Single-particle cryo-EM reveals conformational variability of the oligomeric VCC β-barrel pore in a lipid bilayer

Abstract

Vibrio cholerae cytolysin (VCC) is a water-soluble, membrane-damaging, pore-forming toxin (PFT) secreted by pathogenic V. cholerae, which causes eukaryotic cell death by altering the plasma membrane permeability. VCC self-assembles on the cell surface and undergoes a dramatic conformational change from prepore to heptameric pore structure. Over the past few years, several high-resolution structures of detergent-solubilized PFTs have been characterized. However, high-resolution structural characterization of small β-PFTs in a lipid environment is still rare. Therefore, we used single-particle cryo-EM to characterize the structure of the VCC oligomer in large unilamellar vesicles, which is the first atomic-resolution cryo-EM structure of VCC. From our study, we were able to provide the first documented visualization of the rim domain amino acid residues of VCC interacting with lipid membrane. Furthermore, cryo-EM characterization of lipid bilayer–embedded VCC suggests interesting conformational variabilities, especially in the transmembrane channel, which could have a potential impact on the pore architecture and assist us in understanding the pore formation mechanism.

Cryo-EM-based structural insights into supramolecular assemblies of γ-Hemolysin from Staphylococcus aureus reveal the pore formation mechanism

“…The copyright holder for this preprint this version posted September 15, 2022. ; https://doi.org/10.1101/2022.09.14.507916 doi: bioRxiv preprint Recently, several high resolution cryo-EM-based structural studies have targeted PFTs in detergent 28,32,45 , lipid nanodiscs 46,47 , and liposome environment 29,[48][49][50] to gain structural insights into key residues proximal to the lipid environment. However, this is the first such study on a bicomponent β-PFT in which we provided structural insights into the critical lipidbinding residues.…”

“…Several recent cryo-EM based structural studies have attempted to illuminate the mechanism of oligomeric assembly and pore formation by PFTs in a near-native environment. In addition to mapping the atomic structures of most stable oligomeric states, solution state studies so far also have had the advantage of trapping intermediate pre-pore assemblies 28,29 , capturing the pore complex in a model membrane [29][30][31][32] , as well as unearthing rather uncommon oligomeric stoichiometries 33,34 . Therefore, we chose to characterize the solution state structure of bicomponent PFT, γ-HL in a physiologically relevant membrane microenvironment by employing cryo-EM single particle analysis.…”

Cryo-EM reveals the membrane binding phenomenon of EspB, a virulence factor of the Mycobacterial Type VII secretion system

“…However, we were unable to visualize the distinct bilayer of PA vesicles in the class averages corresponding to the side view of EspB 1-332 (Figure 4A). Unlike transporters or various pore-forming toxins (28,29), which embed firmly in between the two leaflets of membrane-bilayer, it is possible that EspB 1-332 , despite having spontaneous binding affinity to PA bilayer, may adhere superficially to the lipid surface. This could potentially lead to averaging out of the signal corresponding to lipid density during the process of particle alignment.…”

Glu289 residue in the pore-forming motif of Vibrio cholerae cytolysin is important for efficient β-barrel pore formation

“…Vibrio cholerae cytolysin (VCC) is a distinct member in the β-PFT family ( 4 , 5 ). VCC is produced by most of the pathogenic strains of V. cholerae , a Gram-negative bacterium that causes severe diarrheal disease cholera ( 6 ).…”

“…Nine out of these charged residues get positioned into the water-filled lumen of the β-barrel pore. These nine residues are as follows: (1) glutamate at position 281 (E281), (2) lysine at position 283 (K283), (3) glutamate at position 289 (E289), ( 4 ) glutamate at position 297 (E297), ( 5 ) aspartate at position 301 (D301), ( 6 ) lysine at position 304 (K304), (7) lysine at position 306 (K306), (8) glutamate at position 308 (E308), and (9) arginine at position 310 (R310) ( Fig. 1 , A – E ).…”