Pore-forming proteins are a ubiquitous class of molecule that function in a variety of roles
ranging from cellular attack to cellular defence. Despite a range of sizes and architectures, the
majority of pore-forming proteins follow the same general mechanism of action, whereby
soluble monomers, secreted by the host cell, bind target membranes and oligomerise to form
transmembrane pores. One class of pore-forming proteins is the membrane attack
complex/perforin-like (MACPF) superfamily, which has members that function in both immune
and pathological roles. Two members of this family are the bacterial toxin intermedilysin (ILY)
and the human immune pore the membrane attack complex (MAC). With roles in highly
diverse processes, these two MACPF proteins are related through their regulation by the
human immune receptor CD59. This small surface receptor, found on a wide range of human
tissues, contributes to a variety of human diseases on dysregulation. Using a range of
biophysical and structural techniques, the work presented in this thesis is focused on
increasing our understanding of how these two disparate yet related pore-forming proteins
function. For ILY, investigation into the structural transitions associated with cholesterol and
CD59 indicated that CD59 is the main driver behind the key conformation changes required
for pore formation and was hypothesised to set the specific curvature of the ILY pore.
Additionally, a novel disulphide-locked mutant which was unable to form membrane inserted
pores was shown to form a homogeneous 35-mer, highlighting it as a promising candidate for
future structural studies. In conjunction to work on ILY, analysis of the mechanism of pore
formation by the MAC via cryo-electron microscopy exhibited its unique process of pore
closure, a result of the heterogeneous nature of this complex. Building on a previously
published structure, the resolution of both the complete MAC structure and 2 conformational
intermediates was achieved, as well as the first high-resolution map of the membrane-inserted
form of the major MAC constituent C9. These maps formed the basis for both flexible fitting
and atomic modelling of all 5 MAC components, providing new insights into how the MAC is
formed. Understanding how these pore-forming proteins are assembled will be key in aiding
design of new and exciting therapeutics aimed at regulating these important transmembrane
complexes.