Kinetic characterization of the sole nonmuscle myosin-2 from the model organism Drosophila melanogaster
Kinetic characterization of the sole nonmuscle myosin-2 from the model organism Drosophila melanogaster
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DOI：
10.1096/fj.14-266742
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发表时间：
2015-04-01
期刊：
影响因子：
4.8
通讯作者：
Sellers, James R.
中科院分区：
文献类型：
--
作者：
Heissler, Sarah M.;Chinthalapudi, Krishna;Sellers, James R.
非肌肉肌球蛋白-2 是模式生物果蝇中为 F-肌动蛋白细胞骨架提供收缩力的主要酶复合物。尽管肌球蛋白在果蝇发育和体内平衡中发挥着重要作用，但其动力学特征仍然难以捉摸。这项体外研究的目的是对果蝇非肌肉肌球蛋白 2 运动域进行详细的稳态和前稳态动力学表征。动力学特征是缓慢的稳态 ATP 酶活性、对 F-肌动蛋白和 ADP 的高亲和力以及低占空比。对模型生物体非肌肉肌球蛋白-2 补体的总体酶学特征的比较分析表明，果蝇蛋白类似于后生动物而不是原生动物的非肌肉肌球蛋白-2，尽管肌球蛋白运动功能的调节方面是不同的。果蝇非肌肉肌球蛋白 2 对肌球蛋白 2 抑制剂（一种常用的肌球蛋白 2 抑制剂）独特地不敏感。计算机模拟方法与动力学研究表明，果蝇非肌肉肌球蛋白 2 活性位点环开关 2 中的非共有氨基酸 Met466 充当肌球蛋白抑菌素脱敏剂。 M466I突变的引入使蛋白质对肌球蛋白敏感，导致半数最大抑制浓度为36.3+/-4.1μM。总而言之，这些数据表明果蝇非肌肉肌球蛋白-2是真正的分子马达，并在switch-2和肌球蛋白敏感性之间建立了重要联系。
Nonmuscle myosin-2 is the primary enzyme complex powering contractility of the F-actin cytoskeleton in the model organism Drosophila. Despite myosin's essential function in fly development and homeostasis, its kinetic features remain elusive. The purpose of this in vitro study is a detailed steady-state and presteady-state kinetic characterization of the Drosophila nonmuscle myosin-2 motor domain. Kinetic features are a slow steady-state ATPase activity, high affinities for F-actin and ADP, and a low duty ratio. Comparative analysis of the overall enzymatic signatures across the nonmuscle myosin-2 complement from model organisms indicates that the Drosophila protein resembles nonmuscle myosin-2s from metazoa rather than protozoa, though modulatory aspects of myosin motor function are distinct. Drosophila nonmuscle myosin-2 is uniquely insensitive toward blebbistatin, a commonly used myosin-2 inhibitor. An in silico modeling approach together with kinetic studies indicate that the nonconsensus amino acid Met466 in the Drosophila nonmuscle myosin-2 active-site loop switch-2 acts as blebbistatin desensitizer. Introduction of the M466I mutation sensitized the protein for blebbistatin, resulting in a half-maximal inhibitory concentration of 36.3 +/- 4.1 mu M. Together, these data show that Drosophila nonmuscle myosin-2 is a bona fide molecular motor and establish an important link between switch-2 and blebbistatin sensitivity.