title: Single point mutation in Vibrio cholerae cytolysin compromises the membrane pore-formation mechanism of the toxin.
abstract: Vibrio cholerae cytolysin (VCC) belongs to the family of β-barrel pore-forming protein toxins. VCC is secreted by the bacteria as water-soluble monomers, which upon binding to target eukaryotic cells form transmembrane heptameric β-barrel channels. High-resolution 3D structures are described both for the water-soluble monomeric form and the transmembrane oligomeric pore; albeit that our understanding of the mechanistic details of the membrane pore-formation process remains incomplete. Here, we report the characterization of a nonfunctional VCC variant harboring a single point mutation of Ala425Val positioned within a potential membrane-interacting loop in the VCC structure. The mutation appears to affect interaction of the toxin with erythrocytes as well as cholesterol-containing liposome membrane, without affecting the oligomerization ability of the membrane-bound toxin molecules. The membrane-bound oligomers formed by this VCC mutant do not appear to represent the functional pore assembly of the toxin; rather, such assembly could be considered as being trapped in an abortive, nonfunctional oligomeric state. Our results suggest that the Ala425Val mutation in VCC critically compromises its cholesterol-dependent membrane-interaction mechanism and also abrogates the process of functional membrane pore formation by the toxin.
authors.affiliation: Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Mohali, SAS Nagar, Manauli, Punjab, India.
authors.forename: Karan
authors.initials: K
authors.lastname: Paul
authors.forename: Kausik
authors.initials: K
authors.lastname: Chattopadhyay
mesh_terms: D015153:Blotting, Western; D002784:Cholesterol / Q000378:metabolism; D003603:Cytotoxins / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D004910:Erythrocyte Membrane / Q000187:drug effects* / Q000378:metabolism*; D005434:Flow Cytometry; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D008081:Liposomes; D054353:Perforin / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology*; D010713:Phosphatidylcholines / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics*; D011487:Protein Conformation; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014734:Vibrio cholerae / Q000378:metabolism*
chemical_list: D003603:Cytotoxins; D008081:Liposomes; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D054353:Perforin; C024990:asolectin; D002784:Cholesterol