Journal: Commun Biol / Year: 2023 Title: The neoepitope of the complement C5b-9 Membrane Attack Complex is formed by proximity of adjacent ancillary regions of C9. Authors: Charles Bayly-Jones / Bill H T Ho / Corinna Lau / Eleanor W W Leung / Laura D'Andrea / Christopher J Lupton / Susan M Ekkel / Hariprasad Venugopal / James C Whisstock / Tom E Mollnes / ...Authors: Charles Bayly-Jones / Bill H T Ho / Corinna Lau / Eleanor W W Leung / Laura D'Andrea / Christopher J Lupton / Susan M Ekkel / Hariprasad Venugopal / James C Whisstock / Tom E Mollnes / Bradley A Spicer / Michelle A Dunstone / Abstract: The Membrane Attack Complex (MAC) is responsible for forming large β-barrel channels in the membranes of pathogens, such as gram-negative bacteria. Off-target MAC assembly on endogenous tissue is ...The Membrane Attack Complex (MAC) is responsible for forming large β-barrel channels in the membranes of pathogens, such as gram-negative bacteria. 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, but not present in the plasma native C9. For nearly four decades aE11 has been routinely used to study complement, MAC-related inflammation, and pathophysiology. However, the identity of C9 neoepitope remains unknown. Here, we determined the cryo-EM structure of aE11 in complex with polyC9 at 3.2 Å resolution. The aE11 binding site is formed by two separate surfaces of the oligomeric C9 periphery 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, aE11 can recognise a partial epitope in purified monomeric C9 in vitro, albeit weakly. Taken together, our results reveal the structural basis for MAC recognition by aE11.
Entire : Quaternary complex of aE11 Fab and polyC9
Entire
Name: Quaternary complex of aE11 Fab and polyC9
Components
Complex: Quaternary complex of aE11 Fab and polyC9
Protein or peptide: aE11 Fab VH
Protein or peptide: aE11 Fab VL
Protein or peptide: Complement component C9
Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Ligand: beta-D-mannopyranose
Ligand: CALCIUM ION
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Supramolecule #1: Quaternary complex of aE11 Fab and polyC9
Supramolecule
Name: Quaternary complex of aE11 Fab and polyC9 / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#3 Details: Fab fragment of aE11 generated by proteolytic cleavage of aE11 IgG antibody, in complex with recombinant human C9 incubated at 37 degrees Celsius to form homo-oligomeric C9.
Source (natural)
Organism: Homo sapiens (human)
Molecular weight
Theoretical: 2.6 MDa
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Macromolecule #1: aE11 Fab VH
Macromolecule
Name: aE11 Fab VH / type: protein_or_peptide / ID: 1 / Details: Papain-treated IgG / Number of copies: 2 / Enantiomer: LEVO
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
-
Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 0-40 / Average exposure time: 16.0 sec. / Average electron dose: 52.4 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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