The membrane attack complex (MAC) is an important terminal immune effector that targets a range of pathogens. The MAC forms large heterogeneous pores on these pathogens allowing direct delivery of cell wall digesting lysozymes and disruption of membrane gradients. MAC formation requires the sequential assembly of plasma C5b, C6, C7, C8 (a heterotrimer C8α, C8β, C8γ) and then ~18 units of C9 that form the final transmembrane pore. In vitro, C9 can also homo-oligomerise to form the MAC-mimic, polyC9, which can be used to study the structure of the MAC. As such, polyC9 can be used to study how the a commercially important anti-neoantigen antibody, aE11, used for routine histology research, recognises only C9 within the MAC and not the soluble C9. What change does aE11 recognise in the MAC? Is it conformational and does it inform pore assembly? Since aE11 is obtained from a commercial source, small quantities limit the use of common approaches to structure determination.
Here we determine the binding mode of the neoantigen antibody by determining the 4.3 Å cryo-EM reconstruction of polyC9 in complex with the aE11 Fab’. Our findings indicate that the neoantigen consists of an inter-subunit domain-domain contact between the MACPF and TSP domains of neighbouring C9. As the structure and sequence of aE11 is unknown, to confidently assign residue-residue interactions, micro-electron diffraction is being employed to obtain a high-resolution crystal structure of the aE11 Fab.
Our results indicate novel surface topology defined only upon MAC formation is responsible for the neoantigen formation. Two independent loops present in MACPF and TSP domains of adjacent C9 protomers define the discontinuous epitope. This study represents the first insights into the aE11 binding mechanism, highlights the applicability of cryoEM and cryoMED for protein-limited samples and informs future studies of MAC structure and formation.