Stepwise conformational changes of PLY toxin during pore formation shown for one monomer (above, side view) and for three neighbouring monomers (below, view from the pore centre). In the first step, soluble PLY monomers attach to the surface of cholesterol-containing cell membranes via the conserved D4 undecapeptide (Figure 2C) to form circular oligomers of the early prepore. A 90° rotation of D2 moves D1 and D3 towards the membrane in the late prepore. Helix bundles HB1 and HB2 are poised above the membrane surface to refold into 85 Å β-hairpins HP1 and HP2. In the final step of pore formation, both hairpins traverse the hydrophobic membrane core and assemble into a 168-strand, 260 Å β-barrel. Reorganization of the PLY monomer exposes numerous charges on the inside of the β-barrel (Figure 4) that would destabilize the lipid bilayer and repel membrane lipids, resulting in pore opening and cell lysis.