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.

Fig. 1

Formation of MAC on bacterial model membranes. a 3D AFM representation of the endpoint MAC pore structure on supported bilayers composed of an E. coli lipid extract. Inset: Schematic of the MAC, self-assembled from complement proteins C5b6, C7, C8 and C9, embedded within a lipid membrane. b Zoom-in of a single MAC pore (marked with an asterisk in a). c Negative-stain EM of E. coli lipid bilayers deposited on silicon dioxide grids, sequentially incubated with complement proteins C5b6, C7, C8 and C9, resulting in characteristic MAC rings observed in the bilayer membrane. Scale bars: a, c 50 nm, b 25 nm. Height scale (scale inset in b), a, b 20 nm

Fig. 2

C5b-7 initiates MAC formation and recruits C8 and C9 directly from solution. Binding of complement components C5b6, C7 and C8 to supported bacterial model membranes containing PG lipids, as observed by AFM and QCM-D. a AFM images increasingly show protruding features upon addition of C5b6, C7 and C8 to a bilayer formed of DOPE:DOPG (50:50 mol%). Scale bar: 200 nm, height scale: 1 nm. b QCM-D binding assay following the addition of complement proteins, in reverse sequence followed by the forward sequence, to a lipid bilayer formed of DOPC:DOPE:DOPG (47.5:47.5:5 mol%) on a silicon dioxide coated QCM-D sensor. In isolation, complement proteins C7, C8 and C9 do not bind to the membrane, requiring C5b6 to initiate the interaction. Distinct steps in both frequency (Δf) and dissipation (ΔD) upon sequential addition of C5b-7, C8 and C9 demonstrate protein binding to the membrane; wash steps (denoted by dotted red lines) between additions do not lead to a reduction in the signal size, revealing that the protein binding is stable. The dissipation shift increases upon binding, reflecting the relative softness of the protein complexes in the lipid bilayer film

Fig. 3

AFM shows that initial insertion of C9 is rate limiting in MAC assembly. a Time-lapse AFM imaging of MAC assembly on supported bilayers formed of E. coli lipid extract (see Supplementary Movie 1 for full data set). MAC assembly events initiate at distinct time points, with each MAC pore rapidly completing within a few frames; this implies a rate-limiting-step corresponding to initial insertion of C9. Scale bar: 200 nm; height scale (colour scale bar inset in image for t = 0 min): 40 nm. b Number of MAC assemblies detected (see Methods) with time, indicated as grey squares; the solid red line represents fitting with the function A(1–exp(–t/τ_init)), where τ_init = 912 ± 32s represents the characteristic time of MAC pore appearance, highlighted by the dashed green lines. The completion time of each individual MAC is much shorter than τ_init

Fig. 4

Real-time imaging of C9 oligomerization. a AFM image sequence of MAC assembly, cropped from data shown in Fig. 3 and Supplementary Movie 1. C9 oligomerization completes within frames shown (104 s, 6.5 s/frame; 0 s is here approximately defined by the frame preceding detection of the growing MAC). Scale bar: 30 nm, height scale (see colour scale bar in Fig. 1): 16 nm. b The normalised average frame height versus time for a single pore forming event (corresponding to event shown in a), plotted here as a measure for completion of MAC assembly. The red line represents a sigmoidal fit to the data, as a generic and mathematically convenient description of a smooth transition between pore absence and pore completion. The C9 oligomerization time is determined from the width of the transition, highlighted by green dashed lines (see Supplementary Fig. 12 for details). c Distribution of oligomerization times, extracted from n = 33 isolated pore forming events in 6 independent experiments

Fig. 5

Schematic of MAC assembly. Upon formation, C5b6 templates the assembly, recruiting C7 and C8 to the nascent MAC. The C5b-7 complex is irreversibly bound to the membrane. Insertion of the first C9 is the rate limiting step in the MAC assembly; once this barrier has been overcome, subsequent C9 binding/insertion occurs more than two orders of magnitude faster than the initial C9 insertion