The stonefish (Synanceja horrida) is found in the coastal waters of Northern Australia and is amongst the most dangerous of venomous fish, with envenomation leading to haemolysis, oedema, vascular permeability, platelet aggregation and death. Many of the lethal symptoms associated with envenomation by S. horrida have been attributed to stonustoxin, a proteinaceous venom comprised of two 79 kDa subunits, termed α and β, that share 50 % sequence homology. Stonustoxin belongs to a larger group of PRYSPRY domain-containing proteins that play central toxic roles in a number of venomous fish, reptiles, and monotremes. However, despite identification over 50 years ago the structural basis of stonustoxin toxicity has remained unclear.

Here we report the 3.1 Å X-ray crystal structure of stonustoxin. These data reveal a dimer of two homologous proteins, sharing 49 % sequence identity that superpose with an r.m.s deviation of 1.15 Å. Unexpectedly, stonustoxin includes an N-terminal membrane attack complex / perforin-like (MACPF) domain; a region that shares structural similarity to the archetypal MACPF protein perforin, which forms pores that deliver pro-apoptopic granzymes into the cytosol during the immune response. The remainder of the protein comprises a focal adhesion targeting-like domain, a thioredoxin-like domain, and, as previously predicted, a C-terminal PRYSPRY domain. The presence of the MACPF domain was unexpected, but immediately suggests that stonustoxin toxicity and haemolysis is caused by the formation of large β-barrel pores in cell membranes. This is the first high-resolution structure of a MACPF dimer interface in a pore-like conformation, and provides key novel insights into the mechanism of pore formation by the MACPF superfamily.