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| Title | Glu289 residue in the pore-forming motif of Vibrio cholerae cytolysin is important for efficient β-barrel pore formation. |
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| Journal, issue, pages | J Biol Chem, Vol. 298, Issue 10, Page 102441, Year 2022 |
| Publish date | Aug 31, 2022 |
 Authors | Anish Kumar Mondal / Nayanika Sengupta / Mahendra Singh / Rupam Biswas / Kusum Lata / Indrajit Lahiri / Somnath Dutta / Kausik Chattopadhyay /  |
| PubMed Abstract | Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging β-barrel pore-forming toxin. Upon binding to the target membranes, VCC monomers first assemble into oligomeric prepore intermediates and ...Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging β-barrel pore-forming toxin. Upon binding to the target membranes, VCC monomers first assemble into oligomeric prepore intermediates and subsequently transform into transmembrane β-barrel pores. VCC harbors a designated pore-forming motif, which, during oligomeric pore formation, inserts into the membrane and generates a transmembrane β-barrel scaffold. It remains an enigma how the molecular architecture of the pore-forming motif regulates the VCC pore-formation mechanism. Here, we show that a specific pore-forming motif residue, E289, plays crucial regulatory roles in the pore-formation mechanism of VCC. We find that the mutation of E289A drastically compromises pore-forming activity, without affecting the structural integrity and membrane-binding potential of the toxin monomers. Although our single-particle cryo-EM analysis reveals WT-like oligomeric β-barrel pore formation by E289A-VCC in the membrane, we demonstrate that the mutant shows severely delayed kinetics in terms of pore-forming ability that can be rescued with elevated temperature conditions. We find that the pore-formation efficacy of E289A-VCC appears to be more profoundly dependent on temperature than that of the WT toxin. Our results suggest that the E289A mutation traps membrane-bound toxin molecules in the prepore-like intermediate state that is hindered from converting into the functional β-barrel pores by a large energy barrier, thus highlighting the importance of this residue for the pore-formation mechanism of VCC. |
 External links | J Biol Chem / PubMed:36055404 / PubMed Central |
| Methods | EM (single particle) |
| Resolution | 4.7 - 6.4 Å |
| Structure data |  EMDB-33215: Cryo-EM reconstruction of complete transmembrane channel E289A mutant Vibrio cholerae Cytolysin  EMDB-33219: Cryo-EM reconstruction of partial transmembrane channel E289A mutant Vibrio cholerae Cytolysin |
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Vibrio cholerae (bacteria)