Department of Anatomy and Molecular Embryology, Ruhr-University, Bochum, Germany.
Cryo Electron Microscopy, BioQuant, University Hospital, Heidelberg, Germany.
FEBS J. 2018 May;285(9):1715-1729. doi: 10.1111/febs.14442. Epub 2018 Apr 16.
The cyclical interaction between F-actin and myosin in muscle cells generates contractile force. The myosin motor domain hydrolyses ATP, resulting in conformational changes that are amplified by the myosin lever arm that links the motor domain to the rod domain. Recent cryo-electron microscopic data have provided a clear picture of the myosin-ATP-F-actin complex, but structural insights into other stages of the myosin-actin interaction have been less forthcoming. To address this issue, we cross-linked F-actin subunits between Cys374 and Lys191, and separated them by gel filtration. Purified actin-dimers, -trimers and -tetramers retained the ability to polymerize and to stimulate myosin-subfragment 1 (myosin-S1) ATPase activity. To generate stable actin oligomer:myosin-S1 complexes, we blocked actin polymerization with gelsolin and Clostridium botulinum iota toxin-mediated ADP-ribosylation. After polymerization inhibition, actin-trimers and -tetramers retained the ability to stimulate the myosin-S1-ATPase, whereas the actin-dimer showed very little ATPase stimulation. We then analysed the stoichiometry and binding affinity of myosin-S1 to actin oligomers. Actin-trimers and -tetramers bound myosin-S1 in the absence of nucleotide; the trimer contains one myosin-S1 binding site. We calculated a dissociation constant (K ) of 1.1 × 10 m and 1.9 × 10 m for binding of native F-actin and the actin-trimer to myosin-S1, respectively. EM of the actin-trimer:myosin-S1 complex demonstrated the presence of single particles of uniform size. Image reconstruction allowed a reasonable fit of the actin-trimer and myosin-S1 into the obtained density clearly showing binding of one myosin-S1 molecule to the two long-pitch actins of the trimer, supporting the kinetic data.
肌动蛋白和肌球蛋白在肌肉细胞中的周期性相互作用产生收缩力。肌球蛋白的马达结构域水解 ATP，导致构象变化，这些变化通过连接马达结构域和杆状结构域的肌球蛋白杠杆臂放大。最近的低温电子显微镜数据提供了肌球蛋白-ATP-肌动蛋白复合物的清晰图像，但对肌球蛋白-肌动蛋白相互作用的其他阶段的结构见解却较少。为了解决这个问题，我们在 Cys374 和 Lys191 之间交联肌动蛋白亚基，并通过凝胶过滤将它们分离。纯化的肌动蛋白二聚体、三聚体和四聚体保留了聚合和刺激肌球蛋白亚片段 1（肌球蛋白-S1）ATP 酶活性的能力。为了生成稳定的肌动蛋白寡聚体：肌球蛋白-S1 复合物，我们用凝胶电泳和梭菌肉毒毒素介导的 ADP-核糖基化来阻止肌动蛋白聚合。聚合抑制后，肌动蛋白三聚体和四聚体仍能刺激肌球蛋白-S1-ATP 酶，而肌动蛋白二聚体几乎没有刺激 ATP 酶的作用。然后，我们分析了肌球蛋白-S1 与肌动蛋白寡聚体的计量比和结合亲和力。肌动蛋白三聚体和四聚体在没有核苷酸的情况下与肌球蛋白-S1 结合；三聚体含有一个肌球蛋白-S1 结合位点。我们计算出肌球蛋白-S1 与天然肌动蛋白和肌动蛋白三聚体结合的解离常数（K）分别为 1.1×10 和 1.9×10 。肌动蛋白三聚体：肌球蛋白-S1 复合物的 EM 显示存在大小均匀的单个粒子。图像重建允许将肌动蛋白三聚体和肌球蛋白-S1 合理地拟合到获得的密度中，清楚地显示了一个肌球蛋白-S1 分子与三聚体的两个长螺距肌动蛋白的结合，支持了动力学数据。
