The membrane attack complex (MAC) is a potent killing element of the complement system that assembles sequentially on the membrane of gram-negative bacteria and inserts a large beta barrel. Its importance is highlighted by persistent Neisseria meningitis infections in individuals lacking components of this system. Structurally, the MAC is similar to the CDC class of pore forming toxins from gram-positive bacteria and the MACPF toxin, pleurotolysin, from oyster mushrooms. These toxins contain two helical bundles that flank a beta sheet structure and undergo a conformational change to form the barrel of the pore by inserting two transmembrane hairpins (TMHs). The results herein provide insight into the structure of the MAC and a novel region important for the zinc-mediated polymerisation of C9.
The assembled MAC consists of seven individual components: C5b, C6, C7, C8α, C8β, C8γ, and between 12-18 copies of C9. Interestingly, C9 polymerises independently of these components to form a MAC analogue (polyC9) that can be observed by electron microscopy. A pipeline for purifying high-quality polyC9 has been established for use in cryo-electron microscopy. Furthermore, a surface entropy reduction (SER) algorithm was used to improve crystallisability of the C9 monomer. This approach yielded a mutant that lost its propensity for zinc-induced polymerisation while retaining full activity in the presence of the C5b-8 and shown to assemble on membranes.
This study details a rigorous method for production of polyC9 that yields higher resolution structural information and allow for detailed mutational analysis by cryo-EM. Additionally, biochemical characterisation of the SER mutant has identified a region important for polyC9 formation, by an unknown mechanism that can be rescued in the context of a complete MAC. Taken together these results give new insights into how the MAC assembles to kill bacteria.