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. 2023 Feb 16;14(1):890.
doi: 10.1038/s41467-023-36441-z.

Structural basis for membrane attack complex inhibition by CD59

Emma C Couves  1 , Scott Gardner  1 , Tomas B Voisin  1 , Jasmine K Bickel  1   2 , Phillip J Stansfeld  3 , Edward W Tate  2 , Doryen Bubeck  4
Affiliations

Structural basis for membrane attack complex inhibition by CD59

Emma C Couves et al. Nat Commun. .

Abstract

CD59 is an abundant immuno-regulatory receptor that protects human cells from damage during complement activation. Here we show how the receptor binds complement proteins C8 and C9 at the membrane to prevent insertion and polymerization of membrane attack complex (MAC) pores. We present cryo-electron microscopy structures of two inhibited MAC precursors known as C5b8 and C5b9. We discover that in both complexes, CD59 binds the pore-forming β-hairpins of C8 to form an intermolecular β-sheet that prevents membrane perforation. While bound to C8, CD59 deflects the cascading C9 β-hairpins, rerouting their trajectory into the membrane. Preventing insertion of C9 restricts structural transitions of subsequent monomers and indirectly halts MAC polymerization. We combine our structural data with cellular assays and molecular dynamics simulations to explain how the membrane environment impacts the dual roles of CD59 in controlling pore formation of MAC, and as a target of bacterial virulence factors which hijack CD59 to lyse human cells.

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Conflict of interest statement

EWT is a founder and shareholder of Myricx Pharma Ltd. All other authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Schematic of MAC assembly.
The MAC pore is formed from the sequential and stepwise assembly of complement proteins: C5b, grey; C6, blue; C7, orange; C8α, pink; C8β, magenta; C8γ, light purple; C9, alternating monomers are yellow and green. During assembly, complement proteins undergo dramatic structural re-arrangements in which two helical bundles within their MACPF domains unfurl into membrane-inserting β-hairpins. CD59 (cyan) binds at two stages of this assembly process (C5b8 and C5b9) to block membrane perforation and C9 polymerization. Images are rendered from structural models. C5b6 and all MAC assemblies: PDB ID: 6H03; Soluble forms of complement proteins are derived from C6: PDB ID: 3T5O; C8: PDB ID: 3OJY; C9: PDB ID: 6CXO. C7 was derived from an AlphaFold2 prediction: AlphaFold Protein Structure Database P10643.
Fig. 2
Fig. 2. Structure of the C5b8-CD59 complex.
CryoEM map A and model B for the C5b8-CD59 nanodisc complex. Complement proteins are colored (C5b, grey; C6, blue; C7, orange; C8α, pink; C8β, magenta; C8γ light purple). CD59 is cyan. The membrane, not visible in the sharpened map, is schematized for reference. To improve the resolution for CD59, the density for C5b, C6 and C8γ was subtracted from the consensus map (A) and refined. C Model for CD59 and interaction interface with C8α overlayed on the density subtracted focus refined map.
Fig. 3
Fig. 3. CD59 interaction interfaces.
A Ribbon diagram of the C8α-CD59 interface. CD59 (cyan) captures the extending TMH2 residues of C8α (pink). Aromatic residue CD59:F47 binds C8α glycines (G373, G374, G375) to bend the β-hairpin trajectory. The C8α-CD59 interface is further stabilized by a salt bridge between CD59:E58 and C8α:K376, together with a pair of consecutive tyrosine-lysine interactions on either side of the β-sheet (CD59:Y62-C8α:K370; CD59:Y61-C8α:K371). Key residues that mediate the interactions are shown as sticks. B Hydrogen-bonding pattern of backbone atoms within the intermolecular antiparallel β-sheet. C Ribbon diagram of the ILY-CD59 interaction interface (PDB ID: 5IMT). ILY (orange) binds CD59 (cyan) through a β-hairpin extension of domain 4 (D4). The ILY-CD59 interface is comprised of an intermolecular anti-parallel β-sheet, including CD59 residues that engage C8α. D Superposition of the ILY-CD59 crystal structure (PDB ID: 5IMT) with a pose from the atomistic molecular dynamics simulation of GPI-anchored CD59 (tan). E Superposition of the C5b8-CD59 structure with a different pose from the atomistic molecular dynamics simulation of GPI-anchored CD59 (tan). In this position, CD59 is rotated 106° relative to its position in (D). Phosphorous atoms from lipid headgroups are grey spheres, simulated GPI anchor for CD59 is shown in green sticks. Initial and final configurations for the three MD repeats are included in the Supplementary Data Files.
Fig. 4
Fig. 4. Structure of the C5b9-CD59 complex.
CryoEM map A and model B of C5b9-CD59 complexes comprised of 2 molecules of C9 (C91 and C92). (C) A model for the C5b9-CD59 complex comprised of 3 C9 molecules. Complement proteins are colored (C5b, grey; C6, blue; C7, orange; C8α pink, C8β magenta, C8γ light purple); CD59 is cyan. The membrane, not visible in the sharpened map, is schematized for reference. D, E Density for the C5b92-CD59 map after density subtraction and focused refinement (transparent surface) overlayed with the model. D For the terminal C9 (C92, yellow), only the first transmembrane β-hairpin has unfurled while TMH2 (red) remains helical. E CD59 (cyan) deflects both TMH1 and TMH2 from the first C9 (C91, green) to block membrane insertion. F The C91-CD59 interaction interface. The key residues that mediate interactions are shown as sticks. G Electrostatic surface representation of CD59 highlighting positively charged residues (R55, R53, K38) that interact with the C9 glycan extending from N394 (green sticks). Coulombic potential calculated for the CD59 model in B ranging from −10 (red) to +10 (blue) kcal/(mol·e).
Fig. 5
Fig. 5. Influence of the membrane environment on MAC assembly and inhibition.
A, B Cholesterol depletion assays. A representative image (out of 10 randomly selected locations) for each condition is shown. Scale bars, 50 μm. A Complement was activated on CHO cells with a polyclonal anti-CHO IgG antibody. Cells were incubated with C9-depleted human serum supplemented with a chemically labeled fluorescent C9 (C9-Alexafluor 568) capable of forming MAC. B CHO cells treated with MβCD to deplete cholesterol. Complement activation and C9 detection is as described in (A). Insets show a zoomed in view of single cell. Cartoon schematics highlight the pattern of MAC deposition. C CryoEM map of the C5b92-CD59 complex in a lipid nanodisc applying a positive B-factor of +50 Å2 (transparent surface). Surface rendering of the protein model is underlayed. CD59 is cyan, C8β is magenta and the remaining complement proteins are grey. D Map generated from the coarse-grained model of the C5b8-CD59 complex including the GPI anchor (green). Protein components colored as in panel (C). Water molecules in proximity to the membrane are shown as blue spheres. Initial and final configurations for the three MD repeats are included in the Supplementary Data Files.
Fig. 6
Fig. 6. Schematic of MAC assembly and its inhibition by CD59.
A MAC assembles on cholesterol-rich microdomains (red hexagons) in the plasma membrane. While C7 anchors the growing MAC, C8β thins the bilayer and primes the membrane for rupture by C8α (pink). C9 joins the assembly, undergoing discrete structural transitions to form the pore. The MACPF domain of soluble C9 makes an initial contact and aligns the central β-sheet. The pore-forming β-hairpins extend sequentially. TMH1 is followed by TMH2, which allow C9 polymerization to complete the MAC pore. B CD59 is a GPI-anchored cell surface receptor that clusters in cholesterol-rich microdomains. Upon complement activation, CD59 could respond to membrane thinning by C8β and capture C8α as its transmembrane β-hairpins are extending (pink). While bound to C8α, CD59 is positioned to deflect the cascading C9 β-hairpins and divert their membrane trajectory (green). The next C9 molecule (yellow) is able to bind but trapped in an intermediate conformation in which TMH2 remains helical (red) and in which C9 polymerization is halted.
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