CryoEM reveals how the complement membrane attack complex ruptures lipid bilayers

Menny, A.; Serna, Marina; Boyd, Courtney M.; Gardner, Scott; Joseph, Agnel Praveen; Morgan, B. Paul; Topf, Maya; Brooks, Nicholas J.; Bubeck, Doryen

doi:10.1101/392563

Cited by 18 publications

(51 citation statements)

References 66 publications

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“…The C5b6 crystal structure demonstrated that prior to interacting with lipids, C6 membrane-interacting residues remain in their soluble helical form 17 ; however, the complex has been shown to associate with lipid bilayers 16,32 . It is likely that C5b6 membrane-binding is dominated by electrostatic interactions of negatively charged lipid headgroups (such as PG lipids commonly found in Gram-negative bacteria 36 ) with either the thrombospondin (TS)1 domain of C6 37 or by its unfurled membrane interacting -hairpin 16 , both of which expose an interface rich in positive charge. Such lipid headgroup dependence also emerges from studies on MAC binding 29,30 and complement activation on model membranes 38 .…”

Section: Discussionmentioning

confidence: 99%

“…Recruitment of C7 unfurls a lipophilic domain upon binding, while integration of C8 into the assembly is accompanied by an initial insertion into the membrane. The C5b-8 initiator complex then binds C9 and undergoes unidirectional oligomerization (with 18 copies of C9) to complete an 11 nm wide transmembrane pore, as characterized in increasing structural detail by cryo-electron microscopy (cryoEM) [12][13][14][15][16] . Together with crystallographic structures of component proteins, high-resolution cryoEM analyses of the full pore have identified regulatory roles for auxiliary domains 15,17,18 that control the transition from stable proteins in our blood to lethal transmembrane pores.…”

Section: Introductionmentioning

confidence: 99%

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Single-molecule kinetics of pore assembly by the membrane attack complex

Parsons

Stanley

Pyne

et al. 2018

Preprint

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The membrane attack complex (MAC) is a hetero-oligomeric protein assembly that kills pathogens by perforating their cell envelopes. The MAC is formed by sequential assembly of soluble complement proteins C5b, C6, C7, C8 and C9, but little is known about the rate-limiting steps in this process. Here, we use rapid atomic force microscopy (AFM) imaging to show that MAC proteins oligomerize within the membrane, unlike structurally homologous bacterial pore-forming toxins. C5b6 interacts with the lipid bilayer prior to recruiting C7 and C8. We discover that incorporation of the first C9 is the kinetic bottleneck of MAC formation, after which rapid C9 oligomerization completes the pore. This defines the kinetic basis for MAC assembly and provides insight into how human cells are protected from bystander damage by the cell surface receptor CD59, which is offered a maximum temporal window to halt the assembly at the point of C9 insertion.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Single-molecule kinetics of pore assembly by the membrane attack complex

Parsons

Stanley

Pyne

et al. 2018

Preprint

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“…This difference in C6 deposition was not caused by a difference in conversion of C5, as shown by similar amounts of C5a in the supernatant ( S5 ). Since one C5b6 molecule is expected to recruit up to 18 C9 molecules to form a complete MAC pore (10,11), we wanted to analyze the ratio between these components based on the fluorescence measured by flow cytometry. This ratio could indicate whether or not rapid interaction between C5b6 and C7 affects the total amount of C9 molecules per C6 molecule on the surface.…”

Section: Resultsmentioning

confidence: 99%

“…Subsequent association of C7 converts the hydrophilic C5b6 complex into lipophilic C5b-7 that uses an exposed membrane-binding domain of C7 to anchor to the target membrane (7,8). Membrane-bound C5b-7 subsequently recruits one copy of C8 and up to 18 copies of C9, which oligomerize and form of a 1.2 MDa transmembrane MAC pore (C5b-9) (9–11).…”

Section: Introductionmentioning

confidence: 99%

Bacterial killing by complement requires direct anchoring of Membrane Attack Complex precursor C5b-7

Doorduijn

Bardoel

Heesterbeek

et al. 2019

Preprint

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15An important effector function of the human complement system is to directly kill Gram-16 negative bacteria via Membrane Attack Complex (MAC) pores. MAC pores are assembled when 17 surface-bound convertase enzymes convert C5 into C5b, which together with C6, C7, C8 and 18 multiple copies of C9 forms a transmembrane pore that damages the bacterial cell envelope. 19 Recently, we found that bacterial killing by MAC pores requires local conversion of C5 by 20 surface-bound convertases. In this study we aimed to understand why local assembly of MAC 21 pores is essential for bacterial killing. Here, we show that rapid interaction of C7 with C5b6 is 22 required to form bactericidal MAC pores. Binding experiments with fluorescently labelled C6 23 show that C7 prevents release of C5b6 from the bacterial surface. Moreover, trypsin shaving 24 experiments and atomic force microscopy revealed that this rapid interaction between C7 and 25 C5b6 is crucial to efficiently anchor C5b-7 to the bacterial cell envelope and form complete 26 MAC pores. Using complement-resistant clinical E. coli strains, we show that bacterial 27 pathogens can prevent complement-dependent killing by interfering with the anchoring of C5b-7. 28 While C5 convertase assembly was unaffected, these resistant strains blocked efficient anchoring 29 of C5b-7 and thus prevented stable insertion of MAC pores into the bacterial cell envelope. 30 Altogether, these findings provide basic molecular insights into how bactericidal MAC pores are 31 assembled and how bacteria evade MAC-dependent killing. 33When bacteria invade the human body, they are attacked by the immune system that tries to clear 34 the infection. Gram-negative bacteria can be directly killed by the complement system, a family 35 of proteins in blood and bodily fluids, that forms toroid-shaped Membrane Attack Complex 36 (MAC) pores into bacterial membranes (1-4). MAC assembly is initiated when recognition 37 molecules, such as antibodies and lectins, bind to bacterial surface structures and recruit early 38 complement components to the target cell surface. This triggers activation of the complement 39 system, a proteolytic cascade that labels the surface with convertase enzymes that cleave the 40 central components C3 and C5 (5). Conversion of C5 into C5b, by C5 convertases, is a critical 41 first step for the unidirectional step-wise assembly of the MAC pore ( Fig. 1A). Upon formation 42 of C5b, C6 rapidly binds metastable C5b to form a stable C5b6 complex (6). Subsequent 43 association of C7 converts the hydrophilic C5b6 complex into lipophilic C5b-7 that uses an 44 exposed membrane-binding domain of C7 to anchor to the target membrane (7,8). Membrane-45 bound C5b-7 subsequently recruits one copy of C8 and up to 18 copies of C9, which oligomerize 46 and form of a 1.2 MDa transmembrane MAC pore (C5b-9) (9-11). 47Although the molecular steps of MAC assembly have been extensively studied on erythrocytes 48 and model membranes (8,9,(12)(13)(14)(15), the mechanisms by which complement f...

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“…The MAC has been extensively studied, and the structure has been determined at resolutions ranging from 2.51 to 5.6 Å. Many structures of individual components, partial assemblies, and full assemblies are available ( Lovelace et al, 2011 ; Serna et al, 2016 ; Menny et al, 2018 ).…”

Section: Introductionmentioning

confidence: 99%

In vivo Cross-Linking MS of the Complement System MAC Assembled on Live Gram-Positive Bacteria

Khakzad

Happonen²,

Nhieu³

et al. 2021

Front. Genet.

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Protein–protein interactions are central in many biological processes, but they are challenging to characterize, especially in complex samples. Protein cross-linking combined with mass spectrometry (MS) and computational modeling is gaining increased recognition as a viable tool in protein interaction studies. Here, we provide insights into the structure of the multicomponent human complement system membrane attack complex (MAC) using in vivo cross-linking MS combined with computational macromolecular modeling. We developed an affinity procedure followed by chemical cross-linking on human blood plasma using live Streptococcus pyogenes to enrich for native MAC associated with the bacterial surface. In this highly complex sample, we identified over 100 cross-linked lysine–lysine pairs between different MAC components that enabled us to present a quaternary model of the assembled MAC in its native environment. Demonstrating the validity of our approach, this MAC model is supported by existing X-ray crystallographic and electron cryo-microscopic models. This approach allows the study of protein–protein interactions in native environment mimicking their natural milieu. Its high potential in assisting and refining data interpretation in electron cryo-tomographic experiments will be discussed.

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CryoEM reveals how the complement membrane attack complex ruptures lipid bilayers

Cited by 18 publications

References 66 publications

Single-molecule kinetics of pore assembly by the membrane attack complex

Single-molecule kinetics of pore assembly by the membrane attack complex

Bacterial killing by complement requires direct anchoring of Membrane Attack Complex precursor C5b-7

In vivo Cross-Linking MS of the Complement System MAC Assembled on Live Gram-Positive Bacteria

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