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Title: Mechanistic insights in to pore formation by the MACPF superfamily
Author: Boyd, Courtney Margaret
ISNI:       0000 0004 7969 8447
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2018
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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.
Supervisor: Bubeck, Doryen Sponsor: Biotechnology and Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral