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The Membrane Attack Complex (MAC) is responsible for forming large β-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. The cryo-EM structure of monoclonal antibody aE11 bound to polyC9 reveals that the binding site within the Membrane Attack Complex is a quaternary discontinuous epitope formed by two separate surfaces of the oligomeric C9 periphery. [ABSTRACT FROM AUTHOR]

The zinc-dependent metalloprotease meprin α is predominantly expressed in the brush border membrane of proximal tubules in the kidney and enterocytes in the small intestine and colon. In normal tissue homeostasis meprin α performs key roles in inflammation, immunity, and extracellular matrix remodelling. Dysregulated meprin α is associated with acute kidney injury, sepsis, urinary tract infection, metastatic colorectal carcinoma, and inflammatory bowel disease. Accordingly, meprin α is the target of drug discovery programs. In contrast to meprin β, meprin α is secreted into the extracellular space, whereupon it oligomerises to form giant assemblies and is the largest extracellular protease identified to date (~6 MDa). Here, using cryo-electron microscopy, we determine the high-resolution structure of the zymogen and mature form of meprin α, as well as the structure of the active form in complex with a prototype small molecule inhibitor and human fetuin-B. Our data reveal that meprin α forms a giant, flexible, left-handed helical assembly of roughly 22 nm in diameter. We find that oligomerisation improves proteolytic and thermal stability but does not impact substrate specificity or enzymatic activity. Furthermore, structural comparison with meprin β reveal unique features of the active site of meprin α, and helical assembly more broadly. Meprin α is a proteolytic regulator of the extracellular matrix that forms enormous oligomeric filaments of unknown purpose. Here, the authors determine by cryo-EM the structural basis of the meprin supercoiled filament and further characterise a small molecule inhibitor bound to its active site. [ABSTRACT FROM AUTHOR]

The protein NINJ1 drives membrane rupture associated with certain types of cell death. Investigation of NINJ1 reveals mechanistic details of how it functions, raising the possibility of developing new therapeutics.Insights into a mechanism that ruptures the plasma membrane. [ABSTRACT FROM AUTHOR]

A defining property of cytotoxic lymphocytes is their expression and regulated secretion of potent toxins, including the pore-forming protein perforin and serine protease granzymes. Until recently, mechanisms of pore formation and granzyme transfer into the target cell were poorly understood, but advances in structural and cellular biology have now begun to unravel how synergy between perforin and granzymes brings about target cell death. These and other advances are demonstrating the surprisingly broad pathophysiological roles of the perforin-granzyme pathway, and this has important implications for understanding immune homeostasis and for developing immunotherapies for cancer and other diseases. In particular, we are beginning to define and understand a range of human diseases that are associated with a failure to deliver active perforin to target cells. In this Review, we discuss the current understanding of the structural, cellular and clinical aspects of perforin and granzyme biology. [ABSTRACT FROM AUTHOR]

Interleukin 37 (IL-37) and IL-1R8 (SIGIRR or TIR8) are anti-inflammatory orphan members of the IL-1 ligand family and IL-1 receptor family, respectively. Here we demonstrate formation and function of the endogenous ligand-receptor complex IL-37-IL-1R8-IL-18Rα. The tripartite complex assembled rapidly on the surface of peripheral blood mononuclear cells upon stimulation with lipopolysaccharide. Silencing of IL-1R8 or IL-18Rα impaired the anti-inflammatory activity of IL-37. Whereas mice with transgenic expression of IL-37 (IL-37tg mice) with intact IL-1R8 were protected from endotoxemia, IL-1R8-deficient IL-37tg mice were not. Proteomic and transcriptomic investigations revealed that IL-37 used IL-1R8 to harness the anti-inflammatory properties of the signaling molecules Mer, PTEN, STAT3 and p62(dok) and to inhibit the kinases Fyn and TAK1 and the transcription factor NF-κB, as well as mitogen-activated protein kinases. Furthermore, IL-37-IL-1R8 exerted a pseudo-starvational effect on the metabolic checkpoint kinase mTOR. IL-37 thus bound to IL-18Rα and exploited IL-1R8 to activate a multifaceted intracellular anti-inflammatory program. [ABSTRACT FROM AUTHOR]

Natural killer cells and cytotoxic T lymphocytes accomplish the critically important function of killing virus-infected and neoplastic cells. They do this by releasing the pore-forming protein perforin and granzyme proteases from cytoplasmic granules into the cleft formed between the abutting killer and target cell membranes. Perforin, a 67-kilodalton multidomain protein, oligomerizes to form pores that deliver the pro-apoptopic granzymes into the cytosol of the target cell. The importance of perforin is highlighted by the fatal consequences of congenital perforin deficiency, with more than 50 different perforin mutations linked to familial haemophagocytic lymphohistiocytosis (type 2 FHL). Here we elucidate the mechanism of perforin pore formation by determining the X-ray crystal structure of monomeric murine perforin, together with a cryo-electron microscopy reconstruction of the entire perforin pore. Perforin is a thin 'key-shaped' molecule, comprising an amino-terminal membrane attack complex perforin-like (MACPF)/cholesterol dependent cytolysin (CDC) domain followed by an epidermal growth factor (EGF) domain that, together with the extreme carboxy-terminal sequence, forms a central shelf-like structure. A C-terminal C2 domain mediates initial, Ca2+-dependent membrane binding. Most unexpectedly, however, electron microscopy reveals that the orientation of the perforin MACPF domain in the pore is inside-out relative to the subunit arrangement in CDCs. These data reveal remarkable flexibility in the mechanism of action of the conserved MACPF/CDC fold and provide new insights into how related immune defence molecules such as complement proteins assemble into pores. [ABSTRACT FROM AUTHOR]

Gamma-aminobutyric acid (GABA) is synthesized by two isoforms of the pyridoxal 5′-phosphate–dependent enzyme glutamic acid decarboxylase (GAD65 and GAD67). GAD67 is constitutively active and is responsible for basal GABA production. In contrast, GAD65, an autoantigen in type I diabetes, is transiently activated in response to the demand for extra GABA in neurotransmission, and cycles between an active holo form and an inactive apo form. We have determined the crystal structures of N-terminal truncations of both GAD isoforms. The structure of GAD67 shows a tethered loop covering the active site, providing a catalytic environment that sustains GABA production. In contrast, the same catalytic loop is inherently mobile in GAD65. Kinetic studies suggest that mobility in the catalytic loop promotes a side reaction that results in cofactor release and GAD65 autoinactivation. These data reveal the molecular basis for regulation of GABA homeostasis. [ABSTRACT FROM AUTHOR]

AB5 toxins are produced by pathogenic bacteria and consist of enzymatic A subunits that corrupt essential eukaryotic cell functions, and pentameric B subunits that mediate uptake into the target cell. AB5 toxins include the Shiga, cholera and pertussis toxins and a recently discovered fourth family, subtilase cytotoxin, which is produced by certain Shiga toxigenic strains of Escherichia coli. Here we show that the extreme cytotoxicity of this toxin for eukaryotic cells is due to a specific single-site cleavage of the essential endoplasmic reticulum chaperone BiP/GRP78. The A subunit is a subtilase-like serine protease; structural studies revealed an unusually deep active-site cleft, which accounts for its exquisite substrate specificity. A single amino-acid substitution in the BiP target site prevented cleavage, and co-expression of this resistant protein protected transfected cells against the toxin. BiP is a master regulator of endoplasmic reticulum function, and its cleavage by subtilase cytotoxin represents a previously unknown trigger for cell death. [ABSTRACT FROM AUTHOR]

Unusually long major histocompatibility complex (MHC) class I–restricted epitopes are important in immunity, but their 'bulged' conformation represents a potential obstacle to αβ T cell receptor (TCR)–MHC class I docking. To elucidate how such recognition is achieved while still preserving MHC restriction, we have determined here the structure of a TCR in complex with HLA-B*3508 presenting a peptide 13 amino acids in length. This complex was atypical of TCR–peptide–MHC class I interactions, being dominated at the interface by peptide-mediated interactions. The TCR assumed two distinct orientations, swiveling on top of the centrally bulged, rigid peptide such that only limited contacts were made with MHC class I. Although the TCR-peptide recognition resembled an antibody-antigen interaction, the TCR–MHC class I contacts defined a minimal 'generic footprint' of MHC-restriction. Thus our findings simultaneously demonstrate the considerable adaptability of the TCR and the 'shape' of MHC restriction. [ABSTRACT FROM AUTHOR]

Fasciola spp., commonly known as liver fluke, are significant trematode parasites of livestock and humans. They secrete several cathepsin L-like cysteine proteases, some of which differ in enzymatic properties and timing of expression in the parasite's life cycle. A detailed sequence and evolutionary analysis is presented, based on 18 cathepsin L-like enzymes isolated from Fasciola spp. (including a novel clone identified in this study). The enzymes form a monophyletic group which has experienced several gene duplication events over the last ~135 million years, giving rise to the present-day enzymatic repertoire of the parasite. This timing of these duplications appears to correlate with important points in the evolution of the mammalian hosts. Furthermore, the dates suggest that Fasciola hepatica and Fasciola gigantica diverged around 19 million years ago. A novel analysis, based on the pattern of amino acid diversity, was used to identify sites in the enzyme that are predicted to be subject to positive adaptive evolution. Many of these sites occur within the active site cleft of the enzymes, and hence would be expected to lead to differences in substrate specificity. Using homology modeling, with reference to previously obtained biochemical data, we are able to predict S2 subsite specificity for these enzymes: specifically those that can accommodate bulky hydrophobic residues in the P2 position and those that cannot. A number of other positions subject to evolutionary pressure and potentially significant for enzyme function are also identified, including sites anticipated to diminish cystatin binding affinity. [ABSTRACT FROM AUTHOR]

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 β-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. Macrophage-expressed gene 1 (MPEG1) functions within the phagolysosome to damage engulfed microbes, presumably via forming pores in target membranes. In order to provide insights into the mechanism of MPEG1 function and membrane binding, the authors present structures of hexadecameric MPEG1 prepores both in solution and in complex with liposomes. [ABSTRACT FROM AUTHOR]

Conjugation is fundamental for the acquisition of new genetic traits and the development of antibiotic resistance in pathogenic organisms. Here, we show that a hypothetical Clostridium perfringens protein, TcpK, which is encoded by the tetracycline resistance plasmid pCW3, is essential for efficient conjugative DNA transfer. Our studies reveal that TcpK is a member of the winged helix-turn-helix (wHTH) transcription factor superfamily and that it forms a dimer in solution. Furthermore, TcpK specifically binds to a nine-nucleotide sequence that is present as tandem repeats within the pCW3 origin of transfer (oriT). The X-ray crystal structure of the TcpK-TcpK box complex reveals a binding mode centered on and around the β-wing, which is different from what has been previously shown for other wHTH proteins. Structure-guided mutagenesis experiments validate the specific interaction between TcpK and the DNA molecule. Additional studies highlight that the TcpK dimer is important for specific DNA binding. [ABSTRACT FROM AUTHOR]

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. The Complement component 9 (C9) is the pore-forming component of the Membrane Attack Complex which targets pathogens. Here authors use structural biology to compare monomeric C9 to C9 within the polymeric assembly and identify the element which inhibits C9 self-assembly in the absence of the target membrane. [ABSTRACT FROM AUTHOR]

The article presents a study of intermediate states of aeromonas hydrophila toxin aerolysin and its route to pore formation within membranes. It mentions that researchers used single particle cryo-Electron Microscopy to detect three intermediate structures on the pathway including an initial heptameric prepore state, post-prepore structure and a 14 stranded membrane-inserted beta-barrel. Results indicate a marked rotation of the core aerolysin machinery to penetrate membrane-spanning regions.

The article discusses that polymers of serpin proteins underlie diseases, including neurodegenerative disorders. It is said that Alois Alzheimer has offered a description on the abnormal aggregates of the amyloid protein in the brain of a patient who has died of dementia. The known conformational diseases include prion diseases, Parkinson's disease, Huntington's disease and several disorders known as serpinopathies. It is mentioned that the serpin polymer is correctly folded and resembles the relaxed serpin, which does not evoke protective signalling pathway.

Presents a letter to the editor commenting on the post-genomic era that has lead to an explosion in protein expression and structural initiatives from both a biotechnology and an academic perspective.