Functional and structural insights into the interaction and multi-step activation of bacterial ExoY nucleotidyl cyclases toxins by profilin-bound G-actin

Teixeira-Nunes, M.; Retailleau, P.; Raoux-Barbot, D.; Comisso, M.; Missinou, A. A.; Velours, C.; Plancqueel, S.; Ladant, D.; Mechold, U.; Renault, L.

doi:10.1101/2023.01.13.523968

bioRxiv · 10.1101/2023.01.13.523968

Functional and structural insights into the interaction and multi-step activation of bacterial ExoY nucleotidyl cyclases toxins by profilin-bound G-actin

Teixeira-Nunes, M.·Retailleau, P.·Raoux-Barbot, D.·Comisso, M.·Missinou, A. A.·Velours, C.·Plancqueel, S.·Ladant, D.·Mechold, U.·Renault, L.

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Abstract

ExoY virulence factors are members of a family of bacterial nucleotidyl cyclases (NCs) that are activated by specific eukaryotic cofactors and overproduce cyclic purine and pyrimidine nucleotides in host cells. ExoYs are actin-activated NC toxins. Here, we investigate the Vibrio nigripulchritudo Multifunctional-Autoprocessing Repeats-in-ToXin (MARTX) ExoY effector domain (Vn-ExoY) as a model for ExoY-type members that interact with monomeric (G-actin) rather than filamentous (F-actin) actin. Vn-ExoY binds with only modest affinity to free or profilin-bound G-actin, but can capture the G-actin:profilin complex for its own activation by preventing the spontaneous or VASP- or formin-mediated assembly of G-actin:profilin at the barbed ends of F-actin in vitro. This may prolong the lifetime of the cofactor-bound state of Vn-ExoY at sites of active actin cytoskeleton remodelling. A series of high-resolution crystal structures of nucleotide-free, 3-deoxy-ATP- or 3-deoxy-CTP-bound Vn-ExoY, activated by free or profilin-bound G-actin-ATP/-ADP show that the cofactor only partially stabilises the nucleotide-binding pocket (NBP) of all NC toxins. Substrate binding promotes a large, previously-unidentified, closure of their NBP. This confines catalytically important residues of the NC toxins and metal cofactors around the substrate and promotes the recruitment of two metal ions to tightly coordinate the triphosphate moiety of purine or pyrimidine nucleotide substrates. Residues that play an important role in both the purinyl and pyrimidinyl cyclase activity of NC toxins are validated in Vn-ExoY and the distantly-related ExoY from Pseudomonas aeruginosa that interact with F-actin. The data conclusively demonstrate that NC toxins employ a similar two-metal-ion mechanism for catalysing the cyclisation reaction of nucleotides of different sizes. These structural insights into the dynamics of the actin-binding interface of actin-activated ExoYs and the multi-step activation of all NC toxins open up new perspectives for identifying ways to specifically inhibit these bacterial NC enzymes. Author SummaryExoY toxins belong to a family of bacterial nucleotidyl cyclases (NCs) that are injected into eukaryotic cells and bind to specific host cofactors to trigger their toxic, potent NC enzymatic activity. They alter host cell signalling by overproducing purine and pyrimidine cyclic nucleotides, which act as canonical and non-canonical intracellular messengers, respectively. The molecular and mechanistic details underlying the activation and catalytic specificities of NC toxins are only partially understood. Here, we investigate ExoY-type members that are unable to interact with actin filaments for their activation. We show in vitro that such ExoYs capture the actin:profilin complex for activation by disrupting its association with the most dynamic ends of actin filaments. We have captured several structural snapshots along the Vn-ExoY activation pathway by G-actin or G-actin-profilin without or with purine or pyrimidine nucleotide analogues. Our structural data reveal unprecedented mechanistic details of how the active site of all NC toxins is sequentially remodelled by cofactor and substrate binding, how they can accommodate nucleotides of different sizes as substrates, and elucidate important features of their catalytic reaction. These structural insights into the multi-step activation of NC toxins provide new perspectives for identifying ways to specifically inhibit this class of NC enzymes.

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Teixeira-Nunes, M., Retailleau, P., Raoux-Barbot, D., Comisso, M., Missinou, A. A., Velours, C., Plancqueel, S., Ladant, D., Mechold, U., Renault, L.. 2023-01-13. Functional and structural insights into the interaction and multi-step activation of bacterial ExoY nucleotidyl cyclases toxins by profilin-bound G-actin. https://doi.org/10.1101/2023.01.13.523968

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