Cryo-EM structure revealed a novel F-actin binding motif in a <i>Legionella pneumophila</i> lysine fatty acyltransferase.

Abstract

<i>Legionella pneumophila</i> is an opportunistic bacterial pathogen that causes Legionnaires' disease. To establish an intracellular niche conducive to replication, <i>L. pneumophila</i> 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 <i>Legionella</i> 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 (Cryo-EM) 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.