Pleurotolysin is a two-component fungal pore-forming toxin that is a member of the MACPF/CDC pore forming toxin superfamily. The MACPF/CDC pore-forming family includes perforin, from the human immune system and listeriolysin O, a key virulence factor of Listeria monocytogenes. Whilst it is known that MACPF/CDC toxins can form giant membrane embedded β-barrels, little is known about how MACPF toxins initially recognise and bind target membranes. In the case of pleurotolysin, the membrane recognition/binding component (PlyA) binds specifically to sphingomyelin which facilitates docking of the pore-forming component, PlyB.

 Here we put forward evidence that PlyA can exist in a rapid equilibrium between monomer and dimer in solution before contact with the target membrane. To demonstrate this we used analytical size exclusion chromatography, Multi Angle Light Scattering (MALS), X-ray crystallography and analytical ultracentrifugation. We further investigated the putative dimer interface using site-directed mutagenesis including the effect upon haemolytic activity and red blood cell binding. Of particular note, a single point mutation (W6E) was identified at the putative dimerisation interface which was capable of disrupting this equilibrium and resulting in a loss of membrane binding and reduced haemolytic activity.

 These results support the model for the pleurotolysin pore complex and show that PlyA dimerisation occurs before membrane binding. This research also suggests that membrane binding is stabilised by the PlyA dimer allowing for pore formation. This is a membrane binding method that may be used by other families of toxins and membrane binding proteins.