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 Complex-Perforin/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.

Keywords: cytolysin; perforin; pore; stonefish; toxin.

Fig. 1.

The SNTX structure reveals an MACPF/CDC pore-forming heterodimer. (A) Crystal structure of the SNTX heterodimer with SNTX-α (gray) and SNTX-β (blue) shown in cartoon format. (B) The SNTX-domain layout in cartoon format and schematic representation. (C) Schematic representation of the SNTX domain layout. (D) The interaction interface between each SNTX subunit. The Cα-atoms of the interacting residues are colored as per the interaction type, with salt bridges in red, hydrogen bonds in blue, and buried hydrophobics (>20 Ų buried surface area) in green. Interacting residues were calculated by PISA (57). (E) Transmission EM of SNTX pores that are indicated by arrows. (Scale bar: 50 nm.)

Fig. S1.

The SNTX interaction interface. (A) SNTX in surface representation highlighting the electrostatic surface potential of the interface. SNTX-β is rotated ∼180° relative to SNTX-α to expose the two interacting surfaces. Electrostatic surface potential was calculated with QTMG (63). (B) SNTX displayed in surface representation as per A, highlighting the conservation of the interface within SNTX-like proteins of venomous fish. Surface conservation was calculated using the Consurf server (64). (C) Cartoon representation of the FAT domain interface, with selected interacting residues highlighted as sticks.

Fig. S2.

Structural and sequence similarity of SNTX. (A) Alignment of the SNTX-α MACPF/CDC domain with perfringolysin O [rmsd of 2.7 Å over 150 aligned residues; Protein Data Bank (PDB) ID code 1PFO] (16) (B) Alignment of the SNTX-α MACPF/CDC domain with complement component C8β (rmsd of 2.96 Å over 133 aligned residues; PDB ID code 3OJY) (65). (C) Sequence alignment of the SNTX MACPF motif with selected MACPF proteins. (D) Structural alignment of the SNTX-α THX domain with mitochondrial THX3 from Saccharomyces cerevisiae (rmsd of 2.1 Å over 72 aligned residues; PDB ID code 2OE0) (13). (E) The key cysteines of the THX catalytic motif are not conserved in either SNTX subunit. Secondary structural alignments were carried out in PDBeFold (57).

Fig. 2.

SNTX-like proteins form an extensive and ancient third branch of the MACPF/CDC superfamily. (A) The tropical stonefish (S. horrida) highlighting 13 dorsal spines that deliver venom. (B) Close-up view of the venom glands located on either side of a dorsal spine. (C) Rooted Bayesian phylogram of SNTX-like protein sequences from animals and fungi. SNTX-like proteins are defined as proteins sharing significant sequence identity in the SNTX MACPF/CDC domain. Node labels indicate posterior probabilities. The α- and β-subunit sequences are very similar, and the phylogeny indicates that different subunits have evolved multiple times, probably through gene duplication and perhaps, gene conversion. The SNTX-like proteins from venomous species of fish form a sister clade to those from closely related fish groups. Branch lengths in this region of the tree indicate that SNTX-like proteins used as venoms do not vary hugely from those in nonvenomous fish. As such, use of these proteins as toxins may have more to do with expression levels and location than significant evolutionary change. SNTX-like proteins are found in a variety of vertebrates, including the common ostrich, platypus, Tasmanian devil, and coelacanth. The presence of an SNTX-like protein in a member of the Porifera [Amphemidon (Demosponge)] implies that SNTX-like proteins predate the evolution of metazoa. The fact that similar SNTX MACPF/CDC domain proteins are found in fungi implies that this molecule predates animals and was, therefore, present in the ancestors of fungi and animals.

Fig. S3.

Transmission EM of SNTX pores. (A and B) Negative stain transmission EM of SNTX pores formed on incubation with rat erythrocytes. Black and white arrows indicate SNTX pores. (C and D) Negative controls of rat erythrocytes lysed in water. No pores were observed in negative controls.

Fig. S4.

Schematic model of MACPF/CDC pore formation. The core machinery of the MACPF/CDC domain is comprised of a central four-stranded β-sheet and two bundles of α-helices termed TMH1 and TMH2. On association of MACPF/CDC domains, the TMH regions unravel to form a continuous β-sheet that comprises the β-barrel of the pore.

Fig. 3.

Structural basis of SNTX membrane binding and prepore assembly. (A) The PRYSPRY protein/lipid binding pocket is shown as Cα-atom spheres on SNTX-α (orange) and SNTX-β (blue). Residues in the protein/lipid-binding pocket were located by structural alignment [PDBeFold (57)] with the TRIM21:IgG-Fc complex [Protein Data Bank ID code 2IWG (58)]. SNTX PRYSPRY domain residues aligning with the TRIM21 residues that make significant contacts with IgG-Fc are highlighted. The binding pocket is also represented as surface mesh on a zoomed in orientation of the PRYSPRY dimer. (B) The SNTX MACPF/CDC interface within the heterodimer. Hydrogen bonds between strand-β4 of SNTX-α and strand-β1 of SNTX-β are highlighted as red dashed lines, and aligned residues are indicated by green dashed lines. The β-strands are numbered according to their position in the MACPF/CDC central β-sheet. The conserved G208 residue is shown as a sphere at the Cα-atom position, and the electron density difference (2Fo-Fc) map is contoured at 1σ. (C) The SNTX prepore model calculated by extrapolation of 18° of internal symmetry between SNTX subunits. (D) Close-up view of the MACPF/CDC electrostatic interaction at the α/β-heterodimer interface and the β/α-prepore interface (59, 60).

Fig. S5.

Model of the SNTX prepore. (A) The prepore viewed down the β-barrel lumen, highlighting the 18° of rotational symmetry between SNTX subunits. Extrapolation of this internal symmetry forms an SNTX prepore composed of 10 SNTX heterodimers. The position of the symmetry axis is marked by a red circle. (B) Cut-away view of the prepore, highlighting its dimensions and the horizontal alignment of the SNTX molecules. (C) The SNTX β4-α6 binding site. The β4-α6 loop is highlighted in orange, the conserved G208 (SNTX-α) and G209 (SNTX-β) as red spheres, and the electron density difference (2Fo-Fc) map covering the β4-α6 loop contoured to 1σ. (D) The SNTX β4-α6 binding site at the β/α-prepore interface, with the β4-α6 loop in orange, the conserved G209 (SNTX-β) and G208 (SNTX-α) as red spheres, and the 2Fo-Fc map covering the β4-α6 loop contoured to 1σ.