Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States; Biochemistry and Molecular Biophysics Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States.
Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States.
J Struct Biol. 2023 Jun;215(2):107960. doi: 10.1016/j.jsb.2023.107960. Epub 2023 Apr 5.
Spotted fever group Rickettsia undergo actin-based motility inside infected eukaryotic cells using Sca2 (surface cell antigen 2): an ∼ 1800 amino-acid monomeric autotransporter protein that is surface-attached to the bacterium and responsible for the assembly of long unbranched actin tails. Sca2 is the only known functional mimic of eukaryotic formins, yet it shares no sequence similarities to the latter. Using structural and biochemical approaches we have previously shown that Sca2 uses a novel actin assembly mechanism. The first ∼ 400 amino acids fold into helix-loop-helix repeats that form a crescent shape reminiscent of a formin FH2 monomer. Additionally, the N- and C- terminal halves of Sca2 display intramolecular interaction in an end-to-end manner and cooperate for actin assembly, mimicking a formin FH2 dimer. Towards a better structural understanding of this mechanism, we performed single-particle cryo-electron microscopy analysis of Sca2. While high-resolution structural details remain elusive, our model confirms the presence of a formin-like core: Sca2 indeed forms a doughnut shape, similar in diameter to a formin FH2 dimer and can accommodate two actin subunits. Extra electron density, thought to be contributed by the C-terminal repeat domain (CRD), covering one side is also observed. This structural analysis allows us to propose an updated model where nucleation proceeds by encircling two actin subunits, and elongation proceeds either by a formin-like mechanism that necessitates conformational changes in the observed Sca2 model, or via an insertional mechanism akin to that observed in the ParMRC system.
斑点热群立克次体在感染的真核细胞内利用 Sca2（表面细胞抗原 2）进行基于肌动蛋白的运动：一种约 1800 个氨基酸的单体自转运蛋白，它附着在细菌表面，负责组装长而无分支的肌动蛋白尾巴。Sca2 是唯一已知的真核formin 功能模拟物，但与后者没有序列相似性。我们之前使用结构和生化方法表明，Sca2 使用一种新的肌动蛋白组装机制。最初的约 400 个氨基酸折叠成螺旋-环-螺旋重复序列，形成一个新月形，使人联想到formin FH2 单体。此外，Sca2 的 N 端和 C 端两半以端到端的方式显示分子内相互作用，并合作进行肌动蛋白组装，模拟 formin FH2 二聚体。为了更好地理解这种机制的结构，我们对 Sca2 进行了单颗粒冷冻电子显微镜分析。虽然高分辨率的结构细节仍然难以捉摸，但我们的模型证实了存在类似formin 的核心：Sca2 确实形成了一个甜甜圈形状，直径与 formin FH2 二聚体相似，可以容纳两个肌动蛋白亚基。还观察到覆盖一侧的额外电子密度，认为这是由 C 端重复结构域（CRD）贡献的。这种结构分析使我们能够提出一个更新的模型，其中成核通过围绕两个肌动蛋白亚基进行，伸长通过观察到的 Sca2 模型所需的构象变化进行，或者通过类似于 ParMRC 系统观察到的插入机制进行。
