title: 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.
authors.affiliation: Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC, 3800, Australia; Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash University, Melbourne, VIC, 3800, Australia;
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Ellisdon
authors.affiliation: Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC, 3800, Australia; Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash University, Melbourne, VIC, 3800, Australia;
authors.forename: Cyril F
authors.initials: CF
authors.lastname: Reboul
authors.affiliation: Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC, 3800, Australia; Australian Synchrotron, Macromolecular Crystallography, Melbourne, VIC, 3168, Australia;
authors.forename: Santosh
authors.identifier: http://orcid.org/0000-0001-7429-3879
authors.initials: S
authors.lastname: Panjikar
authors.affiliation: Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC, 3800, Australia;
authors.forename: Kitmun
authors.initials: K
authors.lastname: Huynh
authors.affiliation: Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC, 3800, Australia;
authors.forename: Christine A
authors.initials: CA
authors.lastname: Oellig
authors.affiliation: Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC, 3800, Australia; Biomedicine Discovery Institute and Department of Pharmacology, Monash University, Melbourne, VIC, 3800, Australia;
authors.forename: Kelly L
authors.initials: KL
authors.lastname: Winter
authors.affiliation: Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC, 3800, Australia; Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash University, Melbourne, VIC, 3800, Australia; Biomedicine Discovery Institute and Department of Microbiology, Monash University, Melbourne, VIC, 3800, Australia;
authors.forename: Michelle A
authors.identifier: http://orcid.org/0000-0002-6026-648X
authors.initials: MA
authors.lastname: Dunstone
authors.affiliation: Biomedicine Discovery Institute and Department of Pharmacology, Monash University, Melbourne, VIC, 3800, Australia;
authors.forename: Wayne C
authors.initials: WC
authors.lastname: Hodgson
authors.affiliation: Centre for Biodiscovery and Molecular Development of Therapeutics, Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, QLD, 4870, Australia;
authors.forename: Jamie
authors.initials: J
authors.lastname: Seymour
authors.affiliation: Department of Biochemistry and Genetics Otago, University of Otago, Dunedin, 9054 Aotearoa-New Zealand;
authors.forename: Peter K
authors.initials: PK
authors.lastname: Dearden
authors.affiliation: Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104.
authors.forename: Rodney K
authors.initials: RK
authors.lastname: Tweten
authors.affiliation: Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC, 3800, Australia; Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash University, Melbourne, VIC, 3800, Australia; James.Whisstock@monash.edu sheena.mcgowan@monash.edu.
authors.forename: James C
authors.initials: JC
authors.lastname: Whisstock
authors.affiliation: Biomedicine Discovery Institute and Department of Microbiology, Monash University, Melbourne, VIC, 3800, Australia; James.Whisstock@monash.edu sheena.mcgowan@monash.edu.
authors.forename: Sheena
authors.initials: S
authors.lastname: McGowan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000737:chemistry; D015938:Complement Membrane Attack Complex / Q000737:chemistry; D018360:Crystallography, X-Ray; D005397:Fish Venoms / Q000737:chemistry*; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D008969:Molecular Sequence Data; D054353:Perforin / Q000737:chemistry*; D010802:Phylogeny; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry; D012995:Solubility; D040681:Structural Homology, Protein
chemical_list: D015938:Complement Membrane Attack Complex; D005397:Fish Venoms; D021122:Protein Subunits; D054353:Perforin; C073228:stonustoxin; D002784:Cholesterol
keywords: cytolysin; perforin; pore; stonefish; toxin