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Zusammenfassung:
Actin is a highly conserved protein involved in essential cellular processes. As such, it is the target of numerous bacterial protein toxins. ExoY is a virulence factor of the human pathogen Pseudomonas aeruginosa. Inside bacteria, the toxin is inactive, but once it enters target cells, it interacts with filamentous actin (F-actin) and becomes a potent nucleotidyl cyclase (Belyy et al., 2016). Using single-particle cryo-EM, we show that in comparison to the apostate, two flexible regions of ExoY become ordered and interact with F-actin. Our MD simulations and biochemical assays demonstrate that the specific stabilization of these regions leads to allosteric stabilization of the nucleotide-binding pocket and thus activation of the enzyme (Belyy et al., 2021). Our findings pave the road for developing of novel antidotes against toxins of microbial origin. TccC3 is an effector from the insect pathogen Photorhabdus luminescens. Once TccC3 is translocated into the target cell, the enzyme ADP-ribosylates F-actin, resulting in clustering of the cytoskeleton and ultimately cell death (Lang et al., 2010). By combining NMR spectroscopy and cryo-EM we show in atomic detail how TccC3 modifies actin. Binding of TccC3 to its substrate 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 F-actin. This site-specific modification of F-actin prevents its interaction with depolymerization factors, which impairs actin network turnover and leads to steady actin polymerization (Belyy et al., accepted). Our findings reveal a new mechanism of action of a bacterial toxin through specific modification of F-actin.
