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, 286 (20), 17585-92

Structure of Human C8 Protein Provides Mechanistic Insight Into Membrane Pore Formation by Complement

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Structure of Human C8 Protein Provides Mechanistic Insight Into Membrane Pore Formation by Complement

Leslie L Lovelace et al. J Biol Chem.

Abstract

C8 is one of five complement proteins that assemble on bacterial membranes to form the lethal pore-like "membrane attack complex" (MAC) of complement. The MAC consists of one C5b, C6, C7, and C8 and 12-18 molecules of C9. C8 is composed of three genetically distinct subunits, C8α, C8β, and C8γ. The C6, C7, C8α, C8β, and C9 proteins are homologous and together comprise the MAC family of proteins. All contain N- and C-terminal modules and a central 40-kDa membrane attack complex perforin (MACPF) domain that has a key role in forming the MAC pore. Here, we report the 2.5 Å resolution crystal structure of human C8 purified from blood. This is the first structure of a MAC family member and of a human MACPF-containing protein. The structure shows the modules in C8α and C8β are located on the periphery of C8 and not likely to interact with the target membrane. The C8γ subunit, a member of the lipocalin family of proteins that bind and transport small lipophilic molecules, shows no occupancy of its putative ligand-binding site. C8α and C8β are related by a rotation of ∼22° with only a small translational component along the rotation axis. Evolutionary arguments suggest the geometry of binding between these two subunits is similar to the arrangement of C9 molecules within the MAC pore. This leads to a model of the MAC that explains how C8-C9 and C9-C9 interactions could facilitate refolding and insertion of putative MACPF transmembrane β-hairpins to form a circular pore.

Figures

FIGURE 1.
FIGURE 1.
Structure of C8. A, ribbon model showing C8α (red), C8β (blue), and C8γ (green). MACPF domains are in dark colors and modules in light colors. B, view of C8α oriented as in A. The central MACPF β-sheet is blue; α-helices in TMH1 and TMH2 are red, and the remainder of the MACPF domain is gold. The LDLRA domain is violet; EGF-like domain is lavender, N-terminal TSP1 is malachite, and C-terminal TSP1 is green. The upper and lower MACPF subdomains are identified with frames.
FIGURE 2.
FIGURE 2.
Features of the C8α and C8β MACPF domains. Surface of the C8 molecule is shown. β-sheets of the C8α (red) and C8β (blue) MACPF domains are shown as stick models. This view shows “fanning out” of the MACPF subdomains. The upper part likely forms the outer or extracellular part of the pore. The lower part spreads out making the base wider. A stereo version is available online (supplemental Fig. S4).
FIGURE 3.
FIGURE 3.
Superposition of C8α (red) on C8β (blue) based on their upper MACPF subdomains. MACPF domains are in dark colors, and modules are in light colors. This superposition yields a 24° angle between strands of the β-sheets in the lower subdomain. The region with conserved glycine pairs 318–319, 395–396 and 297–298, 375–376 (numbering for C8α and C8β, respectively) is located at the bends of the sheets (circled). Two orthogonal views are shown.
FIGURE 4.
FIGURE 4.
Post-translationally modified residues in C8β. A, central β-sheet is shown in blue, TMHs in red, and the remainder of the MACPF domain in gold. The LDLRA domain is in violet, EGF in lavender, N-terminal TSP1 in malachite, and C-terminal TSP1 in green. Mannosyl tryptophans are clustered together on the periphery of the molecule; phosphothreonine 364 is at the bottom of the structure. B, electron density for mannosylated Trp497 in C8β. The 2FoFc composite omit map is contoured at a 1σ level.
FIGURE 5.
FIGURE 5.
Atomic model of the MAC. The model was generated assuming that the binding mode between C8α and C8β MACPF domains is conserved for C6, C7, and C9. A, view of the modeled C8-C9 complex. Colors correspond to C8α (red), C8β (blue), C8γ (green), and C9 (gold). The putative CD59-binding site in C8α is circled in pink. The nearby position of phosphorylated C8β Thr364 is also shown. A more detailed view of these features is available in supplemental Fig. S7. B, partial model of the MAC. View is of the side facing the membrane. Proteins are colored C6 (cyan), C7 (pink), C8β (blue), C8α (red), C8γ (green), and C9 (gold). Absent are C5b, the CCP and FIM modules of C6 and C7, and the additional N-terminal TSP1 module in C6, the locations of which could not be predicted. Current evidence suggests C5b binds to the C7 FIMs (41); it is assumed the other missing components do not interfere with circular packing of the MACPF domains. The inner diameter of the ring is 110 Å with 80 Å between the protruding loops. The outer diameter is ∼220 Å and the height is 90 Å. This agrees well with EM images of pores formed by poly-C9, which generally have dimensions of ∼100 and 210 Å for the inner and outer diameters, respectively (42). C, molecular envelope of the model shown in B with modules in red and MACPF domains in gold. View is of the side facing away from the membrane. The modules account for a substantial fraction of the surface. It can be speculated that they may partially protect the MACPF domains from proteolytic factors at sites of inflammation as they contain multiple disulfides and carbohydrates.
FIGURE 6.
FIGURE 6.
Comparison of C8 and perforin structures. C8 is in dark colors and perforin in light colors. C8γ is in green, and C8β is omitted. The MACPF/CDC domains are in blue, EGF domains in purple, and remaining domains in brown. The putative membrane location would be horizontal and below the models.

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