Abstract
Actinoporins are eukaryotic pore-forming proteins that create 2-nm pores in natural and model lipid membranes by the self-association of four monomers. The regions that undergo conformational change and form part of the transmembrane pore are currently being defined. It was shown recently that the N-terminal region (residues 10-28) of equinatoxin, an actinoporin from Actinia equina, participates in building of the final pore wall. Assuming that the pore is formed solely by a polypeptide chain, other parts of the toxin should constitute the conductive channel and here we searched for these regions by disulfide scanning mutagenesis. Only double cysteine mutants where the N-terminal segment 1-30 was attached to the beta-sandwich exhibited reduced hemolytic activity upon disulfide formation, showing that other parts of equinatoxin, particularly the beta-sandwich and importantly the C-terminal alpha-helix, do not undergo large conformational rearrangements during the pore formation. The role of the beta-sandwich stability was independently assessed via destabilization of a part of its hydrophobic core by mutations of the buried Trp117. These mutants were considerably less stable than the wild-type but exhibited similar or slightly lower permeabilizing activity. Collectively these results show that a flexible N-terminal region and stable beta-sandwich are pre-requisite for proper pore formation by the actinoporin family.
Highlights
We dedicate this report in the memory of Dr Gianfranco Menestrina
Preparation of Double Cysteine Mutants—The wild-type equinatoxin II (EqtII) is produced in the active form in the cytoplasm of E. coli
Mutagenesis of Tryptophan 117—In summary, by the use of double cysteine mutants we have shown that the major conformational rearrangements of EqtII, which occur during pore formation, are restricted to the N-terminal region
Summary
We dedicate this report in the memory of Dr Gianfranco Menestrina He was a brilliant scientist and a true friend. Pore-forming toxins (PFTs) are a very important group of natural toxins [6], and the best studied examples are bacterial PFTs, because they are important virulence factors in human and animal bacterial disease. In recent years they have been used to study fundamental biological processes such as protein-membrane and protein-protein interactions within the lipid membrane milieu and conformational changes associated with the change of environment from polar to hydrophobic as encountered within the core of lipid membranes. Pores of -PFTs are stable structures that resist heat or detergents, ␣-PFT pores are not stable and have provided little structural information
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
