Interaction of the Eukaryotic Pore-forming Cytolysin Equinatoxin II with Model Membranes: 19F NMR Studies

Anderluh, Gregor; Razpotnik, Andrej; Podlesek, Zdravko; Maček, Peter; Separovic, Frances; Norton, Raymond S.

doi:10.1016/j.jmb.2004.12.058

Cited by 88 publications

(70 citation statements)

References 59 publications

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“…In agreement with our model, in sticholysin II a mutation of the tyrosine equivalent to Tyr 113 (mutant Y111N) largely abolishes hemolytic activity (67). Furthermore, by introducing a 19 F label on EqtII tryptophans, it was shown that Trp 112 is important for SM recognition, as specific NMR chemical shifts were observed upon the addition of SM to PC micelles (31). The preferential engagement of SM by the toxin was inferred also from solid-state NMR data (68).…”

Section: Discussionsupporting

confidence: 78%

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Molecular Determinants of Sphingomyelin Specificity of a Eukaryotic Pore-forming Toxin

Bakrač

Gutiérrez-Aguirre

Podlesek

et al. 2008

Journal of Biological Chemistry

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166

169

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Sphingomyelin (SM) is abundant in the outer leaflet of the cell plasma membrane, with the ability to concentrate in so-called lipid rafts. These specialized cholesterol-rich microdomains not only are associated with many physiological processes but also are exploited as cell entry points by pathogens and protein toxins. SM binding is thus a widespread and important biochemical function, and here we reveal the molecular basis of SM recognition by the membrane-binding eukaryotic cytolysin equinatoxin II (EqtII). The presence of SM in membranes drastically improves the binding and permeabilizing activity of EqtII. Direct binding assays showed that EqtII specifically binds SM, but not other lipids and, curiously, not even phosphatidylcholine, which presents the same phosphorylcholine headgroup. Analysis of the EqtII interfacial binding site predicts that electrostatic interactions do not play an important role in the membrane interaction and that the two most important residues for sphingomyelin recognition are Trp 112 and Tyr 113 exposed on a large loop. Experiments using site-directed mutagenesis, surface plasmon resonance, lipid monolayer, and liposome permeabilization assays clearly showed that the discrimination between sphingomyelin and phosphatidylcholine occurs in the region directly below the phosphorylcholine headgroup. Because the characteristic features of SM chemistry lie in this subinterfacial region, the recognition mechanism may be generic for all SM-specific proteins. Sphingomyelin (SM)8 is an important eukaryotic membrane lipid, located for the most part in the outer leaflet of the plasma membrane in the form of specialized cholesterol-rich microdomains, so-called lipid rafts (1, 2). Many pathogens and toxic proteins employ lipid rafts to invade cells (3, 4), but currently little is known about the molecular details of the recognition mechanism of the lipid components present in the rafts. In the particular case of SM, the specific recognition occurs even though SM exposes the same phosphorylcholine headgroup as the other abundant lipid, phosphatidylcholine. SM-binding proteins are currently exploited as specific markers for cellular SM (5) and are used to identify other proteins involved in sphingolipid metabolism (6).Actinoporins are extremely potent cytolysins produced exclusively by sea anemones (7,8). They may be used to capture prey, in intraspecific aggression, or in preventing adhesion of other organisms (7, 9). The two most studied representatives are EqtII, isolated from the sea anemone Actinia equina, and sticholysin II (StII) from Stichodactyla helianthus. Actinoporins constitute a family of conserved proteins that cause hemolysis of red blood cells by colloid-osmotic lysis and exhibit cytolytic activity against various cell lines (7, 10 -12). Even the most distant members of the family share more than 60% sequence identity and the available threedimensional structures of EqtII and StII are nearly superimposable (13-15). The structure is composed of a tightly folded ␤-sandwich flanked b...

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Section: Discussionsupporting

confidence: 78%

“…Lipid specificity has been poorly addressed in the available structural studies of actinoporins (13-15, 29, 30). Recently, however, it was shown by introducing a 19 F label on EqtII tryptophans that Trp 112 participates in SM recognition, as it exhibits specific NMR chemical shift changes upon the addition of SM to PC micelles (31).…”

mentioning

confidence: 99%

Molecular Determinants of Sphingomyelin Specificity of a Eukaryotic Pore-forming Toxin

Bakrač

Gutiérrez-Aguirre

Podlesek

et al. 2008

Journal of Biological Chemistry

Self Cite

166

169

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“…Several types of cnidarian cytolytic toxins have been isolated and characterized, with the most common and most studied being the actinoporin family of pore-forming toxins (PFTs). 1 This family, highly abundant in sea anemones but found in all cnidarian classes (Hydrozoa, Chyphozoa, and Anthozoa) (1), has been used extensively as a model to study the interactions between proteins and biological or model membranes (7)(8)(9)(10).…”

mentioning

confidence: 99%

Hydralysins, a New Category of β-Pore-forming Toxins in Cnidaria

Sher¹,

Fishman²,

Zhang³

et al. 2005

Journal of Biological Chemistry

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Cnidaria are venomous animals that produce diverse protein and polypeptide toxins, stored and delivered into the prey through the stinging cells, the nematocytes. These include pore-forming cytolytic toxins such as well studied actinoporins. In this work, we have shown that the non-nematocystic paralytic toxins, hydralysins, from the green hydra Chlorohydra viridissima comprise a highly diverse group of ␤-pore-forming proteins, distinct from other cnidarian toxins but similar in activity and structure to bacterial and fungal toxins. Functional characterization of hydralysins reveals that as soluble monomers they are rich in ␤-structure, as revealed by far UV circular dichroism and computational analysis. Hydralysins bind erythrocyte membranes and form discrete pores with an internal diameter of ϳ1.2 nm. The cytolytic effect of hydralysin is cell type-selective, suggesting a specific receptor that is not a phospholipid or carbohydrate. Multiple sequence alignment reveals that hydralysins share a set of conserved sequence motifs with known pore-forming toxins such as aerolysin, ⑀-toxin, ␣-toxin, and LSL and that these sequence motifs are found in and around the poreforming domains of the toxins. The importance of these sequence motifs is revealed by the cloning, expression, and mutagenesis of three hydralysin isoforms that strongly differ in their hemolytic and paralytic activities. The correlation between the paralytic and cytolytic activities of hydralysin suggests that both are a consequence of receptor-mediated pore formation. Hydralysins and their homologues exemplify the wide distribution of ␤-pore formers in biology and provide a useful model for the study of their molecular mode of action.

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“…In order to obtain high incorporation of 5-F-Trp, we have generally used a Trp auxotroph strain that grows only when Trp is present in the medium [35]. As 5-F-Trp proved to be inhibitory for growth, bacteria were first grown in LB medium until they reached mid-exponential phase, then transferred to M9 medium containing 1% Casamino acids with no Trp.…”

Section: Ligand Binding To the Spry Domain-containing Socs Box Proteimentioning

confidence: 99%

Applications of 19F-NMR in Fragment-Based Drug Discovery

et al. 2016

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19 F-NMR has proved to be a valuable tool in fragment-based drug discovery. Its applications include screening libraries of fluorinated fragments, assessing competition among elaborated fragments and identifying the binding poses of promising hits. By observing fluorine in both the ligand and the target protein, useful information can be obtained on not only the binding pose but also the dynamics of ligand-protein interactions. These applications of 19 F-NMR will be illustrated in this review with studies from our fragment-based drug discovery campaigns against protein targets in parasitic and infectious diseases.

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Interaction of the Eukaryotic Pore-forming Cytolysin Equinatoxin II with Model Membranes: 19F NMR Studies

Cited by 88 publications

References 59 publications

Molecular Determinants of Sphingomyelin Specificity of a Eukaryotic Pore-forming Toxin

Molecular Determinants of Sphingomyelin Specificity of a Eukaryotic Pore-forming Toxin

Hydralysins, a New Category of β-Pore-forming Toxins in Cnidaria

Applications of 19F-NMR in Fragment-Based Drug Discovery

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