Structural insights into the interaction and multi-step activation of bacterial ExoY nucleotidyl cyclases toxins by actin-profilin

ExoY virulence factors belong to a family of invasive bacterial nucleotidyl cyclase (NC) toxins that are activated by specific eukaryotic cofactors and overproduce purine and pyrimidine cyclic nucleotides inside eukaryotic host cells. The molecular and mechanistic details underlying their activation and catalytic specificities are only partially understood. ExoY NC toxins use monomeric (G-actin) or polymerized (F-actin) actin to become potent NCs. Here we use the ExoY effector domain (Vn-ExoY) of the Vibrio nigripulchritudo Multifunctional-Autoprocessing Repeats-in-ToXin (MARTX) toxin to study ExoY-like toxins that selectively interact with G-actin. Vn-ExoY interacts with only modest affinity with G-actin but can be potently activated by the G-actin:profilin complex, which is abundant in eukaryotic cells. This interaction inhibits the assembly of actin:profilin onto actin filament barbed-ends in vitro, whether spontaneous or mediated by formin or VASP elongating factors. High-resolution crystal structures of nucleotide-free, 3prime-deoxy-ATP- or 3prime-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 NC toxins. Substrate binding promotes a large closure of their NBP. This constrains catalytically important residues 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. The data conclusively demonstrate that NC toxins employ a similar two-metal-ion mechanism for catalysing the cyclisation reaction of nucleotides of different sizes. ### Competing Interest Statement The authors have declared no competing interest.