RESUMO
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.
Assuntos
Complexo de Ataque à Membrana do Sistema Complemento/química , Complexo de Ataque à Membrana do Sistema Complemento/ultraestrutura , Microscopia Crioeletrônica/métodos , Bicamadas Lipídicas/química , Complemento C6/química , Complemento C6/metabolismo , Complemento C6/ultraestrutura , Complemento C7/química , Complemento C7/metabolismo , Complemento C7/ultraestrutura , Complemento C8/química , Complemento C8/metabolismo , Complemento C8/ultraestrutura , Complemento C9/química , Complemento C9/metabolismo , Complemento C9/ultraestrutura , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Bicamadas Lipídicas/metabolismo , Lipossomos , Modelos Moleculares , Polissacarídeos/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Análise Espectral/métodosRESUMO
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.
Assuntos
Complemento C5b/ultraestrutura , Complemento C6/ultraestrutura , Complemento C7/ultraestrutura , Complemento C8/ultraestrutura , Complemento C9/ultraestrutura , Complexo de Ataque à Membrana do Sistema Complemento/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Cromatografia Líquida , Microscopia Crioeletrônica , Corantes Fluorescentes , Humanos , Processamento de Imagem Assistida por Computador , Espectrometria de Massas , Microscopia Eletrônica , Modelos Moleculares , Estrutura Molecular , Estrutura Secundária de ProteínaRESUMO
The carboxy terminus of human complement component C7 comprises two Factor I-like Modules (FIMs) which are essential for formation of the Membrane Attack Complex, the terminal pathway of the innate immune system. C7-FIMs is a 16.9 kDa, recombinant, disulphide-rich, protein encompassing this C-terminal domain. Using conventional triple resonance experiments 93% of the (1)H, (15)N and (13)C assignment has been achieved, accounting for all assignment apart from a flexible N-terminus cloning artefact and an undefined loop. The chemical shifts have been deposited in the BioMagResBank; Accession No. 15996.
