Disentangling the structural and cellular driving forces of CD59 inhibition of membrane attack complex pore formation

Abstract

The complement Membrane Attack Complex (MAC) is an evolutionary ancient immune effector which deposits on target membranes to lyse cells. MAC is non-selective and is directed to pathogens by upstream complement proteins. However, this lack of specificity can lead to bystander damage of healthy tissues during an immune response. To combat this, host tissues express CD59, a small GPI-anchored protein which directly inhibits MAC pore formation stopping damage to the membrane. CD59 provides critical regulation of MAC, changes to which can have devastating clinical consequences. Over-expression of CD59 confers resistance of tumours to monoclonal antibody therapeutics which activate MAC as a mechanism of tumour killing; whereas loss of CD59 expression can lead to uncontrolled lysis of red blood cells in the rare, but potentially lethal condition, Paroxysmal Nocturnal Haemoglobinuria. Despite clear clinical interest, complex and contradictory literature studies mean a molecular mechanism and exact identification of MAC binding sites remain unknown. This thesis brings together novel advances in sample preparation and image processing in Electron cryo-Microscopy to discover the mechanism of inhibition. The work presented here unambiguously identifies two independent binding sites on CD59 for the transmembrane hairpins of C8a and C9 altering their trajectories such that membrane insertion is no longer favoured. Furthermore, these structures demonstrate CD59 has only an indirect effect on C9 polymerisation. The structures presented are, to our best knowledge, the first high resolution structures of a MAC assembly solved in a membrane environment and forms the basis for understanding the role of the membrane in MAC inhibition. To test these new hypotheses, this thesis also details the early steps taken to set up a robust complement activation procedure to study the cellular control of MAC. Together, these data provide a significant step forward in the understanding of CD59 regulation of MAC which has implications for the design of new compounds to overcome the impact of CD59-related therapeutic resistance.