Cardiovascular Research Institute, Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, United States.
Graduate Group in Biophysics, University of California, San Francisco, San Francisco, United States.
Elife. 2024 Aug 16;13:e81571. doi: 10.7554/eLife.81571.
The M2 proton channel aids in the exit of mature influenza viral particles from the host plasma membrane through its ability to stabilize regions of high negative Gaussian curvature (NGC) that occur at the neck of budding virions. The channels are homo-tetramers that contain a cytoplasm-facing amphipathic helix (AH) that is necessary and sufficient for NGC generation; however, constructs containing the transmembrane spanning helix, which facilitates tetramerization, exhibit enhanced curvature generation. Here, we used all-atom molecular dynamics (MD) simulations to explore the conformational dynamics of M2 channels in lipid bilayers revealing that the AH is dynamic, quickly breaking the fourfold symmetry observed in most structures. Next, we carried out MD simulations with the protein restrained in four- and twofold symmetric conformations to determine the impact on the membrane shape. While each pattern was distinct, all configurations induced pronounced curvature in the outer leaflet, while conversely, the inner leaflets showed minimal curvature and significant lipid tilt around the AHs. The MD-generated profiles at the protein-membrane interface were then extracted and used as boundary conditions in a continuum elastic membrane model to calculate the membrane-bending energy of each conformation embedded in different membrane surfaces characteristic of a budding virus. The calculations show that all three M2 conformations are stabilized in inward-budding, concave spherical caps and destabilized in outward-budding, convex spherical caps, the latter reminiscent of a budding virus. One of the C2-broken symmetry conformations is stabilized by 4 kT in NGC surfaces with the minimum energy conformation occurring at a curvature corresponding to 33 nm radii. In total, our work provides atomistic insight into the curvature sensing capabilities of M2 channels and how enrichment in the nascent viral particle depends on protein shape and membrane geometry.
M2 质子通道通过稳定出芽病毒颈部的高负高斯曲率（NGC）区域，帮助成熟的流感病毒粒子从宿主质膜中逸出。通道是同源四聚体，包含一个面向细胞质的两亲性螺旋（AH），这对于 NGC 的产生是必要且充分的；然而，包含促进四聚体形成的跨膜螺旋的构建体表现出增强的曲率生成。在这里，我们使用全原子分子动力学（MD）模拟来探索 M2 通道在脂质双层中的构象动力学，揭示 AH 是动态的，迅速打破大多数结构中观察到的四元对称。接下来，我们对蛋白质进行了 MD 模拟，以限制在四元和二倍对称构象中，以确定其对膜形状的影响。虽然每种模式都不同，但所有构型都在外层叶中引起明显的曲率，而相反，内层叶显示出最小的曲率和围绕 AH 的显著脂质倾斜。然后从 MD 生成的蛋白质-膜界面轮廓中提取并用作边界条件，用于连续弹性膜模型，以计算嵌入具有出芽病毒特征的不同膜表面的每种构象的膜弯曲能量。计算表明，所有三种 M2 构象都在向内出芽的凹球形帽中稳定，在向外出芽的凸球形帽中不稳定，后者使人联想到出芽病毒。C2 打破对称的构象之一在 NGC 表面稳定 4 kT，最小能量构象发生在曲率对应于 33nm 半径的位置。总的来说，我们的工作提供了对 M2 通道曲率感应能力的原子见解，以及新生病毒粒子的丰度如何取决于蛋白质形状和膜几何形状。
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