Members of the membrane attack complex/perforin (MACPF) superfamily of pore-forming proteins are characterised by a common three-dimensional fold able to puncture lipid membranes. They are found in bacterial and eukaryotes, and include immune effectors, toxins and pathogenic virulence factors. Their conserved pore-forming domain follows the same mechanism whereby two bundles of α-helices unfurl into membrane-spanning β-hairpins.
This thesis provides insights into the effects of MACPF proteins on biological membranes.
Coarse-grain molecular dynamics simulations of the membrane attack complex (MAC) bound to its inhibitor CD59 reveal protein-lipid interactions and local changes in membrane
thickness. These may serve as signals to recruit CD59 or the molecular machinery for MAC clearance. Some bacterial MACPF proteins called cholesterol-dependent cytolysins (CDCs) hijack CD59 on human cells as part of their pore formation pathway. Atomistic simulations of CD59 in a lipid bilayer show that it samples various orientations relative to the membrane, dictating whether its binding site is available for engaging MAC or CDCs, and thus for inhibiting or promoting pore formation. CDCs assemble on cholesterol-rich lipid membranes and undergo sequential conformational changes to puncture bilayers. Site-directed mutagenesis of two CDCs reveals that an amphipathic helix in the pore-forming helical bundles is responsible for tuning the lytic activity of these proteins. Understanding the molecular basis for the function of this helix
will require the high-resolution structure of a CDC late prepore intermediate. The first steps
towards solving this structure by cryo-electron microscopy are presented in this thesis.