Vibrio cholerae cytolysin (VCC), a beta-barrel pore-forming toxin (beta-PFT), induces killing of the target eukaryotic cells by forming heptameric transmembrane beta-barrel pores. Consistent with the beta-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 beta-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 beta-PFT mechanism, it therefore appears that the VCC pre-stem motif plays a critical role toward forming the structural scaffold of the transmembrane beta-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 beta-PFT mode of action. (C) 201 3 Elsevier Inc. All rights reserved.