Stonefish toxin defines an ancient branch of the perforin-like superfamily

Abstract

The lethal factor in stonefish venom is stonustoxin (SNTX), a heterodimeric cytolytic protein that induces cardiovascular collapse in humans and native predators. Here, using X-ray crystallography, we make the unexpected finding that SNTX is a pore-forming member of an ancient branch of the Membrane Attack ComplexPerforin/Cholesterol-Dependent Cytolysin (MACPF/CDC) superfamily. SNTX comprises two homologous subunits (α and β), each of which comprises an N-terminal pore-forming MACPF/CDC domain, a central focal adhesion-targeting domain, a thioredoxin domain, and a C-terminal tripartite motif family-like PRY SPla and the RYanodine Receptor immune recognition domain. Crucially, the structure reveals that the two MACPF domains are in complex with one another and arranged into a stable early prepore-like assembly. These data provide long sought after near-atomic resolution insights into how MACPF/CDC proteins assemble into prepores on the surface of membranes. Furthermore, our analyses reveal that SNTX-like MACPF/CDCs are distributed throughout eukaryotic life and play a broader, possibly immune-related function outside venom.H uman envenoming by the tropical stonefish (Synanceia horrida and related species) results in extreme pain, edema, hypotension, respiratory distress, and on rare occasions, death (1). The lethal factor in stonefish venom is an ∼150-kDa protein termed stonustoxin (SNTX), an unusual example of a vertebrate cytolytic protein complex (2). SNTX is a soluble heterodimeric assembly of two closely related proteins termed SNTX-α and -β that share sequence identity of ∼50% (3). With the exception of a C-terminal PRY SPla and the RYanodine Receptor (PRYSPRY) domain in each protein (4), SNTX shares no obvious sequence similarity to any structurally or functionally characterized molecule. SNTX induces species-specific hemolytic activity (2) by an apparent pore-forming mechanism (5). It induces platelet aggregation (6), and like the closely related Trachynilysin (from Synanceia trachynis), SNTX exhibits activity suggesting that it may function as a neurotoxin (7,8).Because eukaryote pore-forming toxins are relatively rare, we reasoned that SNTX might represent a new exemplar of a vertebrate pore-forming protein. Previous studies had shown that it was possible to purify and crystallize SNTX (9); however, no structure has been reported to date. Accordingly, to address the structural basis for SNTX activity, we determined its X-ray crystal structure.

Nemertean and phoronid genomes reveal lophotrochozoan evolution and the origin of bilaterian heads

“…Among these toxin genes, we also found several genes that have high sequence similarities to the stonefish toxin, stonustoxin. Stonustoxin is a pore-forming protein of the membrane attack complex-perforin/cholesterol-dependent cytolysin superfamily, which is widely distributed among eukaryotes 52 . Wide distribution of this gene in non-toxic taxa suggests that it may play a broader role than envenomation.…”

Exaptation of two ancient immune proteins into a new dimeric pore-forming toxin in snails

“…In our work, SLS, SAXS and NS-EM consistently indicate that native PV2 is a dimer of heterodimers. As far as we know, there is only a single report of another MACPF secreted as a structurally-stable water-soluble dimer (Ellisdon et al, 2015) and not as monomers as the vast majority of MACPF. Interestingly, this dimeric MACPF is also a cytotoxin, the fish stonustoxin (SNTX).…”

“…Interestingly, this dimeric MACPF is also a cytotoxin, the fish stonustoxin (SNTX). However, unlike PV2, SNTX do not have a lectin subunit, or even a carbohydrate-binding domain (Ellisdon et al, 2015). The reason for this dimeric arrangement is still unknown.…”

“…These PFTs are present in bacteria, protists and fungi, but are very rare in animals. In fact, MACPF PFTs were reported only in a vertebrate (stonefish) and two groups of invertebrates: Cnidaria (Anderluh et al, 2014; Ellisdon et al, 2015) and the apple snail Pomacea canaliculata (Dreon et al, 2013).…”

A review on the Scorpaena plumieri fish venom and its bioactive compounds

“…Furthermore, previous reports have shown that the hemolytic effect induced by SNTX was fully prevented by osmotic protectants of adequate size while uncharged molecules of smaller size failed to avert cell lysis [57]. More recently, the pore formation mechanism was directly visualized through transmission electron microscopy of SNTX [58]. …”