Single-molecule kinetics of pore assembly by the membrane attack complex

Abstract

The membrane attack complex (MAC) is a hetero-oligomeric protein assembly that kills pathogens by perforating their cell envelopes. The MAC is formed by sequential assembly of soluble complement proteins C5b, C6, C7, C8 and C9, but little is known about the rate-limiting steps in this process. Here, we use rapid atomic force microscopy (AFM) imaging to show that MAC proteins oligomerize within the membrane, unlike structurally homologous bacterial pore-forming toxins. C5b-7 interacts with the lipid bilayer prior to recruiting C8. We discover that incorporation of the first C9 is the kinetic bottleneck of MAC formation, after which rapid C9 oligomerization completes the pore. This defines the kinetic basis for MAC assembly and provides insight into how human cells are protected from bystander damage by the cell surface receptor CD59, which is offered a maximum temporal window to halt the assembly at the point of C9 insertion.

Imaging live bacteria at the nanoscale: comparison of immobilisation strategies

“…The size and shape of these rings is consistent with cryoEM and AFM data of the whole MAC pore inserted into lipid bilayers. 40,41 Since the immobilisation of living cells and organisms is vital for physiological AFM experiments, this is not the first study trying to achieve this efficiently. But similar techniques are not always reproducible in different studies.…”

How the Membrane Attack Complex Damages the Bacterial Cell Envelope and Kills Gram‐Negative Bacteria

“…Moreover, Parsons et al [30] did not observe any specific insertion of pores into lipid bilayers with C9 alone by AFM. C5b-8 complexes that incorporate C9 on the bacterial OM are responsible for the bactericidal effects of the MAC, but we cannot exclude that C9 enters the periplasm and forms a pore in the IM.…”

“…[24] Moreover, a high density of C3b on the surface changes the substrate specificity of the convertase from C3 into C5. [29,30] However, it is generally accepted that C5b6 needs to recruit C7 to form a stable C5b7 complex that exposes a lipophilic domain that allows the complex to stably bind to the lipid membranes. Despite the fact that C3b and C5b are highly homologous, C5b is not covalently linked to the target surface.…”

“…Interactions of C5b6 with erythrocyte membranes, [60] lipid bilayers, [29,30] and between C3b and C5 have been reported, suggesting that there could be a surface-bound intermediate state of C5b6. Interactions of C5b6 with erythrocyte membranes, [60] lipid bilayers, [29,30] and between C3b and C5 have been reported, suggesting that there could be a surface-bound intermediate state of C5b6.…”

“…Although this might not affect MAC insertion into artificial lipid membranes with a relatively uniform lipid composition, it might affect insertion into the bacterial OM, which has a more complex lipid composition. [64] Parsons et al [30] recently determined the kinetics of MAC assembly on supported lipid bilayers of E. coli lipid extracts using high-speed AFM. [63] The bacterial OM contains a large amount of LPS of varying www.advancedsciencenews.com www.bioessays-journal.com lengths distributed across the OM.…”