Department of Chemistry, New York University, New York, New York, USA.
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA.
J Biol Chem. 2021 Jan-Jun;296:100337. doi: 10.1016/j.jbc.2021.100337. Epub 2021 Jan 27.
Members of the ADF/cofilin family of regulatory proteins bind actin filaments cooperatively, locally change actin subunit conformation and orientation, and sever filaments at "boundaries" between bare and cofilin-occupied segments. A cluster of bound cofilin introduces two distinct classes of boundaries due to the intrinsic polarity of actin filaments, one at the "pointed" end side and the other at the "barbed" end-side of the cluster; severing occurs more readily at the pointed end side of the cluster ("fast-severing" boundary) than the barbed end side ("slow-severing" boundary). A recent electron-cryomicroscopy (cryo-EM) model of the slow-severing boundary revealed structural "defects" at the interface that potentially contribute to severing. However, the structure of the fast-severing boundary remains uncertain. Here, we use extensive molecular dynamics simulations to produce atomic resolution models of both severing boundaries. Our equilibrated simulation model of the slow-severing boundary is consistent with the cryo-EM structural model. Simulations indicate that actin subunits at both boundaries adopt structures intermediate between those of bare and cofilin-bound actin subunits. These "intermediate" states have compromised intersubunit contacts, but those at the slow-severing boundary are stabilized by cofilin bridging interactions, accounting for its lower fragmentation probability. Simulations where cofilin proteins are removed from cofilactin filaments favor a mechanism in which a cluster of two contiguously bound cofilins is needed to fully stabilize the cofilactin conformation, promote cooperative binding interactions, and accelerate filament severing. Together, these studies provide a molecular-scale foundation for developing coarse-grained and theoretical descriptions of cofilin-mediated actin filament severing.
ADF/cofilin 家族的调节蛋白成员协同结合肌动蛋白丝，局部改变肌动蛋白亚基构象和取向，并在无遮蔽和 cofilin 占据的片段之间的“边界”处切割丝。由于肌动蛋白丝的固有极性，一个 cofilin 结合簇引入了两种不同类型的边界，一种在“尖端”侧，另一种在簇的“棘突”侧；在簇的尖端侧（“快速切割”边界）比棘突侧（“缓慢切割”边界）更容易发生切割。最近的电子低温显微镜（cryo-EM）缓慢切割边界模型揭示了界面处的结构“缺陷”，这些缺陷可能有助于切割。然而，快速切割边界的结构仍然不确定。在这里，我们使用广泛的分子动力学模拟来产生两种切割边界的原子分辨率模型。我们的缓慢切割边界的平衡模拟模型与 cryo-EM 结构模型一致。模拟表明，两个边界的肌动蛋白亚基都采用了介于无遮蔽和 cofilin 结合的肌动蛋白亚基之间的结构。这些“中间”状态破坏了亚基间的接触，但在缓慢切割边界，cofilin 桥接相互作用稳定了这些状态，从而降低了其碎片化的概率。从 cofilactin 丝中去除 cofilin 蛋白的模拟实验支持这样一种机制，即需要两个连续结合的 cofilin 蛋白簇来完全稳定 cofilactin 构象，促进协同结合相互作用，并加速丝的切割。这些研究共同为开发基于粗粒化和理论的 cofilin 介导的肌动蛋白丝切割描述提供了分子尺度的基础。
