Assembly and regulation of the membrane attack complex based on structures of C5b6 and sC5b9

Abstract

Activation of the complement system results in formation of membrane attack complexes (MACs), pores that disrupt lipid bilayers and lyse bacteria and other pathogens. Here, we present the crystal structure of the first assembly intermediate, C5b6, together with a cryo-electron microscopy reconstruction of a soluble, regulated form of the pore, sC5b9. Cleavage of C5 to C5b results in marked conformational changes, distinct from those observed in the homologous C3-to-C3b transition. C6 captures this conformation, which is preserved in the larger sC5b9 assembly. Together with antibody labeling, these structures reveal that complement components associate through sideways alignment of the central MAC-perforin (MACPF) domains, resulting in a C5b6-C7-C8β-C8α-C9 arc. Soluble regulatory proteins below the arc indicate a potential dual mechanism in protection from pore formation. These results provide a structural framework for understanding MAC pore formation and regulation, processes important for fighting infections and preventing complement-mediated tissue damage.

Figure 1. The Structure of C5b6

(A) A cartoon and surface representation of C5b6 in two orientations. C5b is colored in cyan, and C6 is colored by domain boundaries. (B) A schematic representation of the domain architecture of C5b. (C) A schematic representation of the domain architecture of C6. (D) A cartoon representation of C5b (cyan) superimposed onto C5 (blue; PDB code 3CU7). C5a is colored red. (E) A cartoon representation of C5b (cyan) superimposed onto C3b (purple; PDB code 2I07). (F) A cartoon representation of C6 from the C5b-C6 complex (brown) superimposed onto free C6 (green; PDB code 3T5O), based on their MACPF domains.

Figure 2. Interface of the C5b6 Complex

(A) The interface between C5b and C6 with the complex “spread” apart. C5b is cyan, with the C6 footprint colored according to the contacting C6 domains. C6 is colored as in Figure 1, and the footprint of C5b is cyan. (B) Hemolytic activity of C6 linker mutants expressed as a percentage relative to recombinant wild-type C6. Each sample was tested at six different concen trations, and standard errors were determined with the use of a nonlinear fitting program (GraFit 5.0). (C) A close-up of the C5b-C6 interaction shows the extensive interface between TED and the C6 linker. Mutated residues tested in (B) are shown as spheres. The unique β-hairpin of C5 (TED) that interacts with the linker is highlighted in red. (D) Structure-based alignment of C5 and C3; the unique insertion in C5 is boxed.

Figure 3. Cryo-EM Structure of sC5b9

(A) MAC components (brown surface) consist of an arc-shaped crescent with a single protrusion (brown arrow), while regulatory domains form a butterfly-like structure (gray surface) below. (B) Class averages of negatively stained sC5b9. (C) Additional density present in the antiC9:sC5b9 averages identifies C9 in the complex (black arrow). (D) Raw images of antiC9:sC5b9. Black arrow indicates antibody. (E) Pseudoatomic model for MAC components consists of C5b6 (C5b, cyan; C6, blue), and C8 (C8α, dark gray; C8β, green; C8γ, light gray solid surface) crystal structures and models for C7 (orange) and C9 (purple). The dashed orange line indicates a ridge connecting the arc with C345C. Cyan and blue asterisks indicate unmodeled density near C345C of C5b and CCP1 of C6, respectively. Scale bars represent 160 Å (B–D).

Figure 4. A Model for MAC Formation

The complement terminal pathway is initiated by the cleavage of C5 to C5b. C6 traps a labile conformation of the C5b TED domain to form C5b6, a platform for the stepwise assembly of components C7, C8, and C9. Regulatory proteins in the plasma block MAC assembly in solution by binding exposed hydrophobic regions and sterically inhibit C9 oligomerization. Binding of C5b8 to membranes recruits multiple C9 molecules whose MACPF domains arrange to form a β-barrel pore similar to that of CDCs.