ABSTRACT
The membrane attack complex (MAC) is one of the immune system's first responders. Complement proteins assemble on target membranes to form pores that lyse pathogens and impact tissue homeostasis of self-cells. How MAC disrupts the membrane barrier remains unclear. Here we use electron cryo-microscopy and flicker spectroscopy to show that MAC interacts with lipid bilayers in two distinct ways. Whereas C6 and C7 associate with the outer leaflet and reduce the energy for membrane bending, C8 and C9 traverse the bilayer increasing membrane rigidity. CryoEM reconstructions reveal plasticity of the MAC pore and demonstrate how C5b6 acts as a platform, directing assembly of a giant ß-barrel whose structure is supported by a glycan scaffold. Our work provides a structural basis for understanding how ß-pore forming proteins breach the membrane and reveals a mechanism for how MAC kills pathogens and regulates cell functions.
Subject(s)
Complement Membrane Attack Complex/chemistry , Complement Membrane Attack Complex/ultrastructure , Cryoelectron Microscopy/methods , Lipid Bilayers/chemistry , Complement C6/chemistry , Complement C6/metabolism , Complement C6/ultrastructure , Complement C7/chemistry , Complement C7/metabolism , Complement C7/ultrastructure , Complement C8/chemistry , Complement C8/metabolism , Complement C8/ultrastructure , Complement C9/chemistry , Complement C9/metabolism , Complement C9/ultrastructure , Complement Membrane Attack Complex/metabolism , Humans , Image Processing, Computer-Assisted , Lipid Bilayers/metabolism , Liposomes , Models, Molecular , Polysaccharides/metabolism , Protein Conformation , Protein Interaction Domains and Motifs , Spectrum Analysis/methodsABSTRACT
In response to complement activation, the membrane attack complex (MAC) assembles from fluid-phase proteins to form pores in lipid bilayers. MAC directly lyses pathogens by a 'multi-hit' mechanism; however, sublytic MAC pores on host cells activate signalling pathways. Previous studies have described the structures of individual MAC components and subcomplexes; however, the molecular details of its assembly and mechanism of action remain unresolved. Here we report the electron cryo-microscopy structure of human MAC at subnanometre resolution. Structural analyses define the stoichiometry of the complete pore and identify a network of interaction interfaces that determine its assembly mechanism. MAC adopts a 'split-washer' configuration, in contrast to the predicted closed ring observed for perforin and cholesterol-dependent cytolysins. Assembly precursors partially penetrate the lipid bilayer, resulting in an irregular ß-barrel pore. Our results demonstrate how differences in symmetric and asymmetric components of the MAC underpin a molecular basis for pore formation and suggest a mechanism of action that extends beyond membrane penetration.
Subject(s)
Complement C5b/ultrastructure , Complement C6/ultrastructure , Complement C7/ultrastructure , Complement C8/ultrastructure , Complement C9/ultrastructure , Complement Membrane Attack Complex/ultrastructure , Multiprotein Complexes/ultrastructure , Chromatography, Liquid , Cryoelectron Microscopy , Fluorescent Dyes , Humans , Image Processing, Computer-Assisted , Mass Spectrometry , Microscopy, Electron , Models, Molecular , Molecular Structure , Protein Structure, SecondaryABSTRACT
The sera from three C8 alpha-gamma deficient patients previously reported to have a selective C8 alpha-gamma defect were analyzed by SDS-PAGE and Western blot using two polyclonal antisera to C8 alpha-gamma and a monoclonal antibody to C8 alpha. All three sera exhibited C8 alpha-gamma bands that dissociated into alpha and gamma chains under reducing conditions. Quantitation of the alpha-gamma subunit in these sera by a sensitive ELISA revealed an amount approximately 1% of that found in normal human serum. A similar assay performed with a specific antiserum to C8 beta showed unexpectedly low levels of C8 beta in these sera, which were confirmed by hemolytic titration of C8 beta. The remarkable differences between C8 alpha-gamma and C8 beta in the C8 alpha-gamma deficient sera was that in spite of their comparable immunochemical levels, C8 beta still exhibited functional activity whereas C8 alpha-gamma was totally inactive. That the residual C8 alpha-gamma was inactive was also proved by its inability to show lytic bands in an overlay system after SDS-PAGE and subsequent removal of SDS. The implications of these findings for a novel concept of C8 deficiency are discussed.
