MAC was solubilized with detergent and purified for structural studies, as described previously 15. The lipid composition of vesicles was selected based on the stoichiometric homogeneity of deposited pores. The human MAC pore was formed on liposomes from individual complement proteins. In combination with flicker spectroscopy, we show how MAC assembly impacts biophysical properties of the bilayer and resolve the mechanisms of membrane interaction and MAC activity. In this study, we used cryoEM to determine the structures of two MAC conformations at near atomic resolution and derive a nearly complete atomic model for the pore.

Based on structural similarity with bacterial homologs, the giant (110 Å diameter) β-barrel pore is formed when helical bundles in the Membrane Attack Complex-Perforin (MACPF) domains transform into transmembrane β-hairpins (TMH) 17, 18, 19, although a molecular mechanism for how this occurs is not currently understood. Previous electron cryo-microscopy (cryoEM) reconstructions of the complex revealed that the final MAC is comprised of 6 polypeptide chains (C5b, C6, C7, C8α, C8β, and C8γ) together with 18 C9 monomers that are arranged in a split-washer configuration 15, 16. C9 molecules associate with C5b8 in the membrane to form C5b9 and polymerize to complete the MAC pore 14. C8 irreversibly binds the nascent complex, resulting in the membrane-inserted C5b8 13. In the presence of membranes, C5b6 (a complex comprised of C5b and C6 11) binds C7 to form the lipophilic MAC precursor C5b7 12. MAC assembles from soluble complement proteins in an obligate sequential pathway 10. Therefore, developing a molecular model of how MAC forms on target cells is essential for understanding the immune response to microbes and for the development of therapeutics that regulate complement activity. Complement activation and MAC formation also contributes to killing of cancer cells during immunotherapy treatments 9. Deficiency of CD59 causes the lethal blood disorder Paroxysmal Nocturnal Hemoglobinuria, a disease characterized by thrombosis and chronic hemolysis 8. Host cells are protected from bystander damage by the GPI-anchored receptor CD59, the only membrane-bound inhibitor of MAC 7.

This innate immune effector is essential for fighting bacteria of the genus Neisseria 5 with genetic deficiencies in component proteins leading to recurrent infections 6. The MAC pore targets a wide range of pathogens, forming on and killing Gram-negative bacteria 2, enveloped viruses 3, and parasites 4. The multiprotein membrane attack complex is a bactericidal weapon of the innate immune system that also modulates inflammation and proliferation when formed on self-cells 1.

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