The Solvent-Exposed C-Terminus of the Cytolysin A Pore-Forming Toxin Directs Pore Formation and Channel Function in Membranes.
Pore-forming toxins (PFTs) bind to cell membranes and form nanoscale pores that allow leakage of cellular components, resulting in cell death. The water-soluble, monomeric form of these toxins shows a dramatic conformational change during pore formation, as exemplified by crystal structures of the monomer and functional pore of cytolysin A (ClyA). The solvent-exposed, C-terminal residues of the protein are essential for activity, but the mechanism by which this region regulates pore formation remains unknown. We show here that deletion of the C-terminus of ClyA did not alter its ability to bind to the membrane or oligomerize in detergent. However, the truncated toxin lysed erythrocytes poorly, was more susceptible to proteolysis and thermal unfolding, and showed low calcein leakage from small unilamellar vesicles. Using fully atomistic molecular dynamics (MD) simulations, we find that deletion of C-terminal residues from the ClyA monomer significantly altered stability and unfolding trajectories in the transmembrane N-terminal helix, a region that is pivotal in maintaining the structural integrity of the helical bundle. MD simulations of pores with or without the C-terminus showed minor differences, implying that if oligomerization could be induced prior to the addition to vesicles, then an active pore could be generated. Via generation of oligomers in a detergent prior to the addition to vesicles, the truncated toxin could induce calcein leakage from vesicles, albeit to a lower extent. Therefore, regions of pore-forming toxins, not directly involved in the pore structure, are not passive players but have important roles in undergoing the transition through intermediary steps leading to successful pore formation in a membrane environment.
Centre for Biosystems Science and Engineering, ‡Department of Chemical Engineering, and §Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science , Bangalore 560012, India.
Pradeep
P
Sathyanarayana
Centre for Biosystems Science and Engineering, ‡Department of Chemical Engineering, and §Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science , Bangalore 560012, India.
Rajat
R
Desikan
Centre for Biosystems Science and Engineering, ‡Department of Chemical Engineering, and §Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science , Bangalore 560012, India.
K Ganapathy
KG
Ayappa
Centre for Biosystems Science and Engineering, ‡Department of Chemical Engineering, and §Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science , Bangalore 560012, India.
Sandhya S
SS
Visweswariah