title: Pre-pore oligomer formation by Vibrio cholerae cytolysin: insights from a truncated variant lacking the pore-forming pre-stem loop.
abstract: Vibrio cholerae cytolysin (VCC), a β-barrel pore-forming toxin (β-PFT), induces killing of the target eukaryotic cells by forming heptameric transmembrane β-barrel pores. Consistent with the β-PFT mode of action, binding of the VCC toxin monomers with the target cell membrane triggers formation of pre-pore oligomeric intermediates, followed by membrane insertion of the β-strands contributed by the pre-stem motif within the central cytolysin domain of each protomer. It has been shown previously that blocking of membrane insertion of the VCC pre-stem motif arrests conversion of the pre-pore state to the functional transmembrane pore. Consistent with the generalized β-PFT mechanism, it therefore appears that the VCC pre-stem motif plays a critical role toward forming the structural scaffold of the transmembrane β-barrel pore. It is, however, still not known whether the pre-stem motif plays any role in the membrane interaction process, and subsequent pre-pore structure formation by VCC. In this direction, we have constructed a recombinant variant of VCC deleting the pre-stem region, and have characterized the effect(s) of physical absence of the pre-stem motif on the distinct steps of the membrane pore-formation process. Our results show that the deletion of the pre-stem segment does not affect membrane binding and pre-pore oligomer formation by the toxin, but it critically abrogates the functional pore-forming activity of VCC. Present study extends our insights regarding the structure-function mechanism associated with the membrane pore formation by VCC, in the context of the β-PFT mode of action.
authors.affiliation: Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Mohali, Sector 81, SAS Nagar, Manauli 140306, Punjab, India.
authors.forename: Karan
authors.initials: K
authors.lastname: Paul
authors.affiliation: Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Mohali, Sector 81, SAS Nagar, Manauli 140306, Punjab, India. Electronic address: kausik@iisermohali.ac.in.
authors.forename: Kausik
authors.initials: K
authors.lastname: Chattopadhyay
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002462:Cell Membrane / Q000737:chemistry; D002463:Cell Membrane Permeability; D006801:Humans; D008958:Models, Molecular; D054353:Perforin / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014734:Vibrio cholerae
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D054353:Perforin
keywords: Bacterial protein toxin; CD; FRET; PFT; Pore-forming toxin; VCC; Vibrio cholerae cytolysin; circular dichroism; fluorescence resonance energy transfer; pore-forming toxin