Structure of the poly-C9 component of the complement membrane attack complex

Abstract

The membrane attack complex (MAC)/perforin-like protein complement component 9 (C9) is the major component of the MAC, a multi-protein complex that forms pores in the membrane of target pathogens. In contrast to homologous proteins such as perforin and the cholesterol-dependent cytolysins (CDCs), all of which require the membrane for oligomerisation, C9 assembles directly onto the nascent MAC from solution. However, the molecular mechanism of MAC assembly remains to be understood. Here we present the 8 Å cryo-EM structure of a soluble form of the poly-C9 component of the MAC. These data reveal a 22-fold symmetrical arrangement of C9 molecules that yield an 88-strand pore-forming β-barrel. The N-terminal thrombospondin-1 (TSP1) domain forms an unexpectedly extensive part of the oligomerisation interface, thus likely facilitating solution-based assembly. These TSP1 interactions may also explain how additional C9 subunits can be recruited to the growing MAC subsequent to membrane insertion.

Figure 1. The structure of poly-C9.

(a) Top–down view of a C9 trimer in the poly-C9 map and (b) cut through of the poly-C9 map with cartoon (red) of the poly-C9 model. Approximate dimensions and the predicted amphipathic region are indicated. (c) Cartoon of the full poly-C9 pore (alternating red and yellow monomers). The barrel is best modelled with the architecture S=n/2 (ref. 42). (d) Cryo-EM end and side views of poly-C9 in individual images (top) and class averages (bottom). (e,f) With the exception of the mobile region of the MACPF domain (which in poly-C9 has rearranged in order to form the barrel), the crystal structure of C6 (PDB ID: 3T50) fits well into the map, with TMH1 and TMH2 omitted for clarity. In this figure the conserved β-sheet of the MACPF domain is in red, the body of the MACPF domain is in blue, the EGF domain in green, the TSP1 domain in purple and the LDLRA domain in pink (labelled).

Figure 2. Interactions made by the TSP1 domain.

(a) A view of the outside of the globular portion of the poly-C9 map showing the TSP1 domain (purple) located at each subunit interface. The central C9 monomer is coloured as in Fig. 1, with the monomers each side in dark yellow and purple (TSP1 domain). (b) A view from the top showing placement of the TSP1 domain between the C-terminal helix (marked with *) of each MACPF domain.

Figure 3. Comparison between the structure of C6 (PDB ID 3T5O; yellow) and model of poly-C9 (red/blue).

The shift of the central bent β-sheet (red) shows (a) an ∼10° rotation of the bottom half of the sheet together with (b) an ∼5.5 Å lateral movement. (c) The HTH region (a pair of α-helices) lines the pore lumen. A trimer is shown with the central monomer coloured red, blue and pink.