Stonefish toxin defines a new branch of the perforin‐like superfamily

The lethal factor in stonefish venom is Stonustoxin (SNTX), a cytolytic protein that induces hypotension and cardiovascular collapse in mammals. Here, we show that SNTX is a pore forming member of an undiscovered branch of the Cholesterol Dependent Cytolysin / Membrane Attack Complex/Perforin (CDC/MACPF) superfamily. Structural analyses suggest that the initial SNTX interaction with a target membrane is mediated by two C‐terminal PRYSPRY domains, which are closely related to the lipid and protein binding domains of the tripartite motif immune protein family. Two N‐terminal CDC/MACPF domains are arranged in a pre‐pore like assembly; these data provide high‐resolution insight into how SNTX and other CDC/MACPF proteins assemble into pores. Finally, our analyses reveal that SNTX‐like CDCs are distributed throughout eukaryotic life, and likely play a broader, and possibly immune‐related function, outside venom.