Zeng Wenjie W, Komaniecki Garrison, Liu Jiaze, Lin Hening, Mao Yuxin
Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, USA.
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.
bioRxiv. 2025 Sep 4:2025.04.18.649563. doi: 10.1101/2025.04.18.649563.
is an opportunistic bacterial pathogen that causes Legionnaires' disease. To establish an intracellular niche conducive to replication, translocates a diverse array of effector proteins that manipulate various host cellular processes, including the actin cytoskeleton. In a screen for effectors that alter actin dynamics, we identified a effector, Lfat1 (lpg1387), which colocalizes with the actin cytoskeleton in eukaryotic cells. Lfat1 specifically binds F-actin through a novel actin-binding domain (ABD). High-resolution cryo-electron microscopy (cryoEM) analysis revealed that this ABD forms a long α-helix hairpin, with its tip interacting with subdomains I and II of two adjacent actin molecules within the F-actin filament. Interestingly, while individual α-helices of the hairpin fail to bind F-actin, co-expression as separate fusion proteins restores binding activity. Furthermore, we demonstrated that Lfat1 exhibits lysine fatty-acyltransferase (KFAT) activity, targeting host small GTPases. These findings establish a foundation for studying the KFAT family of bacterial toxins and uncover a novel F-actin binding motif, providing an alternative F-actin marker with notable flexibility.
是一种导致军团病的机会性细菌病原体。为了建立有利于复制的细胞内生态位，它会转运多种效应蛋白，这些蛋白会操纵各种宿主细胞过程，包括肌动蛋白细胞骨架。在筛选改变肌动蛋白动力学的效应子时，我们鉴定出一种效应子Lfat1（lpg1387），它在真核细胞中与肌动蛋白细胞骨架共定位。Lfat1通过一个新的肌动蛋白结合结构域（ABD）特异性结合F-肌动蛋白。高分辨率冷冻电子显微镜（cryoEM）分析表明，这个ABD形成一个长的α-螺旋发夹结构，其顶端与F-肌动蛋白丝中两个相邻肌动蛋白分子的亚结构域I和II相互作用。有趣的是，虽然发夹结构的单个α-螺旋不能结合F-肌动蛋白，但作为单独的融合蛋白共表达可恢复结合活性。此外，我们证明Lfat1表现出赖氨酸脂肪酰基转移酶（KFAT）活性，靶向宿主小GTP酶。这些发现为研究细菌毒素的KFAT家族奠定了基础，并揭示了一种新的F-肌动蛋白结合基序，提供了一种具有显著灵活性的替代F-肌动蛋白标记物。
