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 know that MACPF/CDC toxins can form giant membrane embedded beta-barrels, less is know about how these proteins initially recognise and bind target membranes. Moreover we know very little about how other pore forming toxins such as actinoporins from sea anemones, bind to membranes.
In the case of pleurotolysin, the membrane recognition/binding component (PlyA) binds specifically to sphingomyelin which then allows docking of the pore forming component, PlyB. Using the pleurotolysin toxin we can study membrane recognition and binding without triggering pore formation. Recent single particle cryo-EM data of the pore complex suggest that the functional conformation of PlyA is a V- shaped dimer. Furthermore, a V-shaped dimer is observed in two different crystal structures of PlyA. However it is unknown whether this PlyA dimer is formed in solution before membrane binding or upon membrane binding.
Here we put forward evidence that PlyA can exist in an equilibrium between monomer and dimer in solution before contact with the target membrane. To demonstrate this we used analytical size exclusion chromatography and multi angle light scattering (MALS). We further investigate the putative dimer interface using site-directed mutagenesis. Some mutants show perturbed haemolytic activity and red blood cell binding although this work is ongoing. To date, our results validate the single particle cryo-EM model for pleurotolysin and suggest that PlyA dimerisation occurs before membrane binding. Future investigation of the dimerisation properties include the use of analytical ultracentrifugation.