Monomer-Monomer Interactions Propagate Structural Transitions Necessary for Pore Formation by the Cholesterol-dependent Cytolysins

Hotze, Eileen M.; Wilson-Kubalek, Elizabeth M.; Farrand, Allison J.; Bentsen, Lori; Parker, Michael W.; Johnson, Arthur E.; Tweten, Rodney K.

doi:10.1074/jbc.m112.380139

Cited by 53 publications

(92 citation statements)

References 28 publications

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“…S5D). Crucially and consistent with this idea, mutagenesis of the equivalent β4-α6 loop structure in CDCs (the β5-region) showed that mobility in this region is essential for initial stable dimer formation as well as subsequent oligomerization events (30,32). Similarly, EM data and mutagenesis data suggest that the equivalent region is also important for assembly of the fungal MACPF protein pleurotolysin (23).…”

Section: Structural Analysis Of the Sntx Dimer In The Context Of A Fusupporting

confidence: 52%

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

Ellisdon

Reboul

Panjikar

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Section: Structural Analysis Of the Sntx Dimer In The Context Of A Fusupporting

confidence: 52%

“…Extensive published experimental data reveal that a key step in prepore assembly is formation of a stable membrane-bound dimer (30). We suggest that the structure of the SNTX heterodimer represents a soluble and stable snapshot of this event.…”

Section: Phylogenetic Analysis Reveals That Sntx Represents An Ancienmentioning

confidence: 99%

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

Ellisdon

Reboul

Panjikar

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…To determine the relative lytic activities of wildtype PFO and its W165T mutant, a hemolytic assay and fluorometric measurements of carboxyfluorescein release from liposomes were performed. In agreement with previous studies (29), PFO evoked hemolysis of sheep erythrocytes in a dose-dependent manner at 37°C (data not shown). In contrast, the W165T point mutation led to loss of the hemolytic activity even at 100 nM PFO (data not shown).…”

Section: Characterization Of the Binding Of Pfo And Pfosupporting

confidence: 81%

“…Further analysis of the W165T variant indicated that this mutation does not block D4 binding to lipid membrane but disturbs the formation of the ring-shaped oligomeric PFO complex by preventing ␤4 from pairing with ␤1 of another membrane-bound PFO monomer. Instead of ringshaped oligomers, linear oligomers were found in the W165T protein (29). The solution structure of PFO shows that D1 is in direct contact with D2 and D3 domains, which undergo major structural changes during incorporation into the membranes and formation of pores.…”

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confidence: 99%

“…Interestingly, an interference in the D1 domain by substitution of Trp-165 with non-aromatic amino acids resulted in loss of lytic activity of PFO (29). Further analysis of the W165T variant indicated that this mutation does not block D4 binding to lipid membrane but disturbs the formation of the ring-shaped oligomeric PFO complex by preventing ␤4 from pairing with ␤1 of another membrane-bound PFO monomer.…”

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Crucial Role of Perfringolysin O D1 Domain in Orchestrating Structural Transitions Leading to Membrane-perforating Pores

Kacprzyk-Stokowiec

Kulma

Traczyk

et al. 2014

Journal of Biological Chemistry

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Background: Pore formation by perfringolysin O consists of well defined stages, but the mechanism is not fully understood. Results: We mapped regions in toxin that undergo stabilization and destabilization upon oligomerization. Conclusion: Properly folded D1 orchestrates structural transitions in other domains necessary for pore formation. Significance: Study of structural changes in cytolysin domains upon binding to membrane is crucial for understanding pore formation mechanism.

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Cholesterol‐dependent cytolysins: The outstanding questions

et al. 2022

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The cholesterol-dependent cytolysins (CDCs) are a major family of bacterial pore-forming proteins secreted as virulence factors by Gram-positive bacterial species. CDCs are produced as soluble, monomeric proteins that bind specifically to cholesterol-rich membranes, where they oligomerize into ring-shaped pores of more than 30 monomers. Understanding the details of the steps the toxin undergoes in converting from monomer to a membrane-spanning pore is a continuing challenge. In this review we summarize what we know about CDCs and highlight the remaining outstanding questions that require answers to obtain a complete picture of how these toxins kill cells. K E Y W O R D S CD59, cholesterol-binding protein, cholesterol-dependent cytolysin, intermedilysin, MACPF, membrane-protein interactions, perfringolysin O, pneumolysin, pore-forming toxin 1 | INTRODUCTION Pore-forming toxins (PFTs), bacterial toxins that act via membrane permeabilization, are typically produced to aid bacterial virulence or survival. Cholesterol-dependent cytolysins (CDCs) are a large family of PFTs observed in numerous Gram-positive bacterial genera including Streptococcus, Clostridium, Listeria, and Gardnerella.

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Monomer-Monomer Interactions Propagate Structural Transitions Necessary for Pore Formation by the Cholesterol-dependent Cytolysins

Cited by 53 publications

References 28 publications

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

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

Crucial Role of Perfringolysin O D1 Domain in Orchestrating Structural Transitions Leading to Membrane-perforating Pores

Cholesterol‐dependent cytolysins: The outstanding questions

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