Activation of the terminal complement pathway results in the formation of a large, multicomponent pore-forming protein known as the membrane attack complex (MAC). Furthermore, the MAC is a member of the MACPF/CDC superfamily of pore forming toxins which include perforin from the human immune system and listeriolysin O, a virulence factor of Listeria monocytogenes. Members of this family form large complexes on lipid membranes that assemble into ring like structures and transition into membrane embedded beta barrels. The MAC is an important part of the innate immune system, and eliminates gram-negative bacteria from the body upon insertion of the pore. 

It is known that a single membrane attack complex consists of a number of protein components: C5b, C6, C7, C8α, C8β, C8γ, and between 12-18 copies of C9. While the structure of a few of these MAC proteins have been solved, little is known about the overall architecture of the MAC, especially how it transitions from a soluble to membrane-inserted form. Interestingly, C9 alone can polymerise in the presence of Zn²⁺ to form a tubular structure (poly-C9) which resembles the structure of membrane-inserted MAC.

Here, we report the characterisation of a mutated C9 that has lost the propensity to form poly-C9 in the presence of Zn²⁺. Further, this mutant is SDS-soluble where most pore-forming toxins, including CDCs and perforin, are resistant to SDS. Despite these differences, the mutant retains full lytic activity in the presence of C9-depleted serum, compared to plasma-purified C9. These results indicate that the mutated C9 is both folded in the correct conformation and capable of forming a complex with the MAC. Using this mutant in the context of ghost membranes we will assess whether this complex correlates with a typical membrane inserted MAC. Altogether these results improve our understanding of MAC assembly and poly-C9 formation.