Molecular Discrimination between Two Conformations of Sphingomyelin in Plasma Membranes.

Sphingomyelin and cholesterol are essential lipids that are enriched in plasma membranes of animal cells, where they interact to regulate membrane properties and many intracellular signaling processes. Despite intense study, the interaction between these lipids in membranes is not well understood. Here, structural and biochemical analyses of ostreolysin A (OlyA), a protein that binds to membranes only when they contain both sphingomyelin and cholesterol, reveal that sphingomyelin adopts two distinct conformations in membranes when cholesterol is present. One conformation, bound by OlyA, is induced by stoichiometric, exothermic interactions with cholesterol, properties that are consistent with sphingomyelin/cholesterol complexes. In its second conformation, sphingomyelin is free from cholesterol and does not bind OlyA. A point mutation abolishes OlyA's ability to discriminate between these two conformations. In cells, levels of sphingomyelin/cholesterol complexes are held constant over a wide range of plasma membrane cholesterol concentrations, enabling precise regulation of the chemical activity of cholesterol.

Assembly dynamics and structure of an aegerolysin, ostreolysin A6.

Ostreolysin A6 (OlyA6) is an oyster mushroom-derived membrane-binding protein that, upon recruitment of its partner protein, pleurotolysin B, forms a cytolytic membrane pore complex. OlyA6 itself is not cytolytic but has been reported to exhibit pro-apoptotic activities in cell culture. Here we report the formation dynamics and the structure of OlyA6 assembly on a lipid membrane containing an OlyA6 high-affinity receptor, ceramide phosphoethanolamine, and cholesterol. High-speed atomic force microscopy revealed the reorganization of OlyA6 dimers from initial random surface coverage to 2D protein crystals composed of hexameric OlyA6 repeat units. Crystal growth took place predominantly in the longitudinal direction by the association of OlyA6 dimers, forming a hexameric unit cell. Molecular-level examination of the OlyA6 crystal elucidated the arrangement of dimers within the unit cell and the structure of the dimer that recruits pleurotolysin B for pore formation.

N-Terminal Region of Vibrio parahemolyticus Thermostable Direct Hemolysin Regulates the Membrane-Damaging Action of the Toxin.

Thermostable direct hemolysin (TDH) of Vibrio parahemolyticus is a membrane-damaging pore-forming toxin with potent cytolytic/cytotoxic activity. TDH exists as a tetramer consisting of protomers with a core ß-sandwich domain, flanked by an 11-amino acid long N-terminal region (NTR). This NTR could not be modeled in the previously determined crystal structure of TDH. Moreover, the functional implication of NTR for the membrane-damaging action of TDH remains unknown. In the present study, we have explored the implications of NTR for the structure-function mechanism of TDH. Our data show that the presence of NTR modulates the physicochemical property of TDH in terms of augmenting the amyloidogenic propensity of the protein. Deletion of NTR compromises the binding of TDH toward target cell membranes and drastically affects the membrane-damaging cytolytic/cytotoxic activity of the toxin. Mutations of aromatic/hydrophobic residues within NTR also confer compromised cell-killing activity. Moreover, covalent trapping of NTR, via an engineered disulfide bond, against the core ß-sandwich domain also abrogates the cytolytic/cytotoxic activity of TDH. This observation suggests that an unrestrained configuration of NTR is crucial for the membrane-damaging action of TDH. On the basis of our study, we propose a model explaining the role of NTR in the membrane-damaging function of TDH.

The Effector Domain Region of the Vibrio vulnificus MARTX Toxin Confers Biphasic Epithelial Barrier Disruption and Is Essential for Systemic Spread from the Intestine.

Vibrio vulnificus causes highly lethal bacterial infections in which the Multifunctional Autoprocessing Repeats-in-Toxins (MARTX) toxin product of the rtxA1 gene is a key virulence factor. MARTX toxins are secreted proteins up to 5208 amino acids in size. Conserved MARTX N- and C-terminal repeat regions work in concert to form pores in eukaryotic cell membranes, through which the toxin's central region of modular effector domains is translocated. Upon inositol hexakisphosphate-induced activation of the of the MARTX cysteine protease domain (CPD) in the eukaryotic cytosol, effector domains are released from the holotoxin by autoproteolytic activity. We previously reported that the native MARTX toxin effector domain repertoire is dispensable for epithelial cellular necrosis in vitro, but essential for cell rounding and apoptosis prior to necrotic cell death. Here we use an intragastric mouse model to demonstrate that the effector domain region is required for bacterial virulence during intragastric infection. The MARTX effector domain region is essential for bacterial dissemination from the intestine, but dissemination occurs in the absence of overt intestinal tissue pathology. We employ an in vitro model of V. vulnificus interaction with polarized colonic epithelial cells to show that the MARTX effector domain region induces rapid intestinal barrier dysfunction and increased paracellular permeability prior to onset of cell lysis. Together, these results negate the inherent assumption that observations of necrosis in vitro directly predict bacterial virulence, and indicate a paradigm shift in our conceptual understanding of MARTX toxin function during intestinal infection. Results implicate the MARTX effector domain region in mediating early bacterial dissemination from the intestine to distal organs-a key step in V. vulnificus foodborne pathogenesis-even before onset of overt intestinal pathology.

Soluble Oligomers of the Pore-forming Toxin Cytolysin A from Escherichia coli Are Off-pathway Products of Pore Assembly.

The α-pore-forming toxin Cytolysin A (ClyA) is responsible for the hemolytic activity of various Escherichia coli and Salmonella enterica strains. Soluble ClyA monomers spontaneously assemble into annular dodecameric pore complexes upon contact with membranes or detergent. At ClyA monomer concentrations above ∼100 nm, the rate-limiting step in detergent- or membrane- induced pore assembly is the unimolecular reaction from the monomer to the assembly-competent protomer, which then oligomerizes rapidly to active pore complexes. In the absence of detergent, ClyA slowly forms soluble oligomers. Here we show that soluble ClyA oligomers cannot form dodecameric pore complexes after the addition of detergent and are hemolytically inactive. In addition, we demonstrate that the natural cysteine pair Cys-87/Cys-285 of ClyA forms a disulfide bond under oxidizing conditions and that both the oxidized and reduced ClyA monomers assemble to active pores via the same pathway in the presence of detergent, in which an unstructured, monomeric intermediate is transiently populated. The results show that the oxidized ClyA monomer assembles to pore complexes about one order of magnitude faster than the reduced monomer because the unstructured intermediate of oxidized ClyA is less stable and dissolves more rapidly than the reduced intermediate. Moreover, we show that oxidized ClyA forms soluble, inactive oligomers in the absence of detergent much faster than the reduced monomer, providing an explanation for several contradictory reports in which oxidized ClyA had been described as inactive.

Crystal structure of Cry6Aa: A novel nematicidal ClyA-type α-pore-forming toxin from Bacillus thuringiensis.

Crystal (Cry) proteins from Bacillus thuringiensis (Bt) are globally used in agriculture as proteinaceous insecticides. Numerous crystal structures have been determined, and most exhibit conserved three-dimensional architectures. Recently, we have identified a novel nematicidal mechanism by which Cry6Aa triggers cell death through a necrosis-signaling pathway via an interaction with the host protease ASP-1. However, we found little sequence conservation of Cry6Aa in our functional study. Here, we report the 1.90 angstrom (Å) resolution structure of the proteolytic form of Cry6Aa (1-396), determined by X-ray crystallography. The structure of Cry6Aa is highly similar to those of the pathogenic toxin family of ClyA-type α-pore-forming toxins (α-PFTs), which are characterized by a bipartite structure comprising a head domain and a tail domain, thus suggesting that Cry6Aa exhibits a previously undescribed nematicidal mode of action. This structure also provides a framework for the functional study of other nematicidal toxins.

Importance of Thr(328) and Thr(369) for functional maintenance of two receptor-binding ß-hairpins of the Bacillus thuringiensis Cry4Ba toxin: Implications for synergistic interactions with Cyt2Aa2.

Bacillus thuringiensis Cry4Ba mosquito-active toxin was previously shown to utilize two critical loop-residues, Tyr(332) and Phe(364) which are respectively located in ß2-ß3 and ß4-ß5 loops, for synergistic interactions with its alternative receptor-Cyt2Aa2. Here, structural analysis of the Cry4Ba-receptor-binding domain revealed that its N-terminal subdomain encompasses ß2-ß3 and ß4-ß5 hairpins which are stabilized by inter-hairpin hydrogen bonding between Thr(328) in ß2 and Thr(369) in ß5. Functional importance of these two side-chains was demonstrated by single-Ala substitutions (T328A and T369A), adversely affecting toxin activity against Aedes aegypti larvae. Unlike toxicity restoration of the inactive E417A/Y455A toxin mutated within another receptor-binding subdomain, defective bioactivity of T328A and T369A mutants cannot be restored by Cyt2Aa2 as also observed for ß2-ß3 (Y332A) and ß4-ß5 (F364A) loop-mutants. ELISA-based analysis further verified a loss in binding of all four bio-inactive mutants (T328A, Y332A, T369A and F364A) to the immobilized Cyt2Aa2. Protein-protein docking suggested that the two critical loop-residues (Tyr(332) and Phe(364)) correspondingly located at ß2-ß3 and ß4-ß5 loops can clearly interact with four counterpart surface-exposed residues of Cyt2Aa2. Altogether, our present data demonstrate structural importance of Thr(328) and Thr(369) toward hydrogen-bonded stabilization of two receptor-binding hairpins (ß2-ß3 and ß4-ß5) for synergistic toxicity of Cry4Ba with Cyt2Aa2.

Mechanistic insights into the first Lygus-active ß-pore forming protein.

The cotton pests Lygus hesperus and Lygus lineolaris can be controlled by expressing Cry51Aa2.834_16 in cotton. Insecticidal activity of pore-forming proteins is generally associated with damage to the midgut epithelium due to pores, and their biological specificity results from a set of key determinants including proteolytic activation and receptor binding. We conducted mechanistic studies to gain insight into how the first Lygus-active ß-pore forming protein variant functions. Biophysical characterization revealed that the full-length Cry51Aa2.834_16 was a stable dimer in solution, and when exposed to Lygus saliva or to trypsin, the protein underwent proteolytic cleavage at the C-terminus of each of the subunits, resulting in dissociation of the dimer to two separate monomers. The monomer showed tight binding to a specific protein in Lygus brush border membranes, and also formed a membrane-associated oligomeric complex both in vitro and in vivo. Chemically cross-linking the ß-hairpin to the Cry51Aa2.834_16 body rendered the protein inactive, but still competent to compete for binding sites with the native protein in vivo. Our study suggests that disassociation of the Cry51Aa2.834_16 dimer into monomeric units with unoccupied head-region and sterically unhindered ß-hairpin is required for brush border membrane binding, oligomerization, and the subsequent steps leading to insect mortality.

Directly Observing the Lipid-Dependent Self-Assembly and Pore-Forming Mechanism of the Cytolytic Toxin Listeriolysin O.

Listeriolysin O (LLO) is the major virulence factor of Listeria monocytogenes and a member of the cholesterol-dependent cytolysin (CDC) family. Gram-positive pathogenic bacteria produce water-soluble CDC monomers that bind cholesterol-dependent to the lipid membrane of the attacked cell or of the phagosome, oligomerize into prepores, and insert into the membrane to form transmembrane pores. However, the mechanisms guiding LLO toward pore formation are poorly understood. Using electron microscopy and time-lapse atomic force microscopy, we show that wild-type LLO binds to membranes, depending on the presence of cholesterol and other lipids. LLO oligomerizes into arc- or slit-shaped assemblies, which merge into complete rings. All three oligomeric assemblies can form transmembrane pores, and their efficiency to form pores depends on the cholesterol and the phospholipid composition of the membrane. Furthermore, the dynamic fusion of arcs, slits, and rings into larger rings and their formation of transmembrane pores does not involve a height difference between prepore and pore. Our results reveal new insights into the pore-forming mechanism and introduce a dynamic model of pore formation by LLO and other CDC pore-forming toxins.

Helicobacter pylori TlyA Forms Amyloid-like Aggregates with Potent Cytotoxic Activity.

Helicobacter pylori is a potent human gastric pathogen. It is known to be associated with several gastroenteric disorders, including gastritis, peptic ulcer, and gastric cancer. The H. pylori genome encodes a gene product TlyA that has been shown to display potent membrane damaging properties and cytotoxic activity. On the basis of such properties, TlyA is considered as a potential virulence factor of H. pylori. In this study, we show that the H. pylori TlyA protein has a strong propensity to convert into the amyloid-like aggregated assemblies, upon exposure to elevated temperatures. Even at the physiological temperature of 37 °C, TlyA shows a strong amyloidogenic property. TlyA aggregates that are generated upon exposure at temperatures of ≥37 °C show prominent binding to dyes like thioflavin T and Nile Red. Transmission electron microscopy also demonstrates the presence of typical amyloid-like fibrils in the TlyA aggregates generated at 37 °C. Conversion of TlyA into the amyloid-like aggregates is found to be associated with major alterations in the secondary and tertiary structural organization of the protein. Finally, our study shows that the preformed amyloid-like aggregates of TlyA are capable of exhibiting potent cytotoxic activities against human gastric adenocarcinoma cells. Altogether, such a propensity of H. pylori TlyA to convert into the amyloid-like aggregated assemblies with cytotoxic activity suggests potential implications for the virulence functionality of the protein.

Decreasing Transmembrane Segment Length Greatly Decreases Perfringolysin O Pore Size.

Perfringolysin O (PFO) is a transmembrane (TM) ß-barrel protein that inserts into mammalian cell membranes. Once inserted into membranes, PFO assembles into pore-forming oligomers containing 30-50 PFO monomers. These form a pore of up to 300 Å, far exceeding the size of most other proteinaceous pores. In this study, we found that altering PFO TM segment length can alter the size of PFO pores. A PFO mutant with lengthened TM segments oligomerized to a similar extent as wild-type PFO, and exhibited pore-forming activity and a pore size very similar to wild-type PFO as measured by electron microscopy and a leakage assay. In contrast, PFO with shortened TM segments exhibited a large reduction in pore-forming activity and pore size. This suggests that the interaction between TM segments can greatly affect the size of pores formed by TM ß-barrel proteins. PFO may be a promising candidate for engineering pore size for various applications.

The effect of gamma sterilization on the insecticidal toxicity of engineered and conventional Bacillus thuringiensis strains.

This study evaluates the effect of gamma radiation on the spore activity, toxicity, and crystal structures of two engineered Bacillus thuringiensis (Bt) strains, TnX and TnY, and the reference Bt strain HD-1. We attempted to identify dosages of cobalt-60 gamma radiation that would inactivate Bt spores but not affect its toxicity. In the radiation dosage range of 10-15 kilogray, no viable spore formation and no significant reduction of the efficiency of Bt against lepidopteran larvae were observed. However, further sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results show that the components of the protoxin are affected by gamma radiation and that some bands are absent after treatment compared with the controls; the change in the protoxin band pattern depends on the type of Bt strain. Furthermore, the spore crystal structure of three Bt strains was studied with scanning electron microscopy and transmission electron microscopy. The results show that there are no changes in the size or shape of the treated Bt spores and crystals compared with the controls. The use of gamma radiation is effective to inactivate the spores of engineered Bt strains while preserving stable Bt toxicity against the target insect larvae.

An engineered ClyA nanopore detects folded target proteins by selective external association and pore entry.

Nanopores have been used in label-free single-molecule studies, including investigations of chemical reactions, nucleic acid analysis, and applications in sensing. Biological nanopores generally perform better than artificial nanopores as sensors, but they have disadvantages including a fixed diameter. Here we introduce a biological nanopore ClyA that is wide enough to sample and distinguish large analyte proteins, which enter the pore lumen. Remarkably, human and bovine thrombins, despite 86% sequence identity, elicit characteristic ionic current blockades, which at -50 mV differ in their main current levels by 26 ± 1 pA. The use of DNA aptamers or hirudin as ligands further distinguished the protein analytes. Finally, we constructed ClyA nanopores decorated with covalently attached aptamers. These nanopores selectively captured and internalized cognate protein analytes but excluded noncognate analytes, in a process that resembles transport by nuclear pores.

The 20-kDa chaperone-like protein of Bacillus thuringiensis ssp. israelensis enhances yield, crystal size and solubility of Cry3A.

AIMS: To determine whether the 20-kDa chaperone-like protein of Bacillus thuringiensis ssp. israelensis enhances synthesis, crystallization and solubility of the Cry3A coleopteran toxin and whether the crystalline inclusions produced are toxic to neonates of the Colorado potato beetle, Leptinotarsa decemlineata. METHODS AND RESULTS: The cry3A gene was expressed in the 4Q7 strain of B. thuringiensis ssp. israelensis in the absence or presence of the 20-kDa gene. The 20-kDa protein enhanced Cry3A yield by 2·7-fold per unit of fermentation medium. Crystal volumes averaged 2·123 and 0·964 µm(3) when synthesized in, respectively, the presence or absence of the 20-kDa protein. Both crystals were soluble at pH 5 and pH 6; however, the larger crystal was 1·7× and 1·5× more soluble at, respectively, pH 7 and pH 10. No significant difference in toxicity against L. decemlineata neonates was observed. CONCLUSIONS: This report demonstrated that the 20-kDa chaperone-like protein enhances yield, volume and solubility of the coleopteran Cry3A crystalline inclusions per unit crystal/spore mixture. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report showing that an accessory protein (20-kDa) could enhance synthesis and crystallization of Cry3A, a finding that could be beneficial for commercial production of this coleopteran-specific insecticidal protein for microbial insecticides and possibly even for transgenic crops.

The role of C-terminus carbohydrate-binding domain of Vibrio cholerae haemolysin/cytolysin in the conversion of the pre-pore ß-barrel oligomer to a functional diffusion channel.

BACKGROUND & OBJECTIVES: Vibrio cholerae cytolysin/hemolysin (VCC) is a 65 kDa pore-forming toxin (PFT) secreted by O1 El Tor and non-O1 strains. The purified toxin, which contains two C-terminus carbohydrate-binding domains in addition to the cytolytic domain at the core, causes lysis of a wide spectrum of eukaryotic cells at picomolar concentrations, apoptogenesis of intestinal and immune cells and accumulation of fluid in rabbit ligated ileal loop. Therefore, it may potentially complement the action of cholera toxin (CT) in diarrheagenic strains that do not produce CT. We showed earlier that ß1-galactosyl-terminated glycoconjugates are strong inhibitors of its pore-forming activity, though carbohydrates are not functional receptors of VCC. Here, we investigate how the 15 kDa C-terminus ß-prism lectin domain contributed to pore formation in erthrocytes. METHODS: VCC was isolated from the culture supernatant of late log phase grown bacteria and purified to homogeneity by chromatography. The 50 kDa truncated variant was generated by restricted proteolysis. Liposome was prepared by sonication of a suspension of phospholipids and calceine release assay was done by spectrofluorometric monitoring of the released dye trapped in liposome. Formation of ß-barrel oligomers in erythrocyte stroma was monitored by scanning electron microscopy. RESULTS: Proteolytic truncation of the C-terminus ß-prism lectin domain decreased hemolytic activity of the toxin by ~800-fold without causing a significant change in pore-forming activity toward synthetic lipid vesicles devoid of incorporated glycoproteins/glycolipids. Truncation at the C-terminus did not impair membrane-binding or assembly to the oligomeric pore. INTERPRETATION & CONCLUSIONS: Our data indicated that the C-terminus domain played a critical role in translocation of the pre-pore oligomeric assembly from the cell surface or lipid-water interface to the hydrocarbon core of the membrane bilayer, signaling the formation of functional diffusion channels.

Carboxy-terminal extension effects on crystal formation and insecticidal properties of Cry15Aa.

Cry15Aa protein, produced by Bacillus thuringiensis serovar thompsoni HD542, in a crystal together with a 40 kDa accompanying protein, is one of a small group of non-typical, less well-studied members of the Cry family of insecticidal proteins, and may provide an alternative for the more commonly used Cry proteins in insect pest management. In this study we examined the role of the C-terminal part of Cry15Aa and of the 40 kDa protein in crystal formation in recombinant B. thuringiensis. The contribution of the 40 kDa protein and of the Cry15Aa carboxy-terminal sequence for crystal formation, crystal solubilization, and insecticidal properties was assessed. No significant differences in toxicity against Cydia pomonella, before or after in vitro solubilization of crystal-spore preparations, were found. Although the 40 kDa protein significantly contributes to in vitro solubility and in vivo crystal formation of Cry15Aa, no direct evidence for involvement of the 40 kDa protein in toxicity of Cry15Aa was found.

Three-dimensional structure of different functional forms of the Vibrio cholerae hemolysin oligomer: a cryo-electron microscopic study.

Vibrio cholerae hemolysin (HlyA) is a 65-kDa water-soluble pore-forming toxin that causes lysis of eukaryotic cells by destroying selective permeability of the plasma membrane bilayer. The HlyA monomer self-assembles on the target cell surface to the more stable beta-barrel amphipathic heptamer, which inserts into the membrane bilayer to form a diffusion channel. Deletion of the 15-kDa beta-prism lectin domain at the C terminus generates a 50-kDa hemolysin variant (HlyA50) with an approximately 1,000-fold decrease in hemolytic activity. Because functional differences are eventually dictated by structural differences, we determined three-dimensional structures of 65- and 50-kDa HlyA oligomers, using cryo-electron microscopy and single-particle methods. Our study clearly shows that the HlyA oligomer has sevenfold symmetry but that the HlyA50 oligomer is an asymmetric molecule. The HlyA oligomer has bowl-like, arm-like, and ring-like domains. The bowl-like domain is coupled with the ring-like domain, and seven side openings are present just beneath the ring-like domain. Although a central channel is present in both HlyA and HlyA50 oligomers, they differ in pore size as well as in shape of the molecules and channel. These structural differences may be relevant to the striking difference in efficiencies of functional channel formation by the two toxin forms.

Hyperproduction of chitinase influences crystal toxin synthesis and sporulation of Bacillus thuringiensis.

Bacillus thuringiensis HD-73 was transformed with the endochitinase gene chiA74 under the control of a strong promoter (pcytA) and a 5' mRNA stabilizing (STAB-SD) sequence (HD-73-pEBchiA74). Expression levels were compared with those observed from the wild type strain (HD-73) and the recombinant HD-73 strain expressing chiA74 under the control of its native promoter (HD-73-pEHchiA74). The chitinolytic activity of HD-73-pEBchiA74 was markedly elevated, being ~58- and 362-fold higher than, respectively, HD-73-pEHchiA74 and parental HD-73, representing the highest levels of chitinase expression in recombinant B. thuringiensis reported to date. Parasporal crystals measured under transmission electron microscopy showed that HD-73 produced crystals of 1.235 (+/-0.214) and 1.356 (+/-0.247) mum in length when the bacterium was grown in respectively, NBS and NBS with glucose. Otherwise, HD-73-pEBchiA74 synthesized crystals of 1.250 (+/-0.222) and 1.139 (+/-0.202) mum in length when cultivated in NBS and NBS with glucose, respectively, values that showed a diminution of ~10 and 20% compared with crystals produced by HD-73-pEHchiA74 grown under the same conditions. Comparison of viable spore counts per ml showed that HD-73-pEBchiA74 produced fewest viable spores (1.5 x 10(9), 1.3 x 10(9)), compared to HD-73-pEHchiA74 (4.9 x 10(9), 5.3 x 10(9)) and HD-73 (6.8 x 10(9), 8.8 x 10(9)) when grown in NBS and NBS supplemented with glucose, respectively. No change in cellular protease activity was observed despite the overproduction of the chitinase.

Characterization of native Bacillus thuringiensis strains and selection of an isolate active against Spodoptera frugiperda and Peridroma saucia.

Twelve Bacillus thuringiensis (Bt) strains, isolated from larvae and soil samples in Argentina, were molecularly and phenotypically characterized and their insecticidal activities against Spodoptera frugiperda and Peridroma saucia were determined. One isolate--Bt RT--produced more than 93% mortality on first instar larvae of both species, which was higher than that produced by the reference strain Bt 4D1. Bt RT carried a different cry gene profile than Bt 4D1. Scanning electron microscopy showed the presence of bipyramidal and cuboidal crystals. Phenotypic characterization revealed lytic enzymes that could contribute to Bt pathogenicity.

High-resolution cryo-EM structures of the E. coli hemolysin ClyA oligomers.

Pore-forming proteins (PFPs) represent a functionally important protein family, that are found in organisms from viruses to humans. As a major branch of PFPs, bacteria pore-forming toxins (PFTs) permeabilize membranes and usually cause the death of target cells. E. coli hemolysin ClyA is the first member with the pore complex structure solved among α-PFTs, employing α-helices as transmembrane elements. ClyA is proposed to form pores composed of various numbers of protomers. With high-resolution cryo-EM structures, we observe that ClyA pore complexes can exist as newly confirmed oligomers of a tridecamer and a tetradecamer, at estimated resolutions of 3.2 Å and 4.3 Å, respectively. The 2.8 Å cryo-EM structure of a dodecamer dramatically improves the existing structural model. Structural analysis indicates that protomers from distinct oligomers resemble each other and neighboring protomers adopt a conserved interaction mode. We also show a stabilized intermediate state of ClyA during the transition process from soluble monomers to pore complexes. Unexpectedly, even without the formation of mature pore complexes, ClyA can permeabilize membranes and allow leakage of particles less than ~400 Daltons. In addition, we are the first to show that ClyA forms pore complexes in the presence of cholesterol within artificial liposomes. These findings provide new mechanistic insights into the dynamic process of pore assembly for the prototypical α-PFT ClyA.