Department of Chemistry and Biochemistry, University of California, UCLA, Los Angeles, CA 90095, USA.
California NanoSystems Institute (CNSI), UCLA, Los Angeles, CA 90095, USA.
Structure. 2020 May 5;28(5):586-593.e3. doi: 10.1016/j.str.2020.04.004. Epub 2020 Apr 28.
Detailed molecular information on G-actin assembly into filaments (F-actin), and their structure, dynamics, and interactions, is essential for understanding their cellular functions. Previous studies indicate that a flexible DNase I binding loop (D-loop, residues 40-50) plays a major role in actin's conformational dynamics. Phalloidin, a "gold standard" for actin filament staining, stabilizes them and affects the D-loop. Using disulfide crosslinking in yeast actin D-loop mutant Q41C/V45C, light-scattering measurements, and cryoelectron microscopy reconstructions, we probed the constraints of D-loop dynamics and its contribution to F-actin formation/stability. Our data support a model of residues 41-45 distances that facilitate G- to F-actin transition. We report also a 3.3-Å resolution structure of phalloidin-bound F-actin in the ADP-Pi-like (ADP-BeFx) state. This shows the phalloidin-binding site on F-actin and how the relative movement between its two protofilaments is restricted by it. Together, our results provide molecular details of F-actin structure and D-loop dynamics.
详细的分子信息表明 G 肌动蛋白组装成纤维（F-肌动蛋白），以及它们的结构、动力学和相互作用，对于理解它们的细胞功能至关重要。先前的研究表明，一个灵活的 DNase I 结合环（D 环，残基 40-50）在肌动蛋白的构象动力学中起着重要作用。鬼笔环肽是肌动蛋白丝染色的“金标准”，它稳定肌动蛋白丝并影响 D 环。我们使用酵母肌动蛋白 D 环突变体 Q41C/V45C 中的二硫键交联、光散射测量和低温电子显微镜重建，探测 D 环动力学的约束及其对 F-肌动蛋白形成/稳定性的贡献。我们的数据支持一个促进 G-肌动蛋白向 F-肌动蛋白转变的残基 41-45 距离模型。我们还报告了鬼笔环肽结合的 F-肌动蛋白在 ADP-Pi 样（ADP-BeFx）状态下的 3.3 Å 分辨率结构。这显示了 F-肌动蛋白上的鬼笔环肽结合位点，以及它的两个原纤维之间的相对运动如何受到限制。总之，我们的结果提供了 F-肌动蛋白结构和 D 环动力学的分子细节。
