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bioRxiv · 10.1101/2022.07.06.498960

The neoepitope of the complement C5b-9 Membrane Attack Complex is formed by proximity of adjacent ancillary regions of C9

Bayly-Jones, C.·Ho, B. H. T.·Lau, C.·Leung, E. W. W.·D'Andrea, L.·Lupton, C. J.·Ekkel, S. M.·Venugopal, H.·Whisstock, J. C.·Mollnes, T. E.·Spicer, B. A.·Dunstone, M. A.
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Abstract

The terminal C5b-9 complement complex (TCC) exists in two forms; the soluble sC5b-9 and the solid-phase inserted Membrane Attack Complex (MAC). The MAC is responsible for forming large {beta}-barrel channels in the membranes of pathogens and can target cells such as gram-negative bacteria. In addition, off-target MAC assembly on endogenous tissue is associated with inflammatory diseases and cancer. Accordingly, a human C5b-9 specific antibody, aE11, has been developed that detects a neoepitope exposed in C9 when it is incorporated into the C5b-9 complex and in polyC9, but not present in the plasma native C9. For nearly four decades aE11 has been routinely used to study complement, MACrelated inflammation, and pathophysiology. However, the identity of this C9 neoepitope remains unknown. Herein, we determined the cryo-EM structure of aE11 in complex with polyC9 at 3.2 [A]resolution. The aE11 binding site revealed that the neoepitope was formed by two separate surfaces on the pore periphery, rather than a conformational change in the protein structure, and is therefore a discontinuous quaternary epitope. These surfaces are contributed by portions of the adjacent TSP1, LDLRA and MACPF domains of two neighbouring C9 protomers. By substituting key antibody interacting residues to the murine orthologue, we validated the unusual binding modality of aE11. Furthermore, our observations indicated that aE11 can recognise a partial epitope in purified monomeric C9, albeit binding was two orders of magnitude weaker. Taken together, our results reveal the basis for MAC recognition by aE11 and that the C9 neoepitope can be formed without substantial conformational rearrangements adding insight into how aE11 is used to quantify MAC formation in disease.

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Bayly-Jones, C., Ho, B. H. T., Lau, C., Leung, E. W. W., D'Andrea, L., Lupton, C. J., Ekkel, S. M., Venugopal, H., Whisstock, J. C., Mollnes, T. E., Spicer, B. A., Dunstone, M. A.. 2022-07-06. The neoepitope of the complement C5b-9 Membrane Attack Complex is formed by proximity of adjacent ancillary regions of C9. https://doi.org/10.1101/2022.07.06.498960

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