MacEwen Melissa J S, Rusnac Domnita-Valeria, Ermias Henok, Locke Timothy M, Gizinski Hayden E, Dexter Joseph P, Sancak Yasemin
Department of Pharmacology, University of Washington, Seattle, WA 98195, USA.
Data Science Initiative and Department of Human Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA.
iScience. 2023 Feb 4;26(4):106146. doi: 10.1016/j.isci.2023.106146. eCollection 2023 Apr 21.
Activation of myosin light chain kinase (MLCK) by calcium ions (Ca) 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 Ca 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 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.
钙离子（Ca）和钙调蛋白（CaM）对肌球蛋白轻链激酶（MLCK）的激活在包括血管平滑肌收缩和细胞运动在内的众多细胞功能中起着重要作用。尽管进行了广泛的生化分析，但激活机制的某些方面仍存在争议，并且针对Ca和CaM与MLCK的结合提出了相互竞争的理论模型。这些模型对于平衡稳态是可解析求解的，并产生了不同的预测，无论赋予参数的数值如何，这些预测都成立。这些预测构成了最近提出的用于模型判别多部分实验策略的基础。在这里，我们通过使用荧光共振能量转移（FRET）系统测量CaM-MLCK结合来实施该策略。根据数学模型对结合数据的解释表明，CaM与MLCK结合存在部分有序的机制。使用评估CaM-MLCK结合的正交方法收集的补充数据进一步支持了这一结论。
