MACPF/CDC pore forming proteins show a unique ability to self-assemble from soluble monomeric proteins into oligomeric rings that change conformation and insert into the target cell membrane. The MACPF/CDC family has been shown to form giant beta-barrel pores that oligomerise, typically using the same unit, and are capable of passive transport of whole soluble proteins across lipid membranes.
The current hypothesis suggests that oligomer assembly is mediated by: 1) binding to a lipid membrane and, 2) planar diffusion upon the target membrane. However, to date, this model is only consistent with pore forming proteins, such as CDCs and perforin, that have dedicated membrane binding domains. In contrast, the proteins of the Membrane Attack Complex (MAC) lack any membrane binding region, but can target a wide range of eukaryotic and bacterial surfaces. However, this precludes the MAC using membrane binding for assembly.
Here we show the first X-ray structure of the soluble C9 component of the MAC and compare this to the near atomic single particle cryo-EM structure of the 22-subunit polyC9. Together these structures show that a 22 amino acid region within the TMH1 loop obstructs oligomer assembly at the oligomer interface. Disulphide trap mutants demonstrate that TMH1 obstructs elongation and need to move position prior to binding of the next C9 unit in the oligomer assembly pathway.
These results challenge the existing dogma in MACPF/CDC pore assembly—that assembly is dependent on membrane binding and requiring lateral diffusion. Instead, movements of the TMH1 and possibly TMH2 of C9 drive MAC assembly. Accordingly, these results explain how the C9 component is able to self-oligomerise into the MAC without the need for lateral diffusion on a membrane and may explain the MAC’s role in assembling on highly variable chemistries at the membrane surface of invading pathogens.