Cryo‐EM structure of the octameric pore of Clostridium perfringens β‐toxin
2022; Springer Nature; Volume: 23; Issue: 12 Linguagem: Inglês
10.15252/embr.202254856
ISSN1469-3178
AutoresJulia Bruggisser, Ioan Iacovache, Samuel C. Musson, Matteo T. Degiacomi, Horst Posthaus, Benoît Zuber,
Tópico(s)Streptococcal Infections and Treatments
ResumoReport10 October 2022Open Access Transparent process Cryo-EM structure of the octameric pore of Clostridium perfringens β-toxin Julia Bruggisser Julia Bruggisser orcid.org/0000-0001-6449-0115 Institute of Animal Pathology, Vetsuisse-Faculty, University of Bern, Bern, Switzerland Contribution: Conceptualization, Investigation, Writing - original draft, Writing - review & editing Search for more papers by this author Ioan Iacovache Ioan Iacovache orcid.org/0000-0001-8470-5056 Institute of Anatomy, Medical Faculty, University of Bern, Bern, Switzerland Contribution: Conceptualization, Investigation, Writing - original draft, Writing - review & editing Search for more papers by this author Samuel C Musson Samuel C Musson orcid.org/0000-0002-2189-554X Department of Physics, Durham University, Durham, UK Contribution: Investigation, Writing - review & editing Search for more papers by this author Matteo T Degiacomi Matteo T Degiacomi orcid.org/0000-0003-4672-471X Department of Physics, Durham University, Durham, UK Contribution: Supervision, Investigation, Writing - review & editing Search for more papers by this author Horst Posthaus Corresponding Author Horst Posthaus [email protected] orcid.org/0000-0002-4579-7493 Institute of Animal Pathology, Vetsuisse-Faculty, University of Bern, Bern, Switzerland Contribution: Conceptualization, Supervision, Project administration, Writing - review & editing Search for more papers by this author Benoît Zuber Corresponding Author Benoît Zuber [email protected] orcid.org/0000-0001-7725-5579 Institute of Anatomy, Medical Faculty, University of Bern, Bern, Switzerland Contribution: Conceptualization, Supervision, Project administration, Writing - review & editing Search for more papers by this author Julia Bruggisser Julia Bruggisser orcid.org/0000-0001-6449-0115 Institute of Animal Pathology, Vetsuisse-Faculty, University of Bern, Bern, Switzerland Contribution: Conceptualization, Investigation, Writing - original draft, Writing - review & editing Search for more papers by this author Ioan Iacovache Ioan Iacovache orcid.org/0000-0001-8470-5056 Institute of Anatomy, Medical Faculty, University of Bern, Bern, Switzerland Contribution: Conceptualization, Investigation, Writing - original draft, Writing - review & editing Search for more papers by this author Samuel C Musson Samuel C Musson orcid.org/0000-0002-2189-554X Department of Physics, Durham University, Durham, UK Contribution: Investigation, Writing - review & editing Search for more papers by this author Matteo T Degiacomi Matteo T Degiacomi orcid.org/0000-0003-4672-471X Department of Physics, Durham University, Durham, UK Contribution: Supervision, Investigation, Writing - review & editing Search for more papers by this author Horst Posthaus Corresponding Author Horst Posthaus [email protected] orcid.org/0000-0002-4579-7493 Institute of Animal Pathology, Vetsuisse-Faculty, University of Bern, Bern, Switzerland Contribution: Conceptualization, Supervision, Project administration, Writing - review & editing Search for more papers by this author Benoît Zuber Corresponding Author Benoît Zuber [email protected] orcid.org/0000-0001-7725-5579 Institute of Anatomy, Medical Faculty, University of Bern, Bern, Switzerland Contribution: Conceptualization, Supervision, Project administration, Writing - review & editing Search for more papers by this author Author Information Julia Bruggisser1,†, Ioan Iacovache2,†, Samuel C Musson3, Matteo T Degiacomi3, Horst Posthaus *,1,‡ and Benoît Zuber *,2,‡ 1Institute of Animal Pathology, Vetsuisse-Faculty, University of Bern, Bern, Switzerland 2Institute of Anatomy, Medical Faculty, University of Bern, Bern, Switzerland 3Department of Physics, Durham University, Durham, UK † These authors contributed equally to this work as first authors. ‡ These authors contributed equally to this work as corresponding authors. *Corresponding author. Tel: +41 31 684 23 99; E-mail: [email protected] *Corresponding author. Tel: +41 31 684 84 40; E-mail: [email protected] EMBO Reports (2022)23:e54856https://doi.org/10.15252/embr.202254856 PDFDownload PDF of article text and main figures.PDF PLUSDownload PDF of article text, main figures, expanded view figures and appendix. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Clostridium perfringens is one of the most widely distributed and successful pathogens producing an impressive arsenal of toxins. One of the most potent toxins produced is the C. perfringens β-toxin (CPB). This toxin is the main virulence factor of type C strains. We describe the cryo-electron microscopy (EM) structure of CPB oligomer. We show that CPB forms homo-octameric pores like the hetero-oligomeric pores of the bi-component leukocidins, with important differences in the receptor binding region and the N-terminal latch domain. Intriguingly, the octameric CPB pore complex contains a second 16-stranded β-barrel protrusion atop of the cap domain that is formed by the N-termini of the eight protomers. We propose that CPB, together with the newly identified Epx toxins, is a member a new subclass of the hemolysin-like family. In addition, we show that the β-barrel protrusion domain can be modified without affecting the pore-forming ability, thus making the pore particularly attractive for macromolecule sensing and nanotechnology. The cryo-EM structure of the octameric pore of CPB will facilitate future developments in both nanotechnology and basic research. Synopsis Clostridium perfringens β-toxin (CPB) is a beta-pore-forming toxin of the hemolysin family and an essential virulence factor of type C strains causing fatal necrotic enteritis in animals and humans. This study reports the cryo-electron microscopy structure of the oligomeric CPB-pore which can facilitate future developments in both nanotechnology and basic research. CPB is the prototype of a novel hemolysin subfamily The N-termini of the eight protomers form a β-barrel protrusion atop of the cap domain The membrane binding domain contains two unique flexible loops which are most likely involved in receptor specificity of CPB Introduction One of the most common and evolutionary conserved bacterial virulence mechanisms is the secretion of protein toxins that disrupt cellular membranes by pore formation. Such pore-forming toxins (PFTs) are used by the pathogens to invade, survive, and disseminate in their hosts. Despite their large diversity, bacterial PFTs share common features. Most are secreted as water-soluble monomers and bind to target cells via membrane receptors. Receptor binding leads to an increase in the local concentration, oligomerization, and insertion of a stable pore in the cell membrane. This allows uncontrolled exchanges between the extracellular and intracellular milieus, disturbs cellular homeostasis, and leads to diverse reactions ranging from defense mechanisms to cell death (Dal Peraro & van der Goot, 2016). Because of their nearly universal presence in bacterial pathogens, common structures and mechanisms used by PFTs are promising targets for novel anti-virulence strategies as alternatives or supplementation to increasingly ineffective antibiotic treatments. Moreover, PFTs have gathered much interest in the scientific community beyond bacterial infections. The nano-sized pores that they form are used for "sensing" biomolecules. Many nanopore applications are based on α-hemolysin (Hla), the prototype hemolysin-like β-PFT secreted by Staphylococcus aureus (Kasianowicz et al, 1996) as well as aerolysin from Aeromonas hydrophila (Cao et al, 2019). The human and animal pathogen Clostridium perfringens causes many severe diseases such as wound infections, septicemia, food poisoning, enterotoxemia, and enteritis (Songer, 1996, 2010; Kiu & Hall, 2018). The bacterium can produce a large arsenal of exotoxins, in particular PFTs that cross the membrane as a β-barrel (β-PFT). The largest group among them, with currently 11 known members, are the hemolysin-like β-PFTs (Popoff & Bouvet, 2009; Popoff, 2014; Mehdizadeh Gohari et al, 2015; Lacey et al, 2019). Despite their widespread use by the pathogen, their role in diseases is incompletely understood. One of the most potent toxins produced by C. perfringens is the β-toxin (CPB; Uzal et al, 2014). CPB is secreted by C. perfringens type C strains and is essential in the pathogenesis of a lethal necrotic enteritis in many animal species and humans (Uzal et al, 2014). CPB contributes to the intestinal damages by targeting endothelial cells and potentially thrombocytes, leading to vascular damage and hemorrhage (Posthaus et al, 2020). Based on sequence homology between mature CPB and other bacterial toxins (Appendix Fig S1 and S2), the toxin is a member of the hemolysin-like family of β-PFTs. Within this group, CPB is most closely related to C. perfringens δ-toxin (46% identity) and NetB (39% identity). In addition, it shows a 41% identity with the newly discovered enterococcal EPX4 toxin (Xiong et al, 2022). It shares lower sequence homology to the staphylococcal toxins Hla (26% identity) and the bi-component leukocidins (Notredame et al, 2000; Robert & Gouet, 2014). Recently, we determined the molecular basis for the specificity of CPB toward endothelial and leukocytic cells by showing that PECAM-1/CD31, an endothelial and leukocytic adhesion molecule, serves as its cellular receptor (Bruggisser et al, 2020; Tarek et al, 2021). So far however, no structural information has been available for CPB. In the present study, we describe the cryo-electron microscopy (EM) structure of CPB in SMA discs, which likely represents the membrane-inserted pore form, at near atomic resolution. We show that CPB forms a homo-oligomeric pore with a novel N-terminal β-barrel replacing the typical hemolysin latch domain. We propose that the N-terminal β-hairpin stabilizes the monomeric protein in solution and influences the pore conductivity. Our results have important implications for comparative studies on related toxins and the rational design of novel anti-virulence strategies against clostridial diseases. In addition, the unique features of the N-terminal β-barrel make CPB an attractive candidate for applications in nanotechnology. Results and Discussion Formation of the CPB oligomeric pore To investigate the pore formed by CPB by single particle cryo-EM, we screened for a suitable detergent for oligomerization. CPB spontaneously assembles into SDS-resistant oligomeric species when stored in solution leading to partial precipitation of the protein. Purification in the presence of cholate followed by detergent removal and exchange with 2.5% SMA (Knowles et al, 2009) led to a homogeneous distribution of pores suitable for single particle cryo-EM (Fig EV1A–G). Interestingly, the synthetic nanodiscs did not require addition of lipids suggesting that the SMA is able to directly wrap around the β-barrel of the CPB pore keeping the complex soluble. Particle distribution, orientation, and sample quality in SMA were better than either in detergent or inserted in protein-based nanodiscs (Fig EV2A–D). The average diameter of the particles was ~100 Å. Two-dimensional classification of the particles revealed CPB pores with eight-fold symmetry. Further refinement and postprocessing resulted in an electron density map at an estimated 3.8 Å resolution (Figs 1A and C, and EV2E and F). This allowed us to unambiguously build a model for the CPB octameric pore except for two loops in the rim domain (Glu76–Ser89, Ala283–Pro287), 4N-terminal amino acids and several amino acids with unresolved side chains (Fig 1B). Figure 1. Structure of the CPB pore Side and top views of the 3.8 Å sharpened cryo-EM map of the Clostridium perfringens β-toxin (CPB) with one protomer highlighted in purple. The map shows an extended 123 Å particle with protrusions on both sides. The diameter of the particles is 97 Å with a visible channel of ~ 30 Å diameter. Model of the CPB oligomer showing a homo-octameric pore in top view and side view. The dimensions of the two β-barrels are indicated as measured from Cα to Cα. One protomer is colored by domains: green—N-terminal β-barrel protrusion; light blue—β-sandwich cap domain; dark blue—rim domain; red—stem domain. Magnified view of the aromatic pocket of the rim domain docked in the cryo-EM map showing the density of the aromatic side chains. Residues are indicated in proximity to their respective densities. Cartoon model of the CPB protomer in the oligomer color coded as in (B) compared to a protomer extracted from the Hla oligomer. The N-terminus of Hla is highlighted in purple. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Oligomerization and solubility of CPB oligomers in different detergents A. Detergent screen showing cholate and deoxycholate as the best candidates for purification and solubilization of Clostridium perfringens β-toxin (CPB) pores. Each detergent was added to the CPB sample for 16 h at 4° and then the sample was centrifuged at 15,000 g for 10 min. The pelleted insoluble fraction was recovered in sample buffer and loaded on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) together with the soluble fraction and the initial (total) fractions. B. Negative stain of representative CPB oligomers after purification in 30 mM cholate with evenly spread oligomers (top) vs after exchanging cholate with 0.1 mM GDN with aggregates and background particles (bottom). C, D. SDS-PAGE gels of His6-CPB purification in the absence (C) and presence (D) of cholate. 10 μl of supernatant (S), flow through (F), and elution (E) fractions were loaded, and gels stained with Coomassie stain. Most oligomers precipitate in the absence of detergent during the purification, whereas after concentration (C) almost all CPB shifted into the oligomeric state in the presence of cholate. E. Table of the detergents used for the screen, their CMC and their concentration. F, G. Characterization of N- and C-terminal tagged CPB constructs by gel electrophoretic analysis under denaturing (F) and native (G) conditions. SDS-PAGE gel analysis of CPB samples (1 μg per lane) showing monomeric CPB in the absence of detergent and SDS-resistant oligomers in the presence of cholate. CPB samples containing cholate were boiled for 5 min at 95°C (+) or not (−). Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Cryo-EM of CPB Electron micrograph of a typical field of view of oligomeric Clostridium perfringens β-toxin (CPB). Automatically picked particles shown in green. Inset showing characteristic 2D class averages, side, tilted, top. Scale bar is 100 nm. FSC showing the resolution of CPB oligomers in different reconstruction conditions with SMA giving the best orientation of the sample on the grid. Refined cryo-EM maps and angular distribution of particles of CPB reconstituted in SMA, BigCHAP or 2N2 nanodiscs. Representative micrographs of the data in panels B and C. Local resolution estimate performed in RELION showing the cap and rim domains at the highest resolution. Position of the NBP at the extremes of the multibody analysis performed in RELION (in green) compared to the CPB map (gray). Download figure Download PowerPoint Molecular architecture of the CPB pore The cryo-EM analysis of CPB clearly showed that like other members of the hemolysin-like family, the CPB protomer is composed of a cap, a rim, and a stem domain (Johnstone et al, 2021). In addition to the prototypical features of the family members, CPB possesses a second β-barrel on top of the cap domain. While the N-terminus of Hla is located inside the cap domain and wraps the vestibule-exposed surface of the adjacent protomer, the CPB N-terminus protrudes from the cap as a short hairpin, which assembles to form a 16-strand β-barrel (Fig 1B and D). We termed it the N-terminal β-barrel protrusion (NBP). The cap domain consists of a β-sandwich composed of two β-sheets and short α-helices. Two strands extend into the lower part of the molecule making up the rim domain. While the cap domain is one of the most conserved features within the hemolysin-like family, interesting differences are found in the rim domain. Unfolded loops in this domain have been shown to bind the toxin receptor in LukGH leukocidin (CD11b) and phosphatidyl choline in both Hla and γ-hemolysin (Trstenjak et al, 2020; Olson et al, 1999; Galdiero & Gouaux, 2004; Monma et al, 2004). Interestingly at position 210, CPB is the only family member to have a bulky aromatic residue, a tyrosine, pointing inside a pocket (Fig 1C). This pocket was shown to bind phosphocholine in Hla (Galdiero & Gouaux, 2004). Furthermore, CPB lacks a four-residue stretch which lines the pocket. Among them a tryptophan which is important for phosphocholine binding in staphylococcal hemolysins (Monma et al, 2004). These differences are compatible with the fact that CPB main receptor is a membrane protein, whereas for both α- and γ-hemolysins the main receptor is phosphatidylcholine (Dal Peraro & van der Goot, 2016; Bruggisser et al, 2020). The loops at the base of the rim domain were well resolved except for two stretches of 14 and 5 residues, respectively (Glu76–Ser89; Ala283–Pro287). Importantly, these stretches are predicted to be surface exposed (Fig EV3A–D). (Trstenjak et al, 2020). We compared the protomer structure in CPB and γ-hemolysin octamers, respectively (Fig EV3E). The rim domain of the two proteins nearly perfectly overlaps except in the membrane-proximal loop region, suggesting that the loops have different functions between the two toxins. This may indicate that this region of CPB could potentially bind its receptor. Indeed, a pairwise flexible alignment of CPB with other hemolysins shows that apart from the NBP the main structural differences are located in the rim domain, in particular in the flexible loops of the rim (Appendix Fig S3). This issue is out of the scope of this paper and will be investigated in subsequent studies. The stem domain is similar to the other family members. It contains a long, curved amphipathic hairpin, which is connected to the cap β-sandwich through two short coils forming the transmembrane β-barrel upon oligomerization. Overall, the CPB octamer forms a 123 Å-long ring-like structure with a widest outer diameter of 97 Å (Fig 1A and B). The channel runs along the 8-fold symmetry axis. It goes through the 25-Å long N-terminal ß-barrel, the central large 72,900 Å3 vestibule, formed by the cap domains, and the transmembrane β-barrel. Click here to expand this figure. Figure EV3. Modeling the missing features of the CPB model and structural comparison of CPB and γ-hemolysin A. Model of Clostridium perfringens δ-toxin (2YGT) using the same color code as in Fig 1 showing the position of its N-terminus folded back as an additional strand with the inset showing a comparison between the N-terminus of the δ-toxin (green) and the N-terminus of Hla (black—4YHD). B. Model of a protomer of CPB extracted from the oligomer structure color coded as before. The missing loops in the rim domain are modeled and shown in gray. The missing loops are only shown as visual guide for the number of missing amino acids in the model as the map quality in those regions is not good enough for model building. C, D. Prediction of the CPB monomer structure by AlphaFold (C) and RosettaFold (D) color coded as before. The predicted N-terminus folds as the N-terminus of δ-toxin. The missing loops in the rim domains fold similarly to our modeling shown in (B) in the case of alphafold algorithm while RosettaFold modeling extends the longer missing loop into a long β-sheet (D). E. Monomers extracted from the C. perfringens β-toxin (CPB) and γ-hemolysin (PDB: 3B07) octamers were aligned (STAMP structural alignment), and the Euclidean distance of their paired Cα atoms measured. In the graph, the black line reports on the distances measured when using our CPB structure. The red line reports on the mean difference when using all structures extracted from our MD simulations for comparison, and the gray shaded region reports on the standard deviation. The protein rendering is colored according to the mean difference of simulated structures. Download figure Download PowerPoint N-terminal β-barrel protrusion To further investigate the NBP and its role, we constructed CPB mutants where the NBP was either deleted (Δ23CPB) or replaced by the equivalent N-termini of S. aureus Hla (Hla-Δ23CPB), γ-hemolysin component B (HlgB-Δ23CPB), or C. perfringens δ-toxin (δ-toxin-Δ23CPB). While the modification of the N-terminus seemed to affect protein solubility in Escherichia coli, with a much lower soluble protein yield for the chimeras when compared to the WT-CPB, the cholate purified proteins still retained their ability to oligomerize and form pores (Fig 2A and B). 2D class averages of the different mutants showed the lack of the NBP density for the Δ23CPB mutant as well as a lack of a structured N-terminus for the Hla-Δ23CPB and HlgB-Δ23CPB. The N-terminus from δ-toxin in the δ-toxin-Δ23CPB chimera seems to form an NBP similar to the WT-CPB, suggesting that the N-terminus of δ-toxin oligomer might also adopt a β-barrel conformation in its oligomeric form (Figs 2C and EV3A). Interestingly, the ability to form the NBP correlates with the cytotoxic activity. The truncated Δ23CPB, HlgB-Δ23CPB, and Hla-Δ23CPB were inactive, whereas activity was fully restored for the δ-toxin-Δ23CPB (Fig 2D). Figure 2. Investigating the N-terminal domain of different hemolysins Characterization of Clostridium perfringens β-toxin (CPB) constructs with different N-termini by gel electrophoretic analysis under denaturing (A) and native (B) conditions. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gel analysis of CPB samples in cholate (1 μg per lane). CPB samples containing cholate were boiled for 5 min at 95°C (+) or not (−). Coomassie stained Blue native PAGE gel (4–16%) of CPB samples (10 μg per lane) showing oligomer formation for all different N-termini constructs. Alignment of CPB N-terminus with N-termini of different hemolysins and cryo-EM 2D classification and average of N-terminally tagged WT CPB (1), Δ23CPB (2), δ-toxin N-terminus Δ23CPB (3), C-terminally tagged WT CPB (4), Hla N-terminus Δ23CPB (5), and HlgB N-terminus Δ23CPB (6). Viability of HEK 293FT/CD31-GFP cells (transduced with CD31-GFP) as a percentage of untreated control cells after incubation with indicated concentrations of toxins (24 h, 37°C). Data (technical replicates) are represented as means (n = 4) ± SD. Download figure Download PowerPoint Channel properties and dynamics To investigate the mechanical and physical properties of the CPB pore, we carried out atomistic molecular dynamics (MD) simulations of it inserted into a lipid bilayer. The CPB channel features four constriction points in its two β-barrels (Fig 3A). The narrowest points have a ~ 6 Å mean radius and are located within the NBP at the level of the positively charged Arg11 and Lys13. The channel reaches a maximum radius of about 20 Å within the vestibule of the cap. Quantifying the root mean square fluctuation (RMSF) of the toxin atoms revealed that the most flexible regions are the NBP and the intracellular turns of the transmembrane β-barrel (Fig 3B). The NBP, while maintaining its overall structure, can oscillate off-axis, whereas the pore turns can squeeze off a perfectly circular symmetry contributing to reducing the local pore radius (Fig EV4A–C). These observations are consistent with EM data obtained by performing a multibody analysis on the CPB particles and could explain the variability observed previously in the channel conductance (Shatursky et al, 2000; Fig EV2F and Movie EV1). Examining charge distribution in the pore lumen revealed a high density of positive charges inside the β-barrel protrusion due to Arg11 and Lys13 (Fig 3C). Potential mean force (PMF) profiles for Na+, Ca2+, and Cl− estimated via umbrella sampling indicated that this region should constitute an energy barrier for cations (4.6 kcal/mol for Na+). The wide cap vestibule features a balanced amount of positive and negative charges, while the transmembrane β-barrel is overall negatively charged, especially due to Glu152 and Glu162. These amino acids, associated with constriction points, determine a highly negative PMF for cations (as low as −24.6 kcal/mol for Ca2+) and a large 10.2 kcal/mol barrier for Cl− in agreement with previous reports suggesting that the CPB pore is mostly permeable to cations (Shatursky et al, 2000; Manich et al, 2008). Figure 3. CPB dynamics and NBP measurements Average pore radius in MD simulations. Above, regions with mean radius < 8 Å (constriction points) are shown in red, > 8 Å and < 10 Å in orange, and > 10 Å in yellow. In the graph, mean values are shown in red, and standard deviations in gray. Polar amino acids at constriction points are annotated. Clostridium perfringens β-toxin (CPB) root mean square fluctuation (RMSF) averaged over the eight CPB chains and three simulation repeats. Above, most mobile regions are shown in yellow, and least mobile in purple. In the graph, mean RMSF values are shown in red, and their standard deviation in gray. The N-terminal β-barrel Protrusion (NBP) is the most mobile region and, along with the flexible intracellular mount of transmembrane β-barrel, is annotated in the graph. Electrostatic properties of the pore internal cavity. Above, negative regions are shown in red and positive ones in blue. In the graph below, potential mean force profiles along the pore axis for Na+, Ca2+, and Cl− ions are shown. The NBP features a small positive region selective to anions, while the transmembrane β-barrel is expected to be highly selective to cations. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. In silico flexibility of the CPB channelFor each conformation in our Clostridium perfringens β-toxin (CPB) simulation, we extracted the coordinates of Cα at each constriction point and fitted them with an ellipse. The constriction point at the intracellular side of the toxin (residues 143 and 147) is the most dynamic, featuring the largest fluctuations in the fitted ellipse. Time evolution of constriction point surface area and aspect ratio of three consecutive 200 ns simulations. Position of all extracted Cα coordinates of each constriction point, aligned so that the each fitted ellipse is centered at the origin and oriented so that its major semiaxis is parallel to the x-axis. Black ellipses fitted to these points represent the average constriction points shape. Only the constriction point at the intracellular side is noticeably elliptical, with Cα atoms featuring larger deviations from the fitted ellipse. Position of the constriction points shown color coded in the cryo-EM map cross section. Download figure Download PowerPoint Comparison with other hemolysins-like β-PFTs In this study, we explored the structure of the membrane inserted oligomeric CPB and showed that it belongs to a new subclass of hemolysin-like β-PFTs that contain an additional β-barrel domain in its extracellular side. After our first version of the manuscript was released on a preprint server, eight novel enterococcal hemolysin-like β-PFTs were reported (Xiong et al, 2022). Interestingly, the structure of two of them was solved and showed that they also adopt an NBP fold atop the cap domain. A structural comparison shows a remarkable structural similarity suggesting that they also belong to the CPB subfamily of hemolysin-like PFTs (Fig EV5A–C). It is of particular interest that the NBP structure of EPX1 toxin has the same fold as CPB while the NBP of EPX4 toxin is reversed. This supports our mutagenesis results that the NBP β-barrel structure can be highly flexible. A detailed comparison of the N-terminal domains with structures of other oligomeric hemolysins is not possible. The N-terminus of the oligomeric γ-hemolysin is disordered (Yamashita et al, 2011). For NetB, oligomer crystallization was only possible after removing the first 20 amino acids of the protein (Savva et al, 2013). For C. perfringens δ-toxin, only the monomer structure is available (Huyet et al, 2013)(Savva et al, 2013). In δ-toxin soluble monomer, unlike S. aureus Hla, the N-terminus adopts a β-hairpin conformation, which extends halfway along the cap β-sandwich and contacts the pre-stem (Fig EV3A). Because of the high sequence similarity between CPB and C. perfringens δ-toxin (Appendix Figs S1 and S2), it is reasonable to assume that the N-terminus of CPB adopts a similar conformation in the water-soluble monomer. To gain insight into the putative structure of CPB soluble monomer, we made use of recent advances in protein structure prediction (Baek et al, 2021; Jumper et al, 2021). Both Rosetta and AlphaFold generated predictions similar to the structure of δ-toxin monomer (Fig EV3C and D) with an N-terminus folded back and forming a β-hairpin. Click here to expand this figure. Figure EV5. The β-toxin subfamily of hemolysin-like β-pore-forming toxins (PFTs) Side by side comparison of protomer structures of CPB, EPX1 (PDB: 7T4E), and EPX4 (PDB: 7T4D) color coded as in Fig 1 (NBP—green, cap—light blue, rim—dark blue, and stem—red). A magnified view of the NBP region showing that while CPB and EPX1 share the
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