.