Structural basis for membrane attack complex inhibition by CD59

CD59 is an abundant immuno-regulatory receptor that protects human cells from damage during complement activation. Here we show how the receptor binds complement proteins C8 and C9 at the membrane to prevent insertion and polymerization of membrane attack complex (MAC) pores. We present cryo-electron microscopy structures of two inhibited MAC precursors known as C5b8 and C5b9. We discover that in both complexes, CD59 binds the pore-forming β-hairpins of C8 to form an intermolecular β-sheet that prevents membrane perforation. While bound to C8, CD59 deflects the cascading C9 β-hairpins, rerouting their trajectory into the membrane. Preventing insertion of C9 restricts structural transitions of subsequent monomers and indirectly halts MAC polymerization. We combine our structural data with cellular assays and molecular dynamics simulations to explain how the membrane environment impacts the dual roles of CD59 in controlling pore formation of MAC, and as a target of bacterial virulence factors which hijack CD59 to lyse human cells

Zeta potential in intact carbonates at reservoir conditions and its impact on oil recovery during controlled salinity waterflooding

It is well known that oil recovery from carbonate reservoirs can be increased by modifying the injected brine composition in a process ‘controlled salinity water-flooding’ (CSW). However, the mineral- to pore- scale processes responsible for improved oil recovery (IOR) during CSW remain ambiguous and there is no method to predict the optimum CSW composition for a given crude-oil-brine rock system. Here we report the first integrated experimental measurements of zeta potential and IOR during CSW obtained at reservoir conditions. The zeta potential is a measure of the electrical potential at mineral-brine and oil-brine interfaces and controls the electrostatic forces acting between these interfaces. We find that the measured zeta potential in clean samples saturated with formation brine is typically positive and becomes more negative with brine dilution irrespective of temperature. After aging and wettability alteration, the zeta potential changes and we suggest a more positive zeta potential indicates a positive zeta potential at the oil-brine interface and vice-versa. Injecting low salinity brine yields IOR when the oil-brine zeta potential is identified to be negative, but no response when it is identified to be positive, consistent with the hypothesis that IOR during CSW is caused by an increase in the repulsive electrostatic force acting between mineral-brine and oil-brine interfaces. We suggest that the optimum brine composition for IOR during CSW should be chosen to yield the largest change in zeta potential at the mineral-brine interface with opposing polarity to the oil-brine interface and can be determined using the experimental method reported here

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

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.Open Acces