The neoepitope of the complement C5b-9 Membrane Attack Complex is formed by proximity of adjacent ancillary regions of C9.

The Membrane Attack Complex (MAC) is responsible for forming large ß-barrel channels in the membranes of pathogens, such as gram-negative bacteria. Off-target MAC assembly on endogenous tissue is associated with inflammatory diseases and cancer. Accordingly, a human C5b-9 specific antibody, aE11, has been developed that detects a neoepitope exposed in C9 when it is incorporated into the C5b-9 complex, but not present in the plasma native C9. For nearly four decades aE11 has been routinely used to study complement, MAC-related inflammation, and pathophysiology. However, the identity of C9 neoepitope remains unknown. Here, we determined the cryo-EM structure of aE11 in complex with polyC9 at 3.2 Å resolution. The aE11 binding site is formed by two separate surfaces of the oligomeric C9 periphery and is therefore a discontinuous quaternary epitope. These surfaces are contributed by portions of the adjacent TSP1, LDLRA, and MACPF domains of two neighbouring C9 protomers. By substituting key antibody interacting residues to the murine orthologue, we validated the unusual binding modality of aE11. Furthermore, aE11 can recognise a partial epitope in purified monomeric C9 in vitro, albeit weakly. Taken together, our results reveal the structural basis for MAC recognition by aE11.

Going full circle: Determining the structures of complement component 9.

Pore forming proteins (PFPs) undergo dramatic conformational changes to punch holes in the target membrane. These PFPs have the ability to self-assemble, by way of oligomerization, and have the capacity to transform from a water soluble state (commonly referred to as fluid phase) to a membrane adhered form. Accordingly, PFPs are metastable, that is they are inert until the right conditions cause the release of potential energy stored in the conformational fold leading to a vast structural rearrangement into a membrane-inserted oligomeric form. However, the metastable state of PFPs poses a problem of leading to aggregation and precipitation in conditions typically required for structural biology techniques. Here, we discuss the protein chemistry of the MACPF protein complement component 9 (C9). C9 is part of a larger complex assembly known as the membrane attack complex (MAC) that has been studied extensively for its ability to form pores in bacteria. An unusual artifact of human C9 is the ability to form a soluble oligomeric state of the channel portion of the MAC, called polyC9. PolyC9 formation does not require the presence of membranes or other complement factors. It is only in recent years that structural studies of the MAC have become successful owing to improved recombinant DNA expression systems and the improvement of high-resolution techniques (both X-ray crystallography and single particle cryo-EM). We discuss the expression and purification of recombinant C9, crystallization of the soluble monomeric form of C9 and the preparation of the oligomeric polyC9.

Branching out the aerolysin, ETX/MTX-2 and Toxin_10 family of pore forming proteins.

Organisms have evolved mechanisms in which cellular membranes can both be targeted and punctured thereby killing the targeted cell. One such mechanism involves the deployment of pore forming proteins (PFPs) which function by oligomerizing on cell membranes and inserting a physical pore spanning the membrane. This pore can lead to cell death by either causing osmotic flux or allowing the delivery of a secondary toxin. Pore forming proteins can be broadly classified into different families depending on the structure of the final pore; either α-PFPs using channels made from α -helices or ß-PFPs using channels made from ß-barrels. There are many different ß-PFPs and an emerging superfamily is the aerolysin-ETX/MTX-2 superfamily. A comparison between the members of this superfamily reveals the pore forming domain is a common module yet the receptor binding region is highly variable. These structural and architectural variations lead to differences in the target recognition and determine the site of activity. Closer investigation of the topology of the family also suggests that the Toxin_10 family of PFPs could be considered as part of the aerolysin-ETX/MTX-2 superfamily. Comparatively, far less is known about how Toxin_10 proteins assemble into the final pore structure than aerolysin-ETX/MTX-2 proteins. This review aims to collate the pore forming protein members and bridge the structural similarities between the aerolysin-ETX/MTX-2 superfamily and the insecticidal Toxin_10 subfamily.

Ancient but Not Forgotten: New Insights Into MPEG1, a Macrophage Perforin-Like Immune Effector.

Macrophage-expressed gene 1 [MPEG1/Perforin-2 (PRF2)] is an ancient metazoan protein belonging to the Membrane Attack Complex/Perforin (MACPF) branch of the MACPF/Cholesterol Dependent Cytolysin (CDC) superfamily of pore-forming proteins (PFPs). MACPF/CDC proteins are a large and extremely diverse superfamily that forms large transmembrane aqueous channels in target membranes. In humans, MACPFs have known roles in immunity and development. Like perforin (PRF) and the membrane attack complex (MAC), MPEG1 is also postulated to perform a role in immunity. Indeed, bioinformatic studies suggest that gene duplications of MPEG1 likely gave rise to PRF and MAC components. Studies reveal partial or complete loss of MPEG1 causes an increased susceptibility to microbial infection in both cells and animals. To this end, MPEG1 expression is upregulated in response to proinflammatory signals such as tumor necrosis factor α (TNFα) and lipopolysaccharides (LPS). Furthermore, germline mutations in MPEG1 have been identified in connection with recurrent pulmonary mycobacterial infections in humans. Structural studies on MPEG1 revealed that it can form oligomeric pre-pores and pores. Strikingly, the unusual domain arrangement within the MPEG1 architecture suggests a novel mechanism of pore formation that may have evolved to guard against unwanted lysis of the host cell. Collectively, the available data suggest that MPEG1 likely functions as an intracellular pore-forming immune effector. Herein, we review the current understanding of MPEG1 evolution, regulation, and function. Furthermore, recent structural studies of MPEG1 are discussed, including the proposed mechanisms of action for MPEG1 bactericidal activity. Lastly limitations, outstanding questions, and implications of MPEG1 models are explored in the context of the broader literature and in light of newly available structural data.

The cryo-EM structure of the acid activatable pore-forming immune effector Macrophage-expressed gene 1.

Macrophage-expressed gene 1 (MPEG1/Perforin-2) is a perforin-like protein that functions within the phagolysosome to damage engulfed microbes. MPEG1 is thought to form pores in target membranes, however, its mode of action remains unknown. We use cryo-Electron Microscopy (cryo-EM) to determine the 2.4 Å structure of a hexadecameric assembly of MPEG1 that displays the expected features of a soluble prepore complex. We further discover that MPEG1 prepore-like assemblies can be induced to perforate membranes through acidification, such as would occur within maturing phagolysosomes. We next solve the 3.6 Å cryo-EM structure of MPEG1 in complex with liposomes. These data reveal that a multi-vesicular body of 12 kDa (MVB12)-associated ß-prism (MABP) domain binds membranes such that the pore-forming machinery of MPEG1 is oriented away from the bound membrane. This unexpected mechanism of membrane interaction suggests that MPEG1 remains bound to the phagolysosome membrane while simultaneously forming pores in engulfed bacterial targets.

The first transmembrane region of complement component-9 acts as a brake on its self-assembly.

Complement component 9 (C9) functions as the pore-forming component of the Membrane Attack Complex (MAC). During MAC assembly, multiple copies of C9 are sequentially recruited to membrane associated C5b8 to form a pore. Here we determined the 2.2 Å crystal structure of monomeric murine C9 and the 3.9 Å resolution cryo EM structure of C9 in a polymeric assembly. Comparison with other MAC proteins reveals that the first transmembrane region (TMH1) in monomeric C9 is uniquely positioned and functions to inhibit its self-assembly in the absence of C5b8. We further show that following C9 recruitment to C5b8, a conformational change in TMH1 permits unidirectional and sequential binding of additional C9 monomers to the growing MAC. This mechanism of pore formation contrasts with related proteins, such as perforin and the cholesterol dependent cytolysins, where it is believed that pre-pore assembly occurs prior to the simultaneous release of the transmembrane regions.

Evaluation of Serum Glycoprotein Biomarker Candidates for Detection of Esophageal Adenocarcinoma and Surveillance of Barrett's Esophagus.

Esophageal adenocarcinoma (EAC) is thought to develop from asymptomatic Barrett's esophagus (BE) with a low annual rate of conversion. Current endoscopy surveillance of BE patients is probably not cost-effective. Previously, we discovered serum glycoprotein biomarker candidates which could discriminate BE patients from EAC. Here, we aimed to validate candidate serum glycoprotein biomarkers in independent cohorts, and to develop a biomarker candidate panel for BE surveillance. Serum glycoprotein biomarker candidates were measured in 301 serum samples collected from Australia (4 states) and the United States (1 clinic) using previously established lectin magnetic bead array (LeMBA) coupled multiple reaction monitoring mass spectrometry (MRM-MS) tier 3 assay. The area under receiver operating characteristic curve (AUROC) was calculated as a measure of discrimination, and multivariate recursive partitioning was used to formulate a multi-marker panel for BE surveillance. Complement C9 (C9), gelsolin (GSN), serum paraoxonase/arylesterase 1 (PON1) and serum paraoxonase/lactonase 3 (PON3) were validated as diagnostic glycoprotein biomarkers in lectin pull-down samples for EAC across both cohorts. A panel of 10 serum glycoprotein biomarker candidates discriminated BE patients not requiring intervention (BE± low grade dysplasia) from those requiring intervention (BE with high grade dysplasia (BE-HGD) or EAC) with an AUROC value of 0.93. Tissue expression of C9 was found to be induced in BE, dysplastic BE and EAC. In longitudinal samples from subjects that have progressed toward EAC, levels of serum C9 were significantly (p < 0.05) increased with disease progression in EPHA (erythroagglutinin from Phaseolus vulgaris) and NPL (Narcissus pseudonarcissus lectin) pull-down samples. The results confirm alteration of complement pathway glycoproteins during BE-EAC pathogenesis. Further prospective clinical validation of the confirmed biomarker candidates in a large cohort is warranted, prior to development of a first-line BE surveillance blood test.

Perforin-A key (shaped) weapon in the immunological arsenal.

Cytotoxic lymphocytes play a key role in immune homeostasis through elimination of virally-infected and transformed target cells. They do this by employing the potent pore-forming protein, perforin, a molecule that permits cytotoxic proteases, such as granzyme B, to enter the target cell cytoplasm. The synergistic activities of perforin and granzymes bring about the destruction of target cells in a process that is now more clearly understood as a result of structural and cellular biology. These data are helping the development of new classes of immunosuppressive molecules for use in treating immune driven disease and in enhancing the success of transplant therapies. This review focuses on structural and biological aspects of perforin function.

Structure of the poly-C9 component of the complement membrane attack complex.

The membrane attack complex (MAC)/perforin-like protein complement component 9 (C9) is the major component of the MAC, a multi-protein complex that forms pores in the membrane of target pathogens. In contrast to homologous proteins such as perforin and the cholesterol-dependent cytolysins (CDCs), all of which require the membrane for oligomerisation, C9 assembles directly onto the nascent MAC from solution. However, the molecular mechanism of MAC assembly remains to be understood. Here we present the 8 Å cryo-EM structure of a soluble form of the poly-C9 component of the MAC. These data reveal a 22-fold symmetrical arrangement of C9 molecules that yield an 88-strand pore-forming ß-barrel. The N-terminal thrombospondin-1 (TSP1) domain forms an unexpectedly extensive part of the oligomerisation interface, thus likely facilitating solution-based assembly. These TSP1 interactions may also explain how additional C9 subunits can be recruited to the growing MAC subsequent to membrane insertion.

Conformational changes during pore formation by the perforin-related protein pleurotolysin.

Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ∼70° opening of the bent and distorted central ß-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane ß-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of ß-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into ß-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted ß-barrel. The intermediate structures of the MACPF domain during refolding into the ß-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters, suggesting why this region is targeted by endogenous inhibitors of MACPF function.