Mechanism of threonine ADP-ribosylation of F-actin by a Tc toxin

Abstract

Tc toxins deliver toxic enzymes into host cells by a unique injection mechanism. One of these enzymes is TccC3, an ADP-ribosyltransferase from Photorhabdus luminescens. Once TccC3 is translocated into the target cell, the enzyme ADP-ribosylates actin, resulting in clustering of the actin cytoskeleton and ultimately cell death. Here, we combine biochemistry, solution and solid-state NMR spectroscopy and cryo-EM to show in atomic detail how TccC3 modifies actin. We find that the ADP-ribosyltransferase does not bind to G-actin but interacts with two consecutive actin subunits of F-actin. The binding of TccC3 to F-actin occurs via an induced-fit mechanism that facilitates access of NAD+ to the nucleotide binding pocket. The following nucleophilic substitution reaction results in the transfer of ADP-ribose to threonine-148 of F-actin. We demonstrate that this site-specific modification of F-actin prevents its interaction with depolymerization factors, such as cofilin, which impairs actin network turnover and leads to steady actin polymerization. Our findings reveal in atomic detail a new mechanism of action of a bacterial toxin through specific targeting and modification of F-actin.

Photorhabdus luminescens TccC3 Toxin Targets the Dynamic Population of F-Actin and Impairs Cell Cortex Integrity

“…Since TMSB4-and PFN-bound G-actin also cannot be utilized as substrates for TccC3, this selectivity allows channeling the activity of the enzyme towards a subpopulation of mostly branched lamellipodial F-actin networks and its immediate derivatives-a relatively small actin pool directly involved in essential activities, such as cell migration, endo-and phagocytosis, and membrane and organelle remodeling. When this manuscript was ready for submission, a structural study published as a bioRxiv preprint independently confirmed our finding that F-and not G-actin is a preferable substrate for TccC3 [53]. Using cryo-EM reconstruction as the main approach, the authors found that the TccC3's binding site on F-actin spans two longitudinally adjacent actin subunits and that the NAD + -binding pocket on the enzyme's active site is shaped by this interaction via an induced-fit mechanism.…”