ERATO Actin Filament Dynamics Project, Japan Science and Technology Corporation, c/o RIKEN Harima Institute at Spring 8, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
J Mol Biol. 2010 Apr 9;397(4):1031-41. doi: 10.1016/j.jmb.2010.02.010. Epub 2010 Feb 12.
AlfA is a recently discovered DNA segregation protein from Bacillus subtilis that is distantly related to actin and the bacterial actin homologues ParM and MreB. Here we show that AlfA mostly forms helical 7/3 filaments, with a repeat of about 180 A, that are arranged in three-dimensional bundles. Other polymorphic structures in the form of two-dimensional rafts or paracrystalline nets were also observed. Here AlfA adopted a 16/7 helical symmetry, with a repeat of about 387 A. Thin polymers consisting of several intertwining filaments also formed. Observed helical symmetries of AlfA filaments differed from those of other members of the actin family: F-actin, ParM, or MreB. Both ATP and guanosine 5'-triphosphate are able to promote rapid AlfA filament formation with almost equal efficiencies. The helical structure is only preserved under physiological salt concentrations and at a pH between 6.4 and 7.4, the physiological range of the cytoplasm of B. subtilis. Polymerization kinetics are extremely rapid and compatible with a cooperative assembly mechanism requiring only two steps: monomer activation followed by elongation, making AlfA one of the most efficient polymerizing motors within the actin family. Phosphate release lags behind polymerization, and time-lapse total internal reflection fluorescence images of AlfA bundles are consistent with treadmilling rather than dynamic microtubule-like instability. High-pressure small angle X-ray scattering experiments reveal that the stability of AlfA filaments is intermediate between the stability of ParM and the stability of F-actin. These results emphasize that actin-like polymerizing machineries have diverged to produce a variety of filament geometries with diverse properties that are tailored for specific biological processes.
AlfA 是一种最近在枯草芽孢杆菌中发现的 DNA 分离蛋白，它与肌动蛋白和细菌肌动蛋白同源物 ParM 和 MreB 有较远的关系。在这里，我们表明 AlfA 主要形成螺旋 7/3 丝，重复约 180A，这些丝排列成三维束。还观察到其他形式的二维筏或准晶网的多态结构。在这里，AlfA 采用了 16/7 的螺旋对称性，重复约 387A。由几个缠绕丝组成的薄聚合物也形成了。观察到的 AlfA 丝的螺旋对称性与肌动蛋白家族的其他成员不同：F-肌动蛋白、ParM 或 MreB。ATP 和鸟苷 5'-三磷酸都能够以几乎相等的效率促进 AlfA 细丝的快速形成。只有在生理盐浓度和 pH 值在 6.4 到 7.4 之间（枯草芽孢杆菌细胞质的生理范围）时，才能保持螺旋结构。聚合动力学非常迅速，与协同组装机制兼容，只需两步：单体激活后伸长，使 AlfA 成为肌动蛋白家族中聚合效率最高的马达之一。磷酸基团的释放滞后于聚合，AlfA 束的时移全内反射荧光图像与踏车行为一致，而不是动态微管样不稳定性。高压小角 X 射线散射实验表明，AlfA 丝的稳定性介于 ParM 和 F-肌动蛋白的稳定性之间。这些结果强调了肌动蛋白样聚合机制已经分化，产生了具有不同特性的各种纤维几何形状，这些特性是为特定的生物过程量身定制的。
