Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; College of Medicine, Mohammed Bin Rashid University of Medicine and Health Sciences, Dubai, United Arab Emirates.
Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.
Biophys J. 2019 Feb 5;116(3):540-550. doi: 10.1016/j.bpj.2018.12.009. Epub 2018 Dec 18.
The Rickettsia ∼1800-amino-acid autotransporter protein surface cell antigen 2 (Sca2) promotes actin polymerization on the surface of the bacterium to drive its movement using an actin comet-tail mechanism. Sca2 mimics eukaryotic formins in that it promotes both actin filament nucleation and elongation and competes with capping protein to generate filaments that are long and unbranched. However, despite these functional similarities, Sca2 is structurally unrelated to eukaryotic formins and achieves these functions through an entirely different mechanism. Thus, while formins are dimeric, Sca2 functions as a monomer. However, Sca2 displays intramolecular interactions and functional cooperativity between its N- and C-terminal domains that are crucial for actin nucleation and elongation. Here, we map the interaction of N- and C- terminal fragments of Sca2 and their contribution to actin binding and nucleation. We find that both the N- and C-terminal regions of Sca2 interact with actin monomers but only weakly, whereas the full-length protein binds two actin monomers with high affinity. Moreover, deletions at both ends of the N- and C-terminal regions disrupt their ability to interact with each other, suggesting that they form a contiguous ring-like structure that wraps around two actin subunits, analogous to the formin homology-2 domain. The discovery of Sca2 as an actin nucleator followed the identification of what appeared to be a repeat of three Wiskott-Aldrich syndrome homology 2 (WH2) domains in the middle of the molecule, consistent with the presence of WH2 domains in most actin nucleators. However, we show here that contrary to previous assumptions, Sca2 does not contain WH2 domains. Instead, our analysis indicates that the region containing the putative WH2 domains is folded as a globular domain that cooperates with other parts of the Sca2 molecule for actin binding and nucleation.
立克次氏体~1800 个氨基酸的自动转运蛋白表面细胞抗原 2（Sca2）通过肌动蛋白彗星尾机制促进细菌表面的肌动蛋白聚合，从而推动其运动。Sca2 模拟真核细胞的formin，既能促进肌动蛋白丝的成核和延伸，又能与盖帽蛋白竞争生成长而无分支的纤维。然而，尽管具有这些功能相似性，但 Sca2 在结构上与真核formin 无关，而是通过完全不同的机制实现这些功能。因此，虽然formin 是二聚体，但 Sca2 作为单体发挥作用。然而，Sca2 显示其 N-和 C-末端结构域之间存在分子内相互作用和功能协同作用，这对于肌动蛋白的成核和延伸至关重要。在这里，我们绘制了 Sca2 的 N-和 C-末端片段的相互作用及其对肌动蛋白结合和成核的贡献图谱。我们发现，Sca2 的 N-和 C-末端区域都与肌动蛋白单体相互作用，但作用较弱，而全长蛋白则以高亲和力结合两个肌动蛋白单体。此外，N-和 C-末端区域的两端缺失会破坏它们相互作用的能力，表明它们形成一个连续的环形结构，类似于formin 同源结构域 2。在鉴定出该分子中部似乎存在三个威特科斯基-奥尔德里奇综合征同源 2（WH2）结构域的重复序列后，发现了 Sca2 作为肌动蛋白成核因子的存在，这与大多数肌动蛋白成核因子中存在 WH2 结构域一致。然而，我们在这里表明，与之前的假设相反，Sca2 不含有 WH2 结构域。相反，我们的分析表明，包含假定的 WH2 结构域的区域被折叠成一个球形结构域，与 Sca2 分子的其他部分合作，用于肌动蛋白结合和成核。
