Stonefish toxin defines an ancient branch of the perforin-like superfamily Andrew M. Ellisdona,b, Cyril F. Reboula,b, Santosh Panjikara,c, Kitmun Huynha, Christine A. Oelliga, Kelly L. Wintera,d, Michelle A. Dunstonea,b,e, Wayne C. Hodgsond, Jamie Seymourf, Peter K. Deardeng, Rodney K. Twetenh, James C. Whisstocka,b,1,2, and Sheena McGowane,1,2 aBiomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC, 3800, Australia; bAustralian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash University, Melbourne, VIC, 3800, Australia; cAustralian Synchrotron, Macromolecular Crystallography, Melbourne, VIC, 3168, Australia; dBiomedicine Discovery Institute and Department of Pharmacology, Monash University, Melbourne, VIC, 3800, Australia; eBiomedicine Discovery Institute and Department of Microbiology, Monash University, Melbourne, VIC, 3800, Australia; fCentre for Biodiscovery and Molecular Development of Therapeutics, Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, QLD, 4870, Australia; gDepartment of Biochemistry and Genetics Otago, University of Otago, Dunedin, 9054 Aotearoa–New Zealand; and hDepartment of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104 Edited by Brenda A. Schulman, St. Jude Children’s Research Hospital, Memphis, TN, and approved November 3, 2015 (received for review April 19, 2015) The lethal factor in stonefish venom is stonustoxin (SNTX), a parallel interface along their entire 115-Å length (2,908 Å2 heterodimeric cytolytic protein that induces cardiovascular collapse buried surface area) (Fig. 1 A–D and Fig. S1). Fold recognition in humans and native predators. Here, using X-ray crystallography, searches reveal that each SNTX protein comprises four do- we make the unexpected finding that SNTX is a pore-forming mains (Fig. 1 B and C) (10). Despite a lack of obvious sequence member of an ancient branch of the Membrane Attack Complex- similarity, the N-terminal domain (residues 1–265) (shown in Perforin/Cholesterol-Dependent Cytolysin (MACPF/CDC) superfamily. greeninFig.1B) is homologous to the Membrane Attack SNTX comprises two homologous subunits (α and β), each of which Complex-Perforin/Cholesterol-Dependent Cytolysin (MACPF/CDC) comprises an N-terminal pore-forming MACPF/CDC domain, a cen- pore-forming domain (Fig. S2). The N-terminal MACPF/CDC tral focal adhesion-targeting domain, a thioredoxin domain, and a domain leads into a focal adhesion-targeting (FAT) domain C-terminal tripartite motif family-like PRY SPla and the RYanodine (266–385) (shown in dark blue in Fig. 1B), with highest structural Receptor immune recognition domain. Crucially, the structure reveals similarity to the human focal adhesion kinase 1 FAT domain that the two MACPF domains are in complex with one another and (rmsd of 2.7 Å over 98 aligned residues) (11). FAT domains are arranged into a stable early prepore-like assembly. These data pro- found in a wide range of proteins and typically perform a scaf- vide long sought after near-atomic resolution insights into how folding role (for example, in the assembly of signaling com- MACPF/CDC proteins assemble into prepores on the surface of mem- in cis branes. Furthermore, our analyses reveal that SNTX-like MACPF/CDCs plexes) (12). In SNTX, the FAT domain makes numerous are distributed throughout eukaryotic life and play a broader, possi- contacts with the MACPF/CDC domain and the thioredoxin – B bly immune-related function outside venom. (THX) domain (386 517) (shown in gray in Fig. 1 and Fig. S2)aswellasextensivein trans interactions at the SNTX-α/β pore | stonefish | toxin | perforin | cytolysin interface (Fig. S1). The THX domain comprises a five-stranded β-sheet and Saccharomyces cerevisiae uman envenoming by the tropical stonefish (Synanceia shares greatest structural similarity with Hhorrida and related species) results in extreme pain, edema, mitochondrial THX3 (rmsd of 2.1 Å over 72 aligned residues) hypotension, respiratory distress, and on rare occasions, death (13). THX domains are typically involved in redox regulation; (1). The lethal factor in stonefish venom is an ∼150-kDa protein termed stonustoxin (SNTX), an unusual example of a vertebrate Significance cytolytic protein complex (2). SNTX is a soluble heterodimeric assembly of two closely related proteins termed SNTX-α and -β Here, we present the structure of the pore-forming toxin sto- that share sequence identity of ∼50% (3). With the exception nustoxin (SNTX), the lethal factor present in stonefish venom. of a C-terminal PRY SPla and the RYanodine Receptor Our work shows that SNTX comprises two homologous sub- (PRYSPRY) domain in each protein (4), SNTX shares no ob- units (α and β), each of which belongs to the perforin super- vious sequence similarity to any structurally or functionally family of pore-forming immune effectors. In SNTX, the α- and characterized molecule. SNTX induces species-specific hemo- β-Membrane Attack Complex-Perforin/Cholesterol-Dependent lytic activity (2) by an apparent pore-forming mechanism (5). It Cytolysin (MACPF/CDC) domains interact and form a prepore- induces platelet aggregation (6), and like the closely related like complex. These data provide, to our knowledge, the first Trachynilysin (from Synanceia trachynis), SNTX exhibits activity high-resolution insights into how MACPF/CDCs interact with suggesting that it may function as a neurotoxin (7, 8). one another during pore formation. Because eukaryote pore-forming toxins are relatively rare, we reasoned that SNTX might represent a new exemplar of a verte- Author contributions: J.C.W. and S.M. designed research; A.M.E., C.F.R., S.P., K.H., C.A.O., K.L.W., J.S., P.K.D., R.K.T., and S.M. performed research; K.L.W., W.C.H., J.S., and S.M. brate pore-forming protein. Previous studies had shown that it was contributed new reagents/analytic tools; A.M.E., C.F.R., M.A.D., P.K.D., R.K.T., J.C.W., possible to purify and crystallize SNTX (9); however, no structure and S.M. analyzed data; and A.M.E., J.C.W., and S.M. wrote the paper. has been reported to date. Accordingly, to address the structural The authors declare no conflict of interest. basis for SNTX activity, we determined its X-ray crystal structure. This article is a PNAS Direct Submission. Data deposition: Crystallography, atomic coordinates, and structure factors have been Results and Discussion deposited in the Protein Data Bank, www.pdb.org (PDB ID code 4WVM). SNTX Is a Heterodimer of Two Distinct Membrane Attack Complex- 1J.C.W. and S.M. contributed equally to this work. Perforin/Cholesterol-Dependent Cytolysin-Like Proteins. We purified 2To whom correspondence may be addressed. Email: [email protected] or SNTX from crude venom and determined its crystal structure [email protected]. to 3.1 Å using anomalous scattering methods (Tables S1 and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. S2). SNTX-α and -β form an obligate dimer with an extensive 1073/pnas.1507622112/-/DCSupplemental. 15360–15365 | PNAS | December 15, 2015 | vol. 112 | no. 50 www.pnas.org/cgi/doi/10.1073/pnas.1507622112 Downloaded by guest on September 23, 2021 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 Å2 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.) however, the SNTX THX domain lacks the canonical catalytic Finally, the PRYSPRY domains (518–703) (shown in blue in residues (Fig. S2). We, therefore, suggest that, in SNTX, the Fig. 1B) of each subunit interact in the heterodimer and are THX domain may play a purely structural role. located distal to the MACPF/CDC domain. The PRYSPRY 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-andβ-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 fromclosely 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 BIOCHEMISTRY proteins are found in fungi implies that this molecule predates animals and was, therefore, present in the ancestors of fungi and animals. Ellisdon et al. PNAS | December 15, 2015 | vol. 112 | no. 50 | 15361 Downloaded by guest on September 23, 2021 domains share greatest structural similarity to the tripartite motif would position the SNTX MACPF/CDC domains appropriately family (TRIM) 21 PRYSPRY domain (rmsd of 1.55 Å over 155 for pore assembly.