Mondal, AK and Sengupta, N and Singh, M and Biswas, R and Lata, K and Lahiri, I and Dutta, S and Chattopadhyay, K (2022) Glu289 residue in the pore-forming motif of Vibrio cholerae cytolysin is important for efficient β-barrel pore formation. In: Journal of Biological Chemistry, 298 (10).
|
PDF
jou_bio_che_298-10_2022.pdf - Published Version Download (5MB) | Preview |
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 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.
| Item Type: | Journal Article |
|---|---|
| Publication: | Journal of Biological Chemistry |
| Publisher: | American Society for Biochemistry and Molecular Biology Inc. |
| Additional Information: | The copyright for this article belongs to the Authors. |
| Keywords: | Activation analysis; Binding energy; Medical imaging; Monomers; Oligomers; Pore size; Toxic materials, Bacterial toxin; Cryo-EM; Membrane pores; Oligomerizations; Pore forming toxins; Prepore; Proteins structures; Trans-membrane domains; Transmembrane domain; Vibrio cholera cytolysin; Vibrio cholerae, Membranes |
| Department/Centre: | Division of Biological Sciences > Molecular Biophysics Unit |
| Date Deposited: | 13 Oct 2022 08:18 |
| Last Modified: | 13 Oct 2022 08:18 |
| URI: | https://eprints.iisc.ac.in/id/eprint/77284 |
Actions (login required)
 |
View Item |
