The Experts below are selected from a list of 14004 Experts worldwide ranked by ideXlab platform
Toshihide Kobayashi - One of the best experts on this subject based on the ideXlab platform.
-
Molecular mechanisms of action of sphingomyelin-specific Pore-Forming Toxin, lysenin
Seminars in Cell and Developmental Biology, 2018Co-Authors: Neval Yilmaz, Akiko Yamaji-hasegawa, Françoise Hullin-matsuda, Toshihide KobayashiAbstract:Lysenin, which is an earthworm Toxin, strongly binds to sphingomyelin (SM). Lysenin oligomerizes on SM-rich domains and can induce cell death by forming pores in the membrane. In this review, the assembly of lysenin on SM-containing membranes is discussed mostly on the basis of the information gained by atomic force microscopy (AFM). AFM data show that lysenin assembles into a hexagonal close packed (hcp) structure by rapid reorganization of its oligomers on an SM/cholesterol membrane. In case of a phase-separated membrane of SM, lysenin induces phase mixing as a result of pore formation in SM-rich domains, and consequently its hcp assembly covers the entire membrane. Besides the lytic action, lysenin is important as an SM marker and its pore has the potential to be used as a biosensor in the future. These points are also highlighted in this review.
-
Assembling of a Pore-Forming Toxin on a Model Membrane
Biophysical Journal, 2014Co-Authors: Neval Yilmaz, Taro Yamada, Peter Greimel, Takayuki Uchihashi, Toshio Ando, Toshihide KobayashiAbstract:The assembling of the sphingomyelin (SM)-binding Pore-Forming Toxin (PFT), lysenin, to SM/cholesterol bilayer was examined by high-speed atomic force microscopy (HS-AFM) [1]. The HS-AFM images of SM/cholesterol bilayer preincubated with lysenin exhibited the hexagonal close packed (hcp) assembly of lysenin oligomers (Fig. 1A). The in-situ AFM images revealed that the formation of the hcp structure took place quickly (Fig. 1B). Before the full coverage of the membrane surface with a stable hcp assembly of lysenin oligomers, most of the oligomers underwent reorganization either by dissociating into monomers or by rapidly diffusing along the membrane in less than a second. The assembling of lysenin oligomers was also followed on SM/DOPC/cholesterol bilayer. Oligomers firstly formed at the edges of the SM-rich domains and covered these domains similarly to the SM/cholesterol bilayer. Our results revealed the dynamic nature of the oligomers of a lipid binding Toxin during its assembling on SM-containing membranes.Reference[1] N. Yilmaz, T. Yamada, P. Greimel, T. Uchihashi, T. Ando, and T. Kobayashi, Biophys. J. 105, 1397-1405 (2013).View Large Image | View Hi-Res Image | Download PowerPoint Slide
-
Real-Time Visualization of a Pore-Forming Toxin Assembling on a Model Membrane
Biophysical Journal, 2013Co-Authors: Neval Yilmaz, Taro Yamada, Takayuki Uchihashi, Toshio Ando, Toshihide KobayashiAbstract:The assembly of the sphingomyelin (SM)-binding Pore-Forming Toxin (PFT), lysenin, on SM/cholesterol bilayer was examined by high-speed atomic force microscopy (HS-AFM). Previous studies suggest that lysenin oligomerizes after binding to SM and forms a honeycomb structure. The HS-AFM images of SM/ cholesterol bilayer preincubated with lysenin exhibited the honeycomb assembly of the lysenin oligomers. The time-lapse AFM images revealed that the honeycomb formation took place quickly. During honeycomb formation most of the oligomers underwent reorganization either by dissociating into monomers or by rapidly diffusing along the membrane in less than a second. In the period of reorganization, the mobile oligomers arranged into a static, well-ordered lattice. Once this static layer was formed, the lysenin molecules were firmly bound to the SM/cholesterol bilayer and the oligomers neither dissociated nor diffused. Our results revealed the dynamic nature of the oligomers of a lipid binding Toxin during honeycomb formation.
-
Lysenin: A sphingomyelin specific Pore-Forming Toxin
Biochimica et biophysica acta, 2007Co-Authors: Hidehiko Shogomori, Toshihide KobayashiAbstract:Sphingomyelin is a major sphingolipid in mammalian cells. Recent results indicate that sphingomyelin is a reservoir of lipid second messengers, ceramide and sphingosine-1-phosphate. Sphingomyelin is also a major component of sphingolipid and cholesterol-rich membrane domains (lipid rafts). Lysenin is a Pore-Forming Toxin that specifically binds sphingomyelin. The binding of lysenin to sphingomyelin is dependent on the membrane distribution of the lipid, i.e. the Toxin selectively binds sphingomyelin clusters. Development of a non-toxic lysenin mutant revealed the spatial and functional heterogeneity of sphingolipid-rich membrane domains.
-
Lysenin: A New Probe for Sphingomyelin
Sphingolipid Biology, 2006Co-Authors: Toshihide Kobayashi, Akiko Yamaji-hasegawaAbstract:Lysenin is a Pore-Forming Toxin that binds to sphingomyelin in a distribution-dependent manner. Studies of this interaction revealed the heterogeneous organization of sphingomyelin in biomembranes while investigations with non-toxic lysenin helped elucidate the spatial and functional heterogeneity of lipid rafts. This chapter summarizes the characterization of lysenin and discusses the possible applications and limitations of this newly developed sphingomyelin probe.
F. Gisou Van Der Goot - One of the best experts on this subject based on the ideXlab platform.
-
Caspase-2 is an initiator caspase responsible for Pore-Forming Toxin-mediated apoptosis
The EMBO journal, 2012Co-Authors: Gergely Imre, Matthias Husmann, Jan Heering, Armelle‐natsuo Takeda, Bernd Thiede, Dagmar Meyer Zu Heringdorf, Douglas R. Green, F. Gisou Van Der Goot, Bhanu Sinha, Volker DötschAbstract:Bacterial pathogens modulate host cell apoptosis to establish a successful infection. Pore-Forming Toxins (PFTs) secreted by pathogenic bacteria are major virulence factors and have been shown to induce various forms of cell death in infected cells. Here we demonstrate that the highly conserved caspase-2 is required for PFT-mediated apoptosis. Despite being the second mammalian caspase to be identified, the role of caspase-2 during apoptosis remains enigmatic. We show that caspase-2 functions as an initiator caspase during Staphylococcus aureus alpha-Toxin- and Aeromonas aerolysin-mediated apoptosis in epithelial cells. Downregulation of caspase-2 leads to a strong inhibition of PFT-mediated apoptosis. Activation of caspase-2 is PIDDosome-independent, and endogenous caspase-2 is recruited to a high-molecular-weight complex in alpha-Toxin-treated cells. Interestingly, prevention of PFT-induced potassium efflux inhibits the formation of caspase-2 complex, leading to its inactivation, thus resisting apoptosis. These results revealed a thus far unknown, obligatory role for caspase-2 as an initiator caspase during PFT-mediated apoptosis. The EMBO Journal (2012) 31, 2615-2628. doi: 10.1038/emboj.2012.93; Published online 24 April 2012
-
Sensitivity of polarized epithelial cells to the Pore-Forming Toxin aerolysin.
Infection and Immunity, 2003Co-Authors: Laurence Abrami, Marc Fivaz, Pierre-etienne Glauser, Nakaba Sugimoto, Chiara Zurzolo, F. Gisou Van Der GootAbstract:Aerolysin is one of the major virulence factors produced by Aeromonas hydrophila, a human pathogen that produces deep wound infection and gastroenteritis. The Toxin interacts with target mammalian cells by binding to the glycan core of glycosylphosphatidyl inositol (GPI)-anchored proteins and subsequently forms a pore in the plasma membrane. Since epithelial cells of the intestine are the primary targets of aerolysin, we investigated its effect on three types of polarized epithelial cells: Caco-2 cells, derived from human intestine; MDCK cells, a well-characterized cell line in terms of protein targeting; and FRT cells, an unusual cell line in that it targets its GPI-anchored proteins to the basolateral plasma membrane in contrast to other epithelial cells, which target them almost exclusively to the apical surface. Surprisingly, we found that all three cell types were sensitive to the Toxin from both the apical and the basolateral sides. Apical sensitivity was always higher, even for FRT cells. In contrast, FRT cells were more sensitive from the basolateral than from the apical side to the related Toxin Clostridium septicum alpha-Toxin, which also binds to GPI-anchored proteins but lacks the lectin binding domain found in aerolysin. These observations are consistent with the notion that a shuttling mechanism involving low-affinity interactions with surface sugars allows aerolysin to gradually move toward the membrane surface, where it can finally encounter the glycan cores of GPI-anchored proteins.
-
Adventures of a Pore-Forming Toxin at the target cell surface
Trends in microbiology, 2000Co-Authors: Laurence Abrami, Marc Fivaz, F. Gisou Van Der GootAbstract:The past three years have shed light on how the Pore-Forming Toxin aerolysin binds to its target cell and then hijacks cellular devices to promote its own polymerization and pore formation. This selective permeabilization of the plasma membrane has unexpected intracellular consequences that might explain the importance of aerolysin in Aeromonas pathogenicity.
-
Dimer Dissociation of the Pore-Forming Toxin Aerolysin Precedes Receptor Binding
The Journal of biological chemistry, 1999Co-Authors: Marc Fivaz, Marie-claire Velluz, F. Gisou Van Der GootAbstract:The Pore-Forming Toxin aerolysin is secreted by Aeromonas hydrophila as an inactive precursor. Based on chemical cross-linking and gel filtration, we show here that proaerolysin exists as a monomer at low concentrations but is dimeric above 0.1 mg/ml. At intermediate concentrations, monomers and dimers appeared to be in rapid equilibrium. All together our data indicate that, at low concentrations, the Toxin is a monomer and that this species is competent for receptor binding. In contrast, a mutant Toxin that forms a covalent dimer was unable to bind to target cells.
-
Increased stability upon heptamerization of the Pore-Forming Toxin aerolysin.
The Journal of biological chemistry, 1999Co-Authors: C. Lesieur, Séverine Frutiger, Graham J. Hughes, Roland Kellner, Franc Pattus, F. Gisou Van Der GootAbstract:Aerolysin is a bacterial Pore-Forming Toxin that is secreted as an inactive precursor, which is then processed at its COOH terminus and finally forms a circular heptameric ring which inserts into membranes to form a pore. We have analyzed the stability of the precursor proaerolysin and the heptameric complex. Equilibrium unfolding induced by urea and guanidinium hydrochloride was monitored by measuring the intrinsic tryptophan fluorescence of the protein. Proaerolysin was found to unfold in two steps corresponding to the unfolding of the large COOH-terminal lobe followed by the unfolding of the small NH(2)-terminal domain. We show that proaerolysin contains two disulfide bridges which strongly contribute to the stability of the Toxin and protect it from proteolytic attack. The stability of aerolysin was greatly enhanced by polymerization into a heptamer. Two regions of the protein, corresponding to amino acids 180-307 and 401-427, were identified, by limited proteolysis, NH(2)-terminal sequencing and matrix-assisted laser desorption ionization-time of flight, as being responsible for stability and maintenance of the heptamer. These regions are presumably involved in monomer/monomer interactions in the heptameric protein and are exclusively composed of beta structure. The stability of the aerolysin heptamer is reminiscent of that of pathogenic, fimbrial protein aggregates found in a variety of neurodegenerative diseases.
Anthony L. Keyburn - One of the best experts on this subject based on the ideXlab platform.
-
structural and functional analysis of the pore forming Toxin netb from clostridium perfringens
Mbio, 2013Co-Authors: Xuxia Yan, Anthony L. Keyburn, Corrine Joy Porter, Simon P Hardy, David L Steer, Ian A Smith, Noelene Sheila Quinsey, Victoria A Hughes, Jackie K CheungAbstract:Clostridium perfringens is an anaerobic bacterium that causes numerous important human and animal diseases, primarily as a result of its ability to produce many different protein Toxins. In chickens, C. perfringens causes necrotic enteritis, a disease of economic importance to the worldwide poultry industry. The secreted Pore-Forming Toxin NetB is a key virulence factor in the pathogenesis of avian necrotic enteritis and is similar to alpha-hemolysin, a β-barrel Pore-Forming Toxin from Staphylococcus aureus. To address the molecular mechanisms underlying NetB-mediated tissue damage, we determined the crystal structure of the monomeric form of NetB to 1.8 A. Structural comparisons with other members of the alpha-hemolysin family revealed significant differences in the conformation of the membrane binding domain. These data suggested that NetB may recognize different membrane receptors or use a different mechanism for membrane-protein interactions. Consistent with this idea, electrophysiological experiments with planar lipid bilayers revealed that NetB formed pores with much larger single-channel conductance than alpha-hemolysin. Channel conductance varied with phospholipid net charge. Furthermore, NetB differed in its ion selectivity, preferring cations over anions. Using hemolysis as a screen, we carried out a random-mutagenesis study that identified several residues that are critical for NetB-induced cell lysis. Mapping of these residues onto the crystal structure revealed that they were clustered in regions predicted to be required for oligomerization or membrane binding. Together these data provide an insight into the mechanism of NetB-mediated pore formation and will contribute to our understanding of the mode of action of this important Toxin. IMPORTANCE Necrotic enteritis is an economically important disease of the worldwide poultry industry and is mediated by Clostridium perfringens strains that produce NetB, a β-Pore-Forming Toxin. We carried out structural and functional studies of NetB to provide a mechanistic insight into its mode of action and to assist in the development of a necrotic enteritis vaccine. We determined the structure of the monomeric form of NetB to 1.8 A, used both site-directed and random mutagenesis to identify key residues that are required for its biological activity, and analyzed pore formation by NetB and its substitution-containing derivatives in planar lipid bilayers.
-
necrotic enteritis derived clostridium perfringens strain with three closely related independently conjugative Toxin and antibiotic resistance plasmids
Mbio, 2011Co-Authors: Anthony L. Keyburn, Trudi L. Bannam, Xuxia Yan, Paul F Harrison, Torsten Seemann, Christopher James Stubenrauch, Lakmini H WeeramantriAbstract:The pathogenesis of avian necrotic enteritis involves NetB, a Pore-Forming Toxin produced by virulent avian isolates of Clostridium perfringens type A. To determine the location and mobility of the netB structural gene, we examined a derivative of the tetracycline-resistant necrotic enteritis strain EHE-NE18, in which netBwas insertionally inactivated by the chloramphen- icol and thiamphenicol resistance gene catP. Both tetracycline and thiamphenicol resistance could be transferred either together or separately to a recipient strain in plate matings. The separate transconjugants could act as donors in subsequent matings, which demonstrated that the tetracycline resistance determinant and the netBgene were present on different conjugative ele- ments. Large plasmids were isolated from the transconjugants and analyzed by high-throughput sequencing. Analysis of the re- sultant data indicated that there were actually three large conjugative plasmids present in the original strain, each with its own Toxin or antibiotic resistance locus. Each plasmid contained a highly conserved 40-kb region that included plasmid replication and transfer regions that were closely related to the 47-kb conjugative tetracycline resistance plasmid pCW3 from C. perfringens. The plasmids were as follows: (i) a conjugative 49-kb tetracycline resistance plasmid that was very similar to pCW3, (ii) a conju- gative 82-kb plasmid that contained the netBgene and other potential virulence genes, and (iii) a 70-kb plasmid that carried the cpb2gene, which encodes a different Pore-Forming Toxin, beta2 Toxin. IMPORTANCE The anaerobic bacterium Clostridium perfringens can cause an avian gastrointestinal disease known as necrotic enteritis. Disease pathogenesis is not well understood, although the plasmid-encoded Pore-Forming Toxin NetB, is an important virulence factor. In this work, we have shown that the plasmid that carries the netBgene is conjugative and has a 40-kb region that is very similar to replication and transfer regions found within each of the sequenced conjugative plasmids from C. perfrin- gens. We also showed that this strain contained two additional large plasmids that were also conjugative and carried a similar 40-kb region. One of these plasmids encoded beta2 Toxin, and the other encoded tetracycline resistance. To our knowledge, this is thefirst report of a bacterial strain that carries three closely related but different independently conjugative plasmids. These results have significant implications for our understanding of the transmission of virulence and antibiotic resistance genes in pathogenic bacteria.
-
NetB, a Pore-Forming Toxin from Necrotic Enteritis Strains of Clostridium perfringens
Toxins, 2010Co-Authors: Anthony L. Keyburn, Trudi L. Bannam, Robert J. Moore, Julian I. RoodAbstract:The Clostridium perfringens necrotic enteritis B-like Toxin (NetB) is a recently discovered member of the β-barrel Pore-Forming Toxin family and is produced by a subset of avian C. perfringens type A strains. NetB is cytotoxic for avian cells and is associated with avian necrotic enteritis. This review examines the current state of knowledge of NetB: its role in pathogenesis, its distribution and expression in C. perfringens and its vaccine potential.
Kausik Chattopadhyay - One of the best experts on this subject based on the ideXlab platform.
-
Revisiting the oligomerization mechanism of Vibrio cholerae cytolysin, a beta-barrel Pore-Forming Toxin
Biochemical and biophysical research communications, 2016Co-Authors: Anand Kumar Rai, Kausik ChattopadhyayAbstract:Vibrio cholerae cytolysin (VCC) is a membrane-damaging beta-barrel Pore-Forming Toxin (beta-PFT). VCC causes permeabilization of the target membranes by forming transmembrane oligomeric beta-barrel pores. Oligomerization is a key step in the mode of action of any beta-PFT, including that of VCC. Earlier studies have identified some of the key residues in VCC that are directly involved in the generation of the inter-protomer contacts, thus playing critical roles in the oligomerization of the membrane-bound Toxin. Analysis of the VCC oligomeric pore structure reveals a potential hydrogen-bond network that appears to connect the sidechain of an asparagine residue (Asn582; located within an inter-domain linker sequence) from one protomer to the backbone CO- and NH-groups of the neighbouring protomer, indirectly through water molecules at most of the inter-protomer interfaces. In the present study, we show that the mutation of Asn582Ala affects the oligomerization and the Pore-Forming activity of VCC in the membrane lipid bilayer of the synthetic lipid vesicles, while the replacement of Asn582Gln results into the restoration of the oligomeric Pore-Forming ability of the Toxin. Using a number of truncated variants of VCC, having deletion in the C-terminal region of the Toxin starting from the Asn582 residue or beyond, we also show that the presence of Asn582 is critically required for the oligomerization of the truncated form of the protein.
-
Structural-Function Mechanism of Vibrio cholerae Cytolysin: a b-barrel Pore-Forming Toxin
The FASEB Journal, 2015Co-Authors: Anand Rai, Kausik ChattopadhyayAbstract:b-barrel Pore-Forming Toxins (b-PFTs) represent an important class of proteins that disrupt membrane permeability of the target cells by forming transmembrane pores. Vibrio cholerae cytolysin (VCC) is a prominent b-PFT that forms heptameric transmembrane pores in the target eukaryotic cells. Our studies have shown that the membrane binding and pore formation by VCC is governed by multiple regulatory mechanisms. Interaction of VCC with the cell surface glycan(s) appears to modulate the efficacy of the process. Physicochemical conditions like pH also appear to regulate interactions of the Toxin with the membranes. It is also evident that VCC may employ distinct structural motifs to mediate interactions with the membrane lipid components. Our study has also explored the sequence of events for the functional pore formation by VCC. We have identified the key amino acid residue(s) in VCC that are crucial to initiate membrane oligomerization. Modification of such residues traps the protein in its membrane-bound ...
-
vibrio cholerae cytolysin structure function mechanism of an atypical β barrel pore forming Toxin
Advances in Experimental Medicine and Biology, 2015Co-Authors: Anand Kumar Rai, Kausik ChattopadhyayAbstract:β-Barrel Pore-Forming Toxins (β-PFTs) represent a unique class of bacterial protein Toxins. β-PFTs act by punching holes in the membrane lipid bilayer of their target host cells. Generalized mechanism of β-PFT mode of action shows unique structural paradigm that involves formation of transmembrane oligomeric β-barrel pores in the target cells. Vibrio cholerae cytolysin (VCC) is a prominent member in the bacterial β-PFT family, and it exhibits common features of the β-PFT mode of action in general. Structure–function mechanism of VCC, however, highlights distinct features that are not commonly documented in the archetypical β-PFT family members. In this review, we present a brief description of our current understanding regarding the mode of action of VCC, in the context of its β-barrel membrane pore formation mechanism.
Peter Macek - One of the best experts on this subject based on the ideXlab platform.
-
Primary and secondary structure of a Pore-Forming Toxin from the sea anemone, Actinia equina L., and its association with lipid vesicles
Biochimica et biophysica acta, 2017Co-Authors: Igor Križaj, Tom Turk, Peter Macek, Gianfranco Menestrina, Franc Gubenšek, Giuseppe Belmonte, Cecilia PederzolliAbstract:The complete amino acid sequence of equinaToxin II, a potent Pore-Forming Toxin with hemolytic, cytotoxic and cardiotoxic activity from the venom of the sea anemone, Actinia equina L., is reported. In addition, circular dicroism was used to estimate the secondary structure of this Toxin either in the water-soluble or in the membrane-anchored form. EquinaToxin II when in water was found to contain about 29-33% of ?-helical structure, 53-58% of ?-strand + ?-turn and 10-16% of random structure. Upon association with phospholipids, in particular with sphingomyelin, a rearrangement of the secondary structure occurs resulting in an increase of the ?-helix content. An amphiphilic ?-helical segment is predicted at the N-terminus, which shares structural homology with membrane active peptides like melittin and viral fusion peptides. In analogy to the behaviour of these peptides we propose that at least part of the ?-helix content increase of equinaToxin II is due to the insertion of its N-terminus into the lipid bilayer. As in the case of melittin, association of 3-4 equinaToxin molecules is necessary to induce membrane permeabilisation. © 1994.
-
molecular determinants of sphingomyelin specificity of a eukaryotic pore forming Toxin
Journal of Biological Chemistry, 2008Co-Authors: Biserka Bakrac, Peter Macek, Ion Gutierrezaguirre, Zdravko Podlesek, Andreas F P Sonnen, Robert J C Gilbert, Jeremy H Lakey, Gregor AnderluhAbstract: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 bind- ing 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 discrim- ination between sphingomyelin and phosphatidylcholine occurs in the region directly below the phosphorylcholine headgroup. Because the characteristic features of SM chemistry lie in this sub- interfacial region, the recognition mechanism may be generic for all SM-specific proteins.
-
Cytotoxic Activity of a Tumor Protease-Activated Pore-Forming Toxin
Bioconjugate chemistry, 2005Co-Authors: Cristina Potrich, Peter Macek, Gianfranco Menestrina, Rossella Tomazzolli, M. Dalla Serra, Gregor Anderluh, Petra Malovrh, Mayra TejucaAbstract:EquinaToxin II is a pore forming Toxin produced by the sea anemone Actinia equina. It is able to kill very unspecifically most cell types by the membrane-perturbing action of an amphiphilic α-helix located at its N-terminal. A normally active N-terminal mutant, containing one single cys in the amphiphilic α-helix, becomes totally inactive when it is bound to avidin via a biotinylated linker. By choosing, as a linker, a peptide containing a tumor protease cleavage site, we were able to construct an enzymatically activable conjugate which should be selective for tumor cells. The introduced cleavage site was designed in order to be digested by both cathepsin B and matrix metalloproteases (MMPs). We confirmed that this conjugate could be activated in vitro by cathepsin B and MMPs. After having measured the enzymatic activity of fibrosarcoma and breast carcinoma cells, we analyzed the cytotoxic effect of the conjugate on the same lines and on human red blood cells (HRBC) as controls. We found that the conjugat...
-
crystal structure of the soluble form of equinaToxin ii a pore forming Toxin from the sea anemone actinia equina
Structure, 2001Co-Authors: Alekos Athanasiadis, Peter Macek, Gregor Anderluh, Dusan TurkAbstract:BACKGROUND: Membrane Pore-Forming Toxins have a remarkable property: they adopt a stable soluble form structure, which, when in contact with a membrane, undergoes a series of transformations, leading to an active, membrane-bound form. In contrast to bacterial Toxins, no structure of a Pore-Forming Toxin from an eukaryotic organism has been determined so far, an indication that structural studies of equinaToxin II (EqtII) may unravel a novel mechanism. RESULTS: The crystal structure of the soluble form of EqtII from the sea anemone Actinia equina has been determined at 1.9 A resolution. EqtII is shown to be a single-domain protein based on a 12 strand beta sandwich fold with a hydrophobic core and a pair of alpha helices, each of which is associated with the face of a beta sheet. CONCLUSIONS: The structure of the 30 N-terminal residues is the largest segment that can adopt a different structure without disrupting the fold of the beta sandwich core. This segment includes a three-turn alpha helix that lies on the surface of a beta sheet and ends in a stretch of three positively charged residues, Lys-30, Arg-31, and Lys-32. On the basis of gathered data, it is suggested that this segment forms the membrane pore, whereas the beta sandwich structure remains unaltered and attaches to a membrane as do other structurally related extrinsic membrane proteins or their domains. The use of a structural data site-directed mutagenesis study should reveal the residues involved in membrane pore formation.
-
crystal structure of the soluble form of equinaToxin ii a pore forming Toxin from the sea anemone actinia equina
Structure, 2001Co-Authors: Alekos Athanasiadis, Peter Macek, Gregor Anderluh, Dusan TurkAbstract:Abstract Background: Membrane pore–forming Toxins have a remarkable property: they adopt a stable soluble form structure, which, when in contact with a membrane, undergoes a series of transformations, leading to an active, membrane-bound form. In contrast to bacterial Toxins, no structure of a Pore-Forming Toxin from an eukaryotic organism has been determined so far, an indication that structural studies of equinaToxin II (EqtII) may unravel a novel mechanism. Results: The crystal structure of the soluble form of EqtII from the sea anemone Actinia equina has been determined at 1.9 A resolution. EqtII is shown to be a single-domain protein based on a 12 strand β sandwich fold with a hydrophobic core and a pair of α helices, each of which is associated with the face of a β sheet. Conclusions: The structure of the 30 N-terminal residues is the largest segment that can adopt a different structure without disrupting the fold of the β sandwich core. This segment includes a three-turn α helix that lies on the surface of a β sheet and ends in a stretch of three positively charged residues, Lys-30, Arg-31, and Lys-32. On the basis of gathered data, it is suggested that this segment forms the membrane pore, whereas the β sandwich structure remains unaltered and attaches to a membrane as do other structurally related extrinsic membrane proteins or their domains. The use of a structural data site-directed mutagenesis study should reveal the residues involved in membrane pore formation.