Mathematical modeling and biochemical analysis support partially ordered calmodulin-myosin light chain kinase binding

• Published in: iScience 2023-04, Vol.26 (4), p.106146-106146, Article 106146
• Main Authors: MacEwen, Melissa J.S., Rusnac, Domnita-Valeria, Ermias, Henok, Locke, Timothy M., Gizinski, Hayden E., Dexter, Joseph P., Sancak, Yasemin
• Format: Article
• Language: English
• Subjects: Biochemical mechanism; Biochemistry; In silico biology
• ISSN: 2589-0042; 2589-0042
• DOI: 10.1016/j.isci.2023.106146

Activation of myosin light chain kinase (MLCK) by calcium ions (Ca2+) and calmodulin (CaM) plays an important role in numerous cellular functions including vascular smooth muscle contraction and cellular motility. Despite extensive biochemical analysis, aspects of the mechanism of activation remain controversial, and competing theoretical models have been proposed for the binding of Ca2+ 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. Interpretation of binding data in light of the mathematical models suggests a partially ordered mechanism for binding CaM to MLCK. Complementary data collected using orthogonal approaches that assess CaM-MLCK binding further support this conclusion. [Display omitted] •Calmodulin (CaM) activation of MLCK is key to many cellular functions•An experimental strategy can distinguish between three models of CaM-MLCK binding•Experimental data support a partially ordered CaM-MLCK binding mechanism Biochemistry; Biochemical mechanism; in silico biology