Mathematical modeling and biochemical analysis support partially ordered CaM-MLCK binding

Activation of myosin light-chain kinase (MLCK) by calcium ions (Ca 2+ ) and calmodulin (CaM) plays an important role in numerous cellular functions including vascular smooth muscle contraction and cellular motility. Despite extensive biochemical analysis of this system, aspects of the mechanism of activation remain controversial, and competing theoretical models have been proposed for the binding of Ca 2+ and CaM to MLCK. The models are analytically solvable for an equilibrium steady state and give rise to distinct predictions that hold regardless of the numerical values assigned to parameters. These predictions form the basis of a recently proposed, multi-part experimental strategy for model discrimination. Here we implement this strategy by measuring CaM-MLCK binding using an in vitro FRET system. This system uses the CaM-binding region of smooth muscle MLCK protein to link two fluorophores to form an MLCK FRET Reporter (FR). Biochemical and biophysical experiments have established that FR can be reliably used to analyze MLCK-CaM binding. We assessed the binding of either wild-type CaM, or mutant CaM with one or more defective EF-hand domains, to FR. Interpretation of binding data in light of the mathematical models suggests a partially ordered mechanism for binding of CaM to MLCK. Complementary data collected using orthogonal approaches that directly quantify CaM-MLCK binding further supports our conclusions.