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Activation of the serum-resident complement system begins a cascade that leads to activation of membrane-resident complement receptors on immune cells, thus coordinating serum and cellular immune responses. Whilst many molecules act to control inappropriate activation, Properdin is the only known positive regulator of the human complement system. By stabilising the alternative pathway C3 convertase it promotes complement self-amplification and persistent activation boosting the magnitude of the serum complement response by all triggers. In this work, we identify a family of tick-derived alternative pathway complement inhibitors, hereafter termed CirpA. Functional and structural characterisation reveals that members of the CirpA family directly bind to properdin, inhibiting its ability to promote complement activation, and leading to potent inhibition of the complement response in a species specific manner. We provide a full functional and structural characterisation of a properdin inhibitor, opening avenues for future therapeutic approaches., Properdin is the only known positive regulator of the human complement system, stabilising the convertase C3 in the alternative pathway of complement activation. Here, the authors report the identification and characterisation of a species-specific properdin inhibitor CirpA, derived from tick saliva.

Folates (also known as vitamin B9) have a critical role in cellular metabolism as the starting point in the synthesis of nucleic acids, amino acids and the universal methylating agent S-adenylsmethionine(1,2). Folate deficiency is associated with a number of developmental, immune and neurological disorders(3–5). Mammals cannot synthesize folates de novo; several systems have therefore evolved to take up folates from the diet and distribute them within the body(3,6). The proton-coupled folate transporter (PCFT) (also known as SLC46A1) mediates folate uptake across the intestinal brush border membrane and the choroid plexus(4,7), and is an important route for the delivery of antifolate drugs in cancer chemotherapy(8–10). How PCFT recognizes folates or antifolate agents is currently unclear. Here we present cryo-electron microscopy structures of PCFT in a substrate-free state and in complex with a new-generation antifolate drug (pemetrexed). Our results provide a structural basis for understanding antifolate recognition and provide insights into the pH-regulated mechanism of folate transport mediated by PCFT.

Accurate Notch signalling is critical for development and homeostasis. Fine‐tuning of Notch–ligand interactions has substantial impact on signalling outputs. Recent structural studies have identified a conserved N‐terminal C2 domain in human Notch ligands which confers phospholipid binding in vitro. Here, we show that Drosophila ligands Delta and Serrate adopt the same C2 domain structure with analogous variations in the loop regions, including the so‐called β1‐2 loop that is involved in phospholipid binding. Mutations in the β1‐2 loop of the Delta C2 domain retain Notch binding but have impaired ability to interact with phospholipids in vitro. To investigate its role in vivo, we deleted five residues within the β1‐2 loop of endogenous Delta. Strikingly, this change compromises ligand function. The modified Delta enhances phenotypes produced by Delta loss‐of‐function alleles and suppresses that of Notch alleles. As the modified protein is present on the cell surface in normal amounts, these results argue that C2 domain phospholipid binding is necessary for robust signalling in vivo fine‐tuning the balance of trans and cis ligand–receptor interactions., Engineered mutations in the conserved N‐terminal C2 domains of endogenous Delta and Serrate show that perturbing one of the protruding loops conferring phospholipid binding leads to defective Notch signalling in vivo, confirming its contribution to normal ligand activity.

Cysteine plays an essential role in cellular redox homoeostasis as a key constituent of the tripeptide glutathione (GSH). A rate limiting step in cellular GSH synthesis is the availability of cysteine. However, circulating cysteine exists in the blood as the oxidised di-peptide cystine, requiring specialised transport systems for its import into the cell. System xc− is a dedicated cystine transporter, importing cystine in exchange for intracellular glutamate. To counteract elevated levels of reactive oxygen species in cancerous cells system xc− is frequently upregulated, making it an attractive target for anticancer therapies. However, the molecular basis for ligand recognition remains elusive, hampering efforts to specifically target this transport system. Here we present the cryo-EM structure of system xc− in both the apo and glutamate bound states. Structural comparisons reveal an allosteric mechanism for ligand discrimination, supported by molecular dynamics and cell-based assays, establishing a mechanism for cystine transport in human cells., System xc- is a cystine transporter that is expressed in the plasma membrane and imports cystine in exchange for intracellular glutamate. Here, the authors present the cryo-EM structure of human system xc- both in the apo form and the glutamate bound state, and further supported by molecular dynamics and cell-based assays they discuss its cystine transport mechanism.

Factor H–related protein (FHR) 1 is one of the five human FHRs that share sequence and structural homology with the alternative pathway complement inhibitor FH. Genetic studies on disease associations and functional analyses indicate that FHR-1 enhances complement activation by competitive inhibition of FH binding to some surfaces and immune proteins. We have recently shown that FHR-1 binds to pentraxin 3. In this study, our aim was to investigate whether FHR-1 binds to another pentraxin, C-reactive protein (CRP), analyze the functional relevance of this interaction, and study the role of FHR-1 in complement activation and regulation. FHR-1 did not bind to native, pentameric CRP, but it bound strongly to monomeric CRP via its C-terminal domains. FHR-1 at high concentration competed with FH for CRP binding, indicating possible complement deregulation also on this ligand. FHR-1 did not inhibit regulation of solid-phase C3 convertase by FH and did not inhibit terminal complement complex formation induced by zymosan. On the contrary, by binding C3b, FHR-1 allowed C3 convertase formation and thereby enhanced complement activation. FHR-1/CRP interactions increased complement activation via the classical and alternative pathways on surfaces such as the extracellular matrix and necrotic cells. Altogether, these results identify CRP as a ligand for FHR-1 and suggest that FHR-1 enhances, rather than inhibits, complement activation, which may explain the protective effect of FHR-1 deficiency in age-related macular degeneration.

Significance The complement system is a crucial antimicrobial system in the human body. However, controlling its regulation is essential, and failure to do so is implicated in a number of clinical inflammatory pathologies leading to great interest in therapeutic complement inhibition. We have identified and characterized a class of complement inhibitors from biting ticks. Utilizing both cryoelectron microscopy and X-ray crystallography we provide a comprehensive understanding of their mechanism of inhibition at the level of the terminal pathway of complement. We present a high-resolution cryoelectron microscopy structure of complement C5, the molecule targeted by the major therapeutic Eculizumab. In addition, we reveal the fold of the CirpT family of tick inhibitors and their unique mode of inhibition., The complement system is a crucial part of innate immune defenses against invading pathogens. The blood-meal of the tick Rhipicephalus pulchellus lasts for days, and the tick must therefore rely on inhibitors to counter complement activation. We have identified a class of inhibitors from tick saliva, the CirpT family, and generated detailed structural data revealing their mechanism of action. We show direct binding of a CirpT to complement C5 and have determined the structure of the C5–CirpT complex by cryoelectron microscopy. This reveals an interaction with the peripheral macro globulin domain 4 (C5_MG4) of C5. To achieve higher resolution detail, the structure of the C5_MG4–CirpT complex was solved by X-ray crystallography (at 2.7 Å). We thus present the fold of the CirpT protein family, and provide detailed mechanistic insights into its inhibitory function. Analysis of the binding interface reveals a mechanism of C5 inhibition, and provides information to expand our biological understanding of the activation of C5, and thus the terminal complement pathway.

Background:There is international interest in the training of psychological therapists to deliver evidence-based treatment for common mental health problems. The UK Improving Access to Psychological Therapies (IAPT) programme, one of the largest training initiatives, relies on competent therapists to successfully deliver cognitive behaviour therapy (CBT) and promote good patient outcome.Aims:To evaluate an IAPT CBT training course by assessing if trainees’ clinical skills improve during training and reach competency standards, and to report patient outcome for submitted training cases. To investigate a possible relationship between trainee competence and patient outcome. To explore professional differences during training.Method:CBT trainee (n = 252) competence was assessed via audio recordings of therapy sessions at the beginning, middle and end of training. Patient pre- to post-treatment outcomes were extracted from submitted training cases (n = 1927). Differences in professional background were examined across competence, academic final grade and tutorial support.Results:CBT trainees attained competence by the end of the course with 77% (anxiety recordings) and 72% (depression recordings) improving reliably. Training cases reported pre- to post-treatment effect sizes of 1.08–2.26 across disorders. CBT competence predicted a small variance in clinical outcome for depression cases. Differences in professional background emerged, with clinical psychologists demonstrating greater competence and higher academic grades. Trainees without a core professional background required more additional support to achieve competence.Conclusions:Part of a new CBT therapist workforce was successfully trained to deliver relatively brief treatment effectively. Trainees without a core profession can be successfully trained to competence, but may need additional support. This has implications for workforce training.

Background:The UK Department of Health Improving Access to Psychological Therapies (IAPT) initiative set out to train a large number of therapists in cognitive behaviour therapies (CBT) for depression and anxiety disorders. Little is currently known about the retention of IAPT CBT trainees, or the use of CBT skills acquired on the course in the workplace after training has finished.Aims:This study set out to conduct a follow-up survey of past CBT trainees on the IAPT High Intensity CBT Course at the Institute of Psychiatry, Psychology and Neuroscience (IoPPN), King's College London (KCL), one of the largest IAPT High Intensity courses in the UK.Method:Past trainees (n= 212) across 6 cohorts (2008-2014 intakes) were contacted and invited to participate in a follow-up survey. A response rate of 92.5% (n= 196) was achieved.Results:The vast majority of IAPT trainees continue to work in IAPT services posttraining (79%) and to practise CBT as their main therapy modality (94%); 61% have become CBT supervisors. A minority (23%) have progressed to other senior roles in the services. Shortcomings are reported in the use of out-of-office CBT interventions, the use of disorder-specific outcome measures and therapy recordings to inform therapy and supervision.Conclusions:Past trainees stay working in IAPT services and continue to use CBT methods taught on the course. Some NICE recommended treatment procedures that are likely to facilitate patients’ recovery are not being routinely implemented across IAPT services. The results have implications for the continued roll out of the IAPT programme, and other future large scale training initiatives.

The structures of SALSA SRCR domains 1 and 8 reveal a cation-dependent mechanism for ligand recognition, contributing to important roles in the immune system and cellular signalling. The cation-binding sites are conserved across all SRCR domains, suggesting conserved functional mechanisms., The scavenger receptor cysteine-rich (SRCR) family of proteins comprises more than 20 membrane-associated and secreted molecules. Characterised by the presence of one or more copies of the ∼110 amino-acid SRCR domain, this class of proteins have widespread functions as antimicrobial molecules, scavenger receptors, and signalling receptors. Despite the high level of structural conservation of SRCR domains, no unifying mechanism for ligand interaction has been described. The SRCR protein SALSA, also known as DMBT1/gp340, is a key player in mucosal immunology. Based on detailed structural data of SALSA SRCR domains 1 and 8, we here reveal a novel universal ligand-binding mechanism for SALSA ligands. The binding interface incorporates a dual cation-binding site, which is highly conserved across the SRCR superfamily. Along with the well-described cation dependency on most SRCR domain–ligand interactions, our data suggest that the binding mechanism described for the SALSA SRCR domains is applicable to all SRCR domains. We thus propose to have identified in SALSA a conserved functional mechanism for the SRCR class of proteins.

Community-based advocacy services are important in enabling victims to escape domestic abuse and rebuild their lives. This study evaluated a domestic abuse service. Two phases of research were conducted following case-file analysis ( n = 86): surveys ( n = 22) and interviews ( n = 12) with victims, and interviews with key individuals ( n = 12) based in related statutory and community organizations. The findings revealed the holistic model of legal, practical, mental health–related, and advocacy components resulted in a range of benefits to victims and enhanced interagency partnership working. Core elements of a successful needs-led, victim-centered service could be distilled.

The physiological roles of the factor H (FH)-related proteins are controversial and poorly understood. Based on genetic studies, FH-related protein 5 (CFHR5) is implicated in glomerular diseases, such as atypical hemolytic uremic syndrome, dense deposit disease, and CFHR5 nephropathy. CFHR5 was also identified in glomerular immune deposits at the protein level. For CFHR5, weak complement regulatory activity and competition for C3b binding with the plasma complement inhibitor FH have been reported, but its function remains elusive. In this study, we identify pentraxin 3 (PTX3) as a novel ligand of CFHR5. Binding of native CFHR5 to PTX3 was detected in human plasma and the interaction was characterized using recombinant proteins. The binding of PTX3 to CFHR5 is of ∼2-fold higher affinity compared with that of FH. CFHR5 dose-dependently inhibited FH binding to PTX3 and also to the monomeric, denatured form of the short pentraxin C–reactive protein. Binding of PTX3 to CFHR5 resulted in increased C1q binding. Additionally, CFHR5 bound to extracellular matrix in vitro in a dose-dependent manner and competed with FH for binding. Altogether, CFHR5 reduced FH binding and its cofactor activity on pentraxins and the extracellular matrix, while at the same time allowed for enhanced C1q binding. Furthermore, CFHR5 allowed formation of the alternative pathway C3 convertase and supported complement activation. Thus, CFHR5 may locally enhance complement activation via interference with the complement-inhibiting function of FH, by enhancement of C1q binding, and by activating complement, thereby contributing to glomerular disease.

Autoantibody formation against Factor H (FH) is found in 7–10% of patients who are diagnosed with atypical haemolytic uraemic syndrome (aHUS). These autoantibodies predominately target the C-terminal cell binding recognition domain of FH and are associated with absence of FHR1. Additional autoantibodies have also been identified in association with aHUS, for example autoantibodies to Factor I. Based on this, and that there are genetic mutations in other complement regulators and activators associated with aHUS, we hypothesised that other complement regulator proteins, particularly surface bound regulators in the kidney, might be the target for autoantibody formation in aHUS. Therefore, we assayed serum derived from 89 patients in the Newcastle aHUS cohort for the presence of autoantibodies to CD46 (membrane cofactor protein, MCP), CD55 (decay accelerating factor, DAF), CD35 (complement receptor type 1, CR1; TP10) and CD59. We also assayed 100 healthy blood donors to establish the normal levels of reactivity towards these proteins in the general population. Recombinant proteins CD46 and CD55 (purified from Escherichia coli) as well as soluble CR1 (CD35) and oligomeric C4BP-CD59 (purified from eukaryotic cell media) were used in ELISA to detect high responders. False positive results were established though Western blot and flow cytometric analysis. After excluding false positive responders to bacterial proteins in the CD46 and CD55 preparations, and responses to blood group antigens in CD35, we found no significant level of patient serum IgG reactivity with CD46, CD55, CD35 or CD59 above that detected in the normal population. These results suggest that membrane anchored complement regulators are not a target for autoantibody generation in aHUS.

Highlights • Protein crystallography plays a major role in integrative structural biology. • XFEL methods bring new opportunities for structural analysis of dynamic systems. • Crystallography and electron microscopy will be complementary into the future., While protein crystallography has, for many years, been the most used method for structural analysis of macromolecular complexes, remarkable recent advances in high-resolution electron cryo-microscopy led to suggestions that ‘the revolution will not be crystallised’. Here we highlight the current success rate, speed and ease of modern crystallographic structure determination and some recent triumphs of both ‘classical’ crystallography and the use of X-ray free electron lasers. We also outline fundamental differences between structure determination using X-ray crystallography and electron microscopy. We suggest that crystallography will continue to co-exist with electron microscopy as part of an integrated array of methods, allowing structural biologists to focus on fundamental biological questions rather than being constrained by the methods available.

BackgroundSubstantial policy, communication and operational gaps exist between mental health services and the police for individuals with enduring mental health needs.AimsTo map and cost pathways through mental health and police services, and to model the cost impact of implementing key policy recommendations.MethodWithin a case-linkage study, we estimated 1-year individual-level healthcare and policing costs. Using decision modelling, we then estimated the potential impact on costs of three recommended service enhancements: street triage, Mental Health Act assessments for all Section 136 detainees and outreach custody link workers.ResultsUnder current care, average 1-year mental health and police costs were £10 812 and £4552 per individual respectively (n = 55). The cost per police incident was £522. Models suggested that each service enhancement would alter per incident costs by between −8% and +6%.ConclusionsRecommended enhancements to care pathways only marginally increase individual-level costs.

Summary The Notch pathway is a core cell-cell signaling system in metazoan organisms with key roles in cell-fate determination, stem cell maintenance, immune system activation, and angiogenesis. Signals are initiated by extracellular interactions of the Notch receptor with Delta/Serrate/Lag-2 (DSL) ligands, whose structure is highly conserved throughout evolution. To date, no structure or activity has been associated with the extreme N termini of the ligands, even though numerous mutations in this region of Jagged-1 ligand lead to human disease. Here, we demonstrate that the N terminus of human Jagged-1 is a C2 phospholipid recognition domain that binds phospholipid bilayers in a calcium-dependent fashion. Furthermore, we show that this activity is shared by a member of the other class of Notch ligands, human Delta-like-1, and the evolutionary distant Drosophila Serrate. Targeted mutagenesis of Jagged-1 C2 domain residues implicated in calcium-dependent phospholipid binding leaves Notch interactions intact but can reduce Notch activation. These results reveal an important and previously unsuspected role for phospholipid recognition in control of this key signaling system., Graphical Abstract, Highlights • Notch ligands contain an N-terminal C2 phospholipid recognition domain • Ca2+-dependent lipid binding by Jagged 1 is required for optimal Notch activation • Lipid binding by Notch ligands is calcium dependent • Notch ligands require bound calcium at the N terminus for full activity, Handford, Lea, and colleagues now discover that the N-terminal portion of Notch ligands consists of a functional, calcium-dependent phospholipid recognition domain, the C2 domain. They also show that Notch-driven activation requires C2 domain activity in addition to canonical Notch-ligand binding.

Summary Pore-forming proteins containing the structurally conserved membrane attack complex/perforin fold play an important role in immunity and host-pathogen interactions. Intermedilysin (ILY) is an archetypal member of a cholesterol-dependent cytolysin subclass that hijacks the complement receptor CD59 to make cytotoxic pores in human cells. ILY directly competes for the membrane attack complex binding site on CD59, rendering cells susceptible to complement lysis. To understand how these bacterial pores form in lipid bilayers and the role CD59 plays in complement regulation, we determined the crystal structure of human CD59 bound to ILY. Here, we show the ILY-CD59 complex at 3.5 Å resolution and identify two interfaces mediating this host-pathogen interaction. An ILY-derived peptide based on the binding site inhibits pore formation in a CD59-containing liposome model system. These data provide insight into how CD59 coordinates ILY monomers, nucleating an early prepore state, and suggest a potential mechanism of inhibition for the complement terminal pathway., Graphical Abstract, Highlights • Crystal structure of the ILY-CD59 complex defines two interfaces • Our two binding interfaces are supported by previous mutagenesis studies • An ILY-derived peptide competes for binding in a liposome model system • Our model provides a structural basis for CD59 nucleation of an ILY early prepore, The crystal structure of human CD59 in complex with the bacterial toxin intermedilysin (ILY) is now presented by Bubeck and colleagues. These results suggest that CD59 positions the cholesterol-binding motif of ILY in proximity to the membrane and nucleates oligomerization of ILY monomers through two binding faces. This model for CD59 function gives insight into the mechanism of cholesterol-dependent cytolysin pore formation and provides a framework for future investigation probing regulation of the complement terminal pathway.

The complement system is a key component regulation influences susceptibility to age-related macular degeneration, meningitis, and kidney disease. Variation includes genomic rearrangements within the complement factor H-related ( CFHR ) locus. Elucidating the mechanism underlying these associations has been hindered by the lack of understanding of the biological role of CFHR proteins. Here we present unique structural data demonstrating that three of the CFHR proteins contain a shared dimerization motif and that this hitherto unrecognized structural property enables formation of both homodimers and heterodimers. Dimerization confers avidity for tissue-bound complement fragments and enables these proteins to efficiently compete with the physiological complement inhibitor, complement factor H (CFH), for ligand binding. Our data demonstrate that these CFHR proteins function as competitive antagonists of CFH to modulate complement activation in vivo and explain why variation in the CFHRs predisposes to disease.

Factor I (fI) is a key serine protease that modulates the complement cascade by regulating the levels of C3 convertases. Human fI circulates in plasma as a heavily N-glycosylated (25–27% w/w) heterodimer composed of two disulphide linked chains, each carrying three N-linked oligosaccharide chains. It had been suggested that the oligosaccharides may have both structural and functional roles in the interactions with the natural substrate and the cofactor during a catalysis. The N-linked glycans of each fI chain were characterised in detail and the analysis revealed a similar composition of the glycan pools with both chains heavily sialylated. Disialylated structures were in excess over monosialylated ones: 55% over 40% for the heavy chain and 62% over 35% for the light chain. The dominant type of glycan identified on both chains was A2G2S2, a biantennary structure with chains terminating in sialic acid linked to galactose. The glycan characterisation facilitated a strategy for the partial deglycosylation of the enzyme. Assessment of the proteolytic activities of the native and partially deglycosylated forms of fI showed that both forms of the enzyme have very similar proteolytic activities against C3(NH3) indicating that the charged glycans of fI do not influence the fI-cofactor–substrate interactions.

Decay-accelerating factor (DAF; CD55) inhibits the complement (C) cascade by dissociating the multimolecular C3 convertase enzymes central to amplificatiom. We have previously demonstrated using surface plasmon resonance (Biacore International) that DAF mediates decay of the alternative pathway C3 convertase, C3bMb, but not of the inactive proenzyme, C3bB, and have shown that the major site of interaction is with the larger cleavage subunit factor B (Bb) subunit. In this study, we dissect these interactions and demonstrate that the second short consensus repeat (SCR) domain of DAF (SCR2) interacts only with Bb, whereas SCR4 interacts with C3b. Despite earlier studies that found SCR3 to be critical to DAF activity, we find that SCR3 dees not directly interact with either subunit. Furthermore, we demonstrate that properdin, a positive regulator of the alternative pathway, does not directly interact with DAF. Extending from studies of binding to decay-accelerating activity, we show that truncated forms of DAF consisting of SCRs 2 and 3 bind the convertase stably via SCR1-Bb interactions but have little functional activity. In contrast, an SCR34 construct mediates decay acceleration, presumably due to SCR4-C3b interactions demonstrated above, because SCR3 alone has no binding or functional effect. We propose that DAF interacts with C3bBb through major sites in SCR2 and SCR4. Binding to Bb via SCR2 increases avidity of binding, concentrating DAF on the active convertase, whereas more transient interactions through SCR4 with C3b directly mediate decay acceleration. These data provide new insights into the mechanisms involved in C3 convertase decay by DAF. Copyright © 2006 by The American Association of Immunologists, Inc.

Centrioles organise centrosomes and template cilia and flagella. Several centriole and centrosome proteins have been linked to microcephaly (MCPH), a neuro-developmental disease associated with small brain size. CPAP (MCPH6) and STIL (MCPH7) are required for centriole assembly, but it is unclear how mutations in them lead to microcephaly. We show that the TCP domain of CPAP constitutes a novel proline recognition domain that forms a 1:1 complex with a short, highly conserved target motif in STIL. Crystal structures of this complex reveal an unusual, all-β structure adopted by the TCP domain and explain how a microcephaly mutation in CPAP compromises complex formation. Through point mutations, we demonstrate that complex formation is essential for centriole duplication in vivo. Our studies provide the first structural insight into how the malfunction of centriole proteins results in human disease and also reveal that the CPAP–STIL interaction constitutes a conserved key step in centriole biogenesis. DOI: http://dx.doi.org/10.7554/eLife.01071.001, eLife digest Organisms—and individual tissues—grow and develop by dividing their cells. However, the process of cell division does not have to be symmetric, and the fates of the cells can be very different if cellular contents, including RNAs or proteins, are exclusively retained in the ‘mother’ or passed to her ‘daughter’. Organelles known as centrioles can play an important part in influencing whether cell division is symmetric or asymmetric. Centrioles contain ordered assemblies of various proteins, and mutations in some of these proteins can cause developmental defects in humans. For example, mutations in the centriolar proteins CPAP and STIL cause a syndrome known as microcephaly, in which the brain is smaller than normal. Although CPAP and STIL are known to bind each other, how they interact on a molecular level to form centrioles—and how this interaction is disrupted in microcephaly—is not well understood. Cottee et al. have now used structural and biochemical assays to explore how these two proteins bind to each other, and have identified specific amino acid residues that enable this interaction. These residues are highly conserved across many organisms, and a mutation in one of them has previously been associated with microcephaly in humans. Now, Cottee et al. demonstrate that this mutation weakens the interaction between CPAP and STIL in vitro. To explore these processes in vivo, Cottee et al. studied mutant fruit flies in which the interactions between CPAP and STIL were weaker than normal, and found that these mutations prevented the normal formation of centrioles. Furthermore, there was a striking correlation between the ability to form centrioles in fruit flies and the ability of CPAP and STIL to bind each other, based on the structural model and in vitro binding studies. Cumulatively, these findings reinforce the importance of CPAP and STIL in centriole formation, and suggest that one reason for the development of microcephaly may be defects in the proper formation of centrioles. DOI: http://dx.doi.org/10.7554/eLife.01071.002

To provide insight into the structural and functional properties of human complement component 5 (C5), we determined its crystal structure at a resolution of 3.1 A. The core of C5 adopted a structure resembling that of C3, with the domain arrangement at the position corresponding to the C3 thioester being very well conserved. However, in contrast to C3, the convertase cleavage site in C5 was ordered and the C345C domain flexibly attached to the core of C5. Binding of the tick C5 inhibitor OmCI to C5 resulted in stabilization of the global conformation of C5 but did not block the convertase cleavage site. The structure of C5 may render possible a structure-based approach for the design of new selective complement inhibitors.

Decay-accelerating factor (DAF, CD55) is a glycophosphatidyl inositol-anchored glycoprotein that regulates the activity of C3 and C5 convertases. In addition to understanding the mechanism of complement inhibition by DAF through structural studies, there is also an interest in the possible therapeutic potential of the molecule. In this report we describe the cloning, expression in Escherichia coli, isolation and membrane-targeting modification of the four short consensus repeat domains of soluble human DAF with an additional C-terminal cysteine residue to permit site-specific modification. The purified refolded recombinant protein was active against both classical and alternative pathway assays of complement activation and had similar biological activity to soluble human DAF expressed in Pichia pastoris. Modification with a membrane-localizing peptide restored cell binding and gave a large increase in antihemolytic potency. These data suggested that the recombinant DAF was correctly folded and suitable for structural studies as well as being the basis for a DAF-derived therapeutic. Crystals of the E. coli-derived protein were obtained and diffracted to 2.2 A, thus permitting the first detailed X-ray crystallography studies on a functionally active human complement regulator protein with direct therapeutic potential.

Factor H binding protein (fHbp) is one of the main antigens of the 4-component meningococcus B (4CMenB) multicomponent vaccine against disease caused by serogroup B Neisseria meningitidis (MenB). fHbp binds the complement down-regulating protein human factor H (hfH), thus resulting in immune evasion. fHbp exists in 3 variant groups with limited cross-protective responses. Previous studies have described the generation of monoclonal antibodies (mAbs) targeting variant-specific regions of fHbp. Here we report for the first time the functional characterization of two mAbs that recognize a wide panel of fHbp variants and subvariants on the MenB surface and that are able to inhibit fHbp binding to hfH. The antigenic regions targeted by the two mAbs were accurately mapped by hydrogen-deuterium exchange mass spectrometry (HDX-MS), revealing partially overlapping epitopes on the N terminus of fHbp. Furthermore, while none of the mAbs had bactericidal activity on its own, a synergistic effect was observed for each of them when tested by the human complement serum bactericidal activity (hSBA) assay in combination with a second nonbactericidal mAb. The bases underlying fHbp variant cross-reactivity, as well as inhibition of hfH binding and cooperativity effect observed for the two mAbs, are discussed in light of the mapped epitopes.

Complement component C8 plays a pivotal role in the formation of the membrane attack complex (MAC), an important antibacterial immune effector. C8 initiates membrane penetration and coordinates MAC pore formation. High-resolution structures of C8 subunits have provided some insight into the function of the C8 heterotrimer; however, there is no structural information describing how the intersubunit organization facilitates MAC assembly. We have determined the structure of C8 by electron microscopy and fitted the C8α-MACPF (membrane attack complex/perforin)-C8γ co-crystal structure and a homology model for C8β-MACPF into the density. Here, we demonstrate that both the C8γ protrusion and the C8α-MACPF region that inserts into the membrane upon activation are accessible., Graphical Abstract Research Highlights ► Complement component C8 initiates membrane penetration of the membrane attack complex, an important innate immune effector. ► The structure of C8 reveals a core domain with a globular protrusion. ► Docking of pseudo-atomic models into the electron microscopy reconstruction indicates that C8γ projects away from the C8αβ core, in which the predicted transmembrane residues of C8α-MACPF and one face of the C8β-MACPF are surface exposed.

Centrioles are among the most beautiful of biological structures. How their highly conserved nine-fold symmetry is generated is a question that has intrigued cell biologists for decades. Two recent structural studies provide the tantalizing suggestion that the self-organizing properties of the SAS-6 protein hold the answer.

In a field where structure has finally begun to have a real impact, a series of new structures over the last two years have further extended our understanding of some of the critical regulatory events of the complement system. Notably, information has begun to flow from larger assemblies of components which allow insight into the often transient assemblies critical to complement regulation at the cell surface. This review will summarise the key structures determined since the last International Complement Workshop and the insights these have given us, before highlighting some questions that still require molecular frameworks to drive understanding.

Factor I (FI) is a crucial inhibitor controlling all complement pathways due to its ability to degrade activated complement proteins C3b and C4b in the presence of cofactors such as factor H, C4b-binding protein, complement receptor 1 or CD46. Complete deficiency of FI, which is synthesized mainly in the liver is rare and leads to complement consumption resulting in recurrent severe infections, glomerulonephritis or autoimmune diseases. Incomplete FI deficiency is in turn associated with atypical haemolytic uremic syndrome, a severe disease characterized by thrombocytopenia, microangiopathic haemolytic anaemia and acute renal failure. Structurally, FI is a 88kDa heterodimer of a heavy chain consisting of one FI-membrane attack complex (FIMAC) domain, one CD5 domain and two low-density lipoprotein receptor domains (LDLr), and a light chain which is a serine protease domain (SP), linked to the heavy chain by a disulfide bond. FI cleaves its in vivo substrates C3b and C4b only in the presence of cofactors, it shows poor enzymatic activity towards synthetic substrates tested so far and it has no natural inhibitor.

Background: The site of Jagged/Serrate ligand recognition by Notch is unknown. Results: Two critical residues involved in an intramolecular hydrophobic interaction across the central β-sheet of EGF12 form a ligand-binding platform. Conclusion: The ligand-binding region is adjacent to a Fringe-sensitive residue involved in modulating Notch activity. Significance: The results have implications for understanding receptor/ligand recognition, Notch regulation by O-glycosylation, and the development of paralogue-specific antibodies., We have mapped a Jagged/Serrate-binding site to specific residues within the 12th EGF domain of human and Drosophila Notch. Two critical residues, involved in a hydrophobic interaction, provide a ligand-binding platform and are adjacent to a Fringe-sensitive residue that modulates Notch activity. Our data suggest that small variations within the binding site fine-tune ligand specificity, which may explain the observed sequence heterogeneity in mammalian Notch paralogues, and should allow the development of paralogue-specific ligand-blocking antibodies. As a proof of principle, we have generated a Notch-1-specific monoclonal antibody that blocks binding, thus paving the way for antibody tools for research and therapeutic applications.

Activation of complement C5 generates the potent anaphylatoxin C5a and leads to pathogen lysis, inflammation and cell damage. The therapeutic potential of C5 inhibition has been demonstrated by eculizumab, one of the world's most expensive drugs. However, the mechanism of C5 activation by C5 convertases remains elusive, thus limiting development of therapeutics. Here we identify and characterize a new protein family of tick-derived C5 inhibitors. Structures of C5 in complex with the new inhibitors, the phase I and phase II inhibitor OmCI, or an eculizumab Fab reveal three distinct binding sites on C5 that all prevent activation of C5. The positions of the inhibitor-binding sites and the ability of all three C5-inhibitor complexes to competitively inhibit the C5 convertase conflict with earlier steric-inhibition models, thus suggesting that a priming event is needed for activation.

This study investigated the social construction of domestic abuse by police officers, specifically in the context of arguments presented to the prosecutor for a decision on whether to proceed with or discontinue the case. Nineteen police files were examined with a particular focus on the MG3, the “Report to Crown Prosecutors for Charging Decision.” Access to such sensitive material is usually denied to researchers; therefore, this study offers unusual insights into the treatment of victims and perpetrators of interpersonal violence by the police. Discourse analysis revealed three dominant speech genres: impartiality, credibility, and the “real” victim. These genres separately and in interaction served to construct domestic abuse cases in ways that did not support the victim’s account. The “dialogic reverberations” of these findings are discussed and the implications of the work for research and practice are considered.

A review of published tetartohedrally twinned macromolecular structures is presented, together with details of the recent structure determination of triclinic tetartohedrally twinned crystals of human complement factor I., Tetartohedral crystal twinning is discussed as a particular case of (pseudo)merohedral twinning when the number of twinned domains is four. Tetartohedrally twinned crystals often possess pseudosymmetry, with the rotational part of the pseudo­symmetry operators coinciding with the twinning operators. Tetartohedrally twinned structures from the literature are reviewed and the recent structure determination of tetartohedrally twinned triclinic crystals of human complement factor I is discussed.

Summary Background and objectives Atypical hemolytic uremic syndrome is a disease associated with mutations in the genes encoding the complement regulators factors H and I. In addition, factor H autoantibodies have been reported in ;10% of patients with atypical hemolytic uremic syndrome. This study searched for the presence of factor I autoantibodies in atypical hemolytic uremic syndrome. Design, setting, participants, & measurements This study screened 175 atypical hemolytic uremic syndrome patients for factor I autoantibodies using ELISA with confirmatory Western blotting. Functional studies using purified immunoglobulin from one patient were subsequently undertaken. ResultsFactor Iautoantibodieswere detectedinthree patients.Inone patient withah ightiterofautoantibody, the titer was tracked over time and was found to have no association with disease activity. This study found evidence of animmune complex of antibody and factor Ii nthis patient,but purifiedIgG,isolated from current serum samples, had only a minor effect on fluid phase and cell surface complement regulation. Genetic analysis of the three patients with factor I autoantibodies revealed that they had two copies of the genes encoding factor H–related proteins 1 and 3 and therefore, did no th ave ad eletion commonly associated with factor H autoantibodies in atypical hemolytic uremic syndrome. Two patients, however, had functionally significant mutations in complement factor H. Conclusions These findings reinforce the concept of multiple concurrent risk factors being associated with atypical hemolytic uremic syndrome but question whether autoantibodies per se predispose to atypical hemolytic uremic syndrome. Clin J Am Soc Nephrol 7: 417–426, 2012. doi: 10.2215/CJN.05750611

The structure of rat CrrY1–4 determined in two distinct crystal forms shows a pronounced bend at the interface between domains 3 and 4., Complement receptor 1-related protein Y (CrrY) is an important cell-surface regulator of complement that is unique to rodent species. The structure of rat CrrY domains 1–4 has been determined in two distinct crystal forms and reveals a 70° bend between domains 3 and 4. Comparisons of this structure with those of other complement regulators suggests that rearrangement of this interface may occur on forming the regulatory complex with C3b.

Objective Nurses need to be competent and confident in performing drug calculations to ensure patient safety. The purpose of this study is to compare an interactive e-drug calculations package, developed using Cognitive Load Theory as its theoretical framework, with traditional handout learning support on nursing students' drug calculation ability, self-efficacy and support material satisfaction. Design A cluster randomised controlled trial comparing the e-package with traditional handout learning support was conducted with a September cohort ( n =137) and a February cohort ( n =92) of second year diploma nursing students. Students from each cohort were geographically dispersed over 3 or 4 independent sites. Measurements Students from each cohort were invited to participate, halfway through their second year, before and after a 12 week clinical practice placement. During their placement the intervention group received the e-drug calculations package while the control group received traditional ‘handout' support material. Drug calculation ability and self-efficacy tests were given to the participants pre- and post-intervention. Participants were given the support material satisfaction scale post-intervention. Results Students in both cohorts randomised to e-learning were more able to perform drug calculations than those receiving the handout (September: mean 48.4% versus 34.7%, p =0.027; February: mean 47.6% versus 38.3%, p =0.024). February cohort students using the e-package were more confident in performing drug calculations than those students using handouts (self-efficacy mean 56.7% versus 45.8%, p =0.022). There was no difference in improved self-efficacy between intervention and control for students in the September cohort. Students who used the package were more satisfied with its use than the students who used the handout (mean 29.6 versus 26.5, p =0.001), particularly with regard to the package enhancing their learning ( p =0.023), being an effective way to learn ( p =0.005), providing practice and feedback ( p p =0.027), user friendly ( p =0.02) and providing learning enjoyment ( p =0.022). Conclusion It is essential that nurses are educated and supported to become, and remain, confident and competent in performing drug calculations. This study found the e-drug calculations package, based on Cognitive Load Theory, to be significantly more effective than a handout in improving students' drug calculation ability and self-efficacy, with students who used the package being significantly more satisfied with its use than students who used the handout. This package could particularly be useful for the continuing professional development of any healthcare professional involved in drug calculations.

Background and aims: CD8 T cells are central to the control of hepatitis C virus (HCV) although the key features of a successful CD8 T cell response remain to be defined. In a cohort of Irish women infected by a single source, a strong association between viral clearance and the human lecucocyte (HLA)-A*03 allele has been described, and the aim of this study was to define the protective nature of the associated CD8 T cell response. Methods: A sequence-led approach was used to identify HLA-A*03-restricted epitopes. We examine the CD8 T cell response associated with this gene and address the likely mechanism underpinning this protective effect in this special cohort, using viral sequencing, T cell assays and analysis of fitness of viral mutants. Results: A strong 'HLA footprint' in a novel NS3 epitope (TVYHGAGTK) was observed. A lysine (K) to arginine (R) substitution at position 9 (K1088R) was seen in a significant number of A*03-positive patients (9/12) compared with the control group (1/33, p=0.0003). Threonine (T) was also substituted with alanine (A) at position 8 (T1087A) more frequently in A*03-positive patients (6/12) compared with controls (2/33, p=0.01), and the double substitution of TK to AR was also observed predominantly in HLA-A*03- positive patients (p=0.004). Epitope-specific CD8 T cell responses were observed in 60% of patients three decades after exposure and the mutants selected in vivo impacted on recognition in vitro. Using HCV replicons matched to the viral sequences, viral fitness was found to be markedly reduced by the K1088R substitution but restored by the second substitution T1087A. Conclusions: It is proposed that at least part of the protective effect of HLA-A*03 results from targeting of this key epitope in a functional site: the requirement for two mutations to balance fitness and escape provides an initial host advantage. This study highlights the potential protective impact of common HLA-A alleles against persistent viruses, with important implications for HCV vaccine studies.

Title. Patient safety: numerical skills and drug calculation abilities of nursing students and Registered Nurses. Aim. This paper is a report of a correlational study of the relations of age, status, experience and drug calculation ability to numerical ability of nursing students and Registered Nurses. Background. Competent numerical and drug calculation skills are essential for nurses as mistakes can put patients’ lives at risk. Method. A cross-sectional study was carried out in 2006 in one United Kingdom university. Validated numerical and drug calculation tests were given to 229 second year nursing students and 44 Registered Nurses attending a non-medical prescribing programme. Results. The numeracy test was failed by 55% of students and 45% of Registered Nurses, while 92% of students and 89% of nurses failed the drug calculation test. Independent of status or experience, older participants (‡35 years) were statistically significantly more able to perform numerical calculations. There was no statistically significant difference between nursing students and Registered Nurses in their overall drug calculation ability, but nurses were statistically significantly more able than students to perform basic numerical calculations and calculations for solids, oral liquids and injections. Both nursing students and Registered Nurses were statistically significantly more able to perform calculations for solids, liquid oral and injections than calculations for drug percentages, drip and infusion rates. Conclusion. To prevent deskilling, Registered Nurses should continue to practise and refresh all the different types of drug calculations as often as possible with regular (self)-testing of their ability. Time should be set aside in curricula for nursing students to learn how to perform basic numerical and drug calculations. This learning should be reinforced through regular practice and assessment.

Background: Students on health and social care degree programmes spend 50% of their time on practice placements. Because of the diversity of settings and the need to evidence their work, it is vital to understand the information and resource needs of placement students. Objectives: The aim of this investigation was to understand the needs of placement students in terms of accessing resources whilst they are in the field in order to inform a guide to meet these needs. Methods: Focus groups were conducted with students on midwifery, social work and post-registration health professions degree programmes on three different sites across the region. Data were analysed using Thematic Content Analysis. Results: Three themes emerged from the data: inequality, user education needs and students’ solutions and strategies. Conclusions: It is essential to speak to placement students in order to understand their needs in terms of accessing and using library resources. The timing and content of information skills training is key to meeting student needs while on placement.

The Notch receptor and its ligands are key components in a core metazoan signaling pathway that regulates the spatial patterning, timing and outcome of many cell-fate decisions. Ligands contain a disulfide-rich Delta/Serrate/LAG-2 (DSL) domain required for Notch trans-activation or cis-inhibition. Here we report the X-ray structure of a receptor binding region of a Notch ligand, the DSL-EGF3 domains of human Jagged-1 (J-1(DSL-EGF3)). The structure reveals a highly conserved face of the DSL domain, and we show, by functional analysis of Drosophila melanogster ligand mutants, that this surface is required for both cis- and trans-regulatory interactions with Notch. We also identify, using NMR, a surface of Notch-1 involved in J-1(DSL-EGF3) binding. Our data imply that cis- and trans-regulation may occur through the formation of structurally distinct complexes that, unexpectedly, involve the same surfaces on both ligand and receptor.

CD97, the archetypal member of the EGF-TM7 protein family, is constitutively expressed on granulocytes and monocytes and rapidly up-regulated on T and B cells following activation. The key isoform of CD97 expressed on leukocytes binds the complement regulatory protein CD55 (also termed decay-accelerating factor). CD97 has been shown recently to mediate co-stimulation of T cells via CD55. Here, we demonstrate that blocking the interaction between CD55 on monocytes and CD97 on T cells leads to inhibition of proliferation and interferon-gamma secretion. This implies that bidirectional interactions between CD97 and CD55 are involved in T cell regulation. Structural studies presented here reveal the molecular basis for this activity. We have solved the structure of EMR2, a very close homolog of CD97, using x-ray crystallography. NMR-based chemical shift mapping of the EMR2-CD55 interaction has allowed us to generate a model for the CD97-CD55 complex. The structure of the complex reveals that the T cell and complement regulatory activities of CD55 occur on opposite faces of the molecule. This suggests that CD55 might simultaneously regulate both the innate and adaptive immune responses, and we have shown that CD55 can still regulate complement when bound to CD97.

The attrition of rape cases from the criminal justice system (CJS) remains high and there is a paucity of research in relation to marginalized groups. Sex workers (SWs) are vulnerable to sexual violence due to the nature of their work. They are also unlikely to report such violence to police for a range of reasons. Two stages of research sought to describe the victim, perpetrator, and offense characteristics of SW rape and to examine the attrition of these cases. All rapes and attempted rapes ( N = 1,146) reported to police in a large city in the South West of England over a 21-year period were examined; 67 cases involved SWs. Data were extracted from police files in line with the variables of interest. Secondary analysis of the total number of SW rapes ( n = 67) resulted in a profile of these cases. A matched pairs study revealed significant differences in victim, perpetrator, and assault characteristics between SW ( n = 62) and non-sex-worker (NSW) samples ( n = 62). Although no significant difference was found in terms of attrition from the CJS, SW cases were observed to secure more convictions for rape than NSW cases. The implications of the findings for practice and future research are discussed.

A polymorphism in complement factor H has recently been associated with age-related macular degeneration (AMD), the leading cause of blindness in the elderly. A histidine rather than a tyrosine at residue position 384 in the mature protein increases the risk of AMD. Here, using a recombinant construct, we show that amino acid 384 is adjacent to a heparin-binding site in CCP7 of factor H and demonstrate that the allotypic variants differentially recognize heparin. This functional alteration may affect binding of factor H to polyanionic patterns on host surfaces, potentially influencing complement activation, immune complex clearance, and inflammation in the macula of AMD patients.

Many enteroviruses bind to the complement control protein decay-accelerating factor (DAF) to facilitate cell entry. We present here a structure for echovirus (EV) type 12 bound to DAF using cryo-negative stain transmission electron microscopy and three-dimensional image reconstruction to 16-A resolution, which we interpreted using the atomic structures of EV11 and DAF. DAF binds to a hypervariable region of the capsid close to the 2-fold symmetry axes in an interaction that involves mostly the short consensus repeat 3 domain of DAF and the capsid protein VP2. A bulge in the density for the short consensus repeat 3 domain suggests that a loop at residues 174-180 rearranges to prevent steric collision between closely packed molecules at the 2-fold symmetry axes. Detailed analysis of receptor interactions between a variety of echoviruses and DAF using surface plasmon resonance and comparison of this structure (and our previous work; Bhella, D., Goodfellow, I. G., Roversi, P., Pettigrew, D., Chaudhry, Y., Evans, D. J., and Lea, S. M. (2004) J. Biol. Chem. 279, 8325-8332) with reconstructions published for EV7 bound to DAF support major differences in receptor recognition among these viruses. However, comparison of the electron density for the two virus.receptor complexes (rather than comparisons of the pseudo-atomic models derived from fitting the coordinates into these densities) suggests that the dramatic differences in interaction affinities/specificities may arise from relatively subtle structural differences rather than from large-scale repositioning of the receptor with respect to the virus surface.

The idea of citizenship dates back to classical antiquity. It was originally concerned to address legitimacy of occupancy in the public sphere. Our empirical study contributes to the project of developing a social psychology of the citizen by focusing on the dynamics of such membership, specifically rights and identities. The authors briefly describe a number of existing psychological models of the citizen. Drawing on the main theoretical principles of discursive psychology, rather than asking, 'who is the citizen?' in terms of mental states, we suggest a shift in focus to the more social question, 'how do people claim citizenship and to what ends?'. We present an analysis of private letters of complaint that formed part of a larger mixed data set used in a recent research programme centred on disputes over Britain's newer travellers' rights of settlement. Specifically our analysis demonstrates how some of the letter writers generate a basis for claims-making by making relevant a citizenship/ governance alignment of identities. We also demonstrate how the entitlements associated with the category citizen are built up and action-oriented rather than flowing from the (unproblematic) assumption of citizenship. Finally we discuss how citizenship can be used for the purposes of inclusion and exclusion.

A cellular receptor for the haemagglutinating enteroviruses (HEV), and the protein that mediates haemagglutination, is the membrane complement regulatory protein decay accelerating factor (DAF; CD55). Although primate DAF is highly conserved, significant differences exist to enable cell lines derived from primates to be utilized for the characterization of the DAF binding phenotype of human enteroviruses. Thus, several distinct DAF-binding phenotypes of a selection of HEVs (viz. coxsackievirus A21 and echoviruses 6, 7, 11-13, 29) were identified from binding and infection assays using a panel of primate cells derived from human, orang-utan, African Green monkey and baboon tissues. These studies complement our recent determination of the crystal structure of SCR(34) of human DAF [Williams, P., Chaudhry, Y., Goodfellow, I. G., Billington, J., Powell, R., Spiller, O. B., Evans, D. J. and Lea, S. (2003). J Biol Chem 278, 10691-10696] and have enabled us to better map the regions of DAF with which enteroviruses interact and, in certain cases, predict specific virus-receptor contacts.

Theories of sex offending have for several years relied upon the notion of cognitive distortions as an important cause of sexual offending. In this study we critique this notion and suggest that the sort of phenomenon addressed by cognitive distortions is better understood by adopting a discursive psychology approach. In this approach, talk is regarded as occasioned and action oriented. Thus 'cognitive distortions' are conceptualized as something people do rather than something that people have. Sessions from a prison-based sex offender treatment programme were taped and transcribed. A discursive psychology analysis was conducted on those sessions relating to offenders' first accounts of their offences. Our analysis suggests that offenders utilize a particular narrative organization to manage their blame and responsibility for the offence. This organization is based on a first part which is oriented to quotidian precursors to the offence and an immediately following second which is oriented to a sudden shift in the definition of the situation. The implications of this analysis are discussed, in relation to the status of cognitive distortions and treatment.

We have used X-ray crystallography to determine the structure of a decay accelerating factor (DAF)-binding, clinic-derived isolate of echovirus 11 (EV11-207). The structures of the capsid proteins closely resemble those of capsid proteins of other picornaviruses. The structure allows us to interpret a series of amino acid changes produced by passaging EV11-207 in different cell lines as highlighting the locations of multiple receptor-binding sites on the virion surface. We suggest that a DAF-binding site is located at the fivefold axes of the virion, while the binding site for a distinct but as yet unidentified receptor is located within the canyon surrounding the virion fivefold axes.

Genome-wide association studies have found variation within the complement factor H gene family links to host susceptibility to meningococcal disease caused by infection with Neisseria meningitidis (Davila et al., 2010). Mechanistic insights have been challenging since variation within this locus is complex and biological roles of the factor H-related proteins, unlike factor H, are incompletely understood. N. meningitidis subverts immune responses by hijacking a host-immune regulator, complement factor H (CFH), to the bacterial surface (Schneider et al., 2006; Madico et al., 2007; Schneider et al., 2009). We demonstrate that complement factor-H related 3 (CFHR3) promotes immune activation by acting as an antagonist of CFH. Conserved sequences between CFH and CFHR3 mean that the bacterium cannot sufficiently distinguish between these two serum proteins to allow it to hijack the regulator alone. The level of protection from complement attack achieved by circulating N. meningitidis therefore depends on the relative levels of CFH and CFHR3 in serum. These data may explain the association between genetic variation in both CFH and CFHR3 and susceptibility to meningococcal disease. DOI: http://dx.doi.org/10.7554/eLife.04008.001, eLife digest Meningitis is a potentially life-threatening condition whereby the membranes that cover and protect the brain and spinal cord become inflamed. Often meningitis is caused by a viral or bacterial infection—such as infection by a bacterium called Neisseria meningitidis, also known as meningococcus. However, not everyone that comes into contact with this bacterium will develop meningitis; 40% of the population is thought to carry N. meningitidis at the back of the nasal cavity and yet show no signs of the disease. It remains unclear why some people exposed to N. meningitidis develop meningitis while others do not; however recent research revealed that part of the immune system called the complement system plays a role in susceptibility to meningitis. The complement system is a collection of small proteins that work together to support the actions of the cells of the immune system. When activated, complement proteins trigger a cascade of events that helps to destroy the pathogen. Several mechanisms exist to keep the complement proteins in check—for example, a protein called complement factor H (or CFH) protects host cells from being attacked by other complement proteins. N. meningitidis can undermine the complement system by expressing a protein that binds to CFH and firmly fixes CFH to its cell surface. While the CFH-binding protein helps explain why some people are unable to mount the appropriate immune response to infection by N. meningitidis, it does not explain why some carriers of the pathogen do not develop meningitis. Now, Caesar et al. have examined a protein called CFH related-3 (or CFHR3), and discovered that CFHR3 competes with CFH for the binding protein on N. meningitidis. CFHR3 is structurally similar to CFH, but it is unable to regulate or silence the complement system. Caesar et al. explain that susceptibility to meningococcal disease is determined by how much CFH and how much CFHR3 each individual has, and that those with less CFHR3 will be more susceptible to N. meningitidis. An individual's genes will affect how much CFH and CFHR3 they have, while the genes of the bacterium can influence how strongly the CFH binding protein binds to either of these human proteins. Caesar et al. suggest that these two factors determine whether or not an individual will develop meningitis or simply carry the bacterium without any ill effects. Caesar et al.'s findings highlight the different ways that people's genes can determine how they respond to an invading pathogen. The findings also suggest that it is important to consider variation in the levels of these complement proteins across a population when planning immunisation schedules. DOI: http://dx.doi.org/10.7554/eLife.04008.002

The Notch signaling pathway is essential for many aspects of development, cell fate determination, and tissue homeostasis. Notch signaling can be modulated by posttranslational modifications to the Notch receptor, which are known to alter both ligand binding and receptor activation. We have modified the ligand-binding region (EGF domains 11-13) of human Notch1 (hN1) with O-fucose and O-glucose glycans and shown by flow cytometry and surface plasmon resonance that the Fringe-catalyzed addition of GlcNAc to the O-fucose at T466 in EGF12 substantially increases binding to Jagged1 and Delta-like 1 (DLL1) ligands. We have subsequently determined the crystal structures of EGF domains 11-13 of hN1 modified with either the O-fucose monosaccharide or the GlcNAc-fucose disaccharide at T466 of EGF12 and observed no change in backbone structure for each variant. Collectively, these data demonstrate a role for GlcNAc in modulating the ligand-binding site in hN1 EGF12, resulting in an increased affinity of this region for ligands Jagged1 and DLL1. We propose that this finding explains the Fringe-catalyzed enhancement of Notch-Delta signaling observed in flies and humans, but suggest that the inhibitory effect of Fringe on Jagged/Serrate mediated signaling involves other regions of Notch.

Neisseria meningitis remains a leading cause of sepsis and meningitis, and vaccines are required to prevent infections by this important human pathogen. Factor H binding protein (fHbp) is a key antigen that elicits protective immunity against the meningococcus and recruits the host complement regulator, fH. As the high affinity interaction between fHbp and fH could impair immune responses, we sought to identify non-functional fHbps that could act as effective immunogens. This was achieved by alanine substitution of fHbps from all three variant groups (V1, V2 and V3 fHbp) of the protein; while some residues affected fH binding in each variant group, the distribution of key amino underlying the interaction with fH differed between the V1, V2 and V3 proteins. The atomic structure of V3 fHbp in complex with fH and of the C-terminal barrel of V2 fHbp provide explanations to the differences in the precise nature of their interactions with fH, and the instability of the V2 protein. To develop transgenic models to assess the efficacy of non-functional fHbps, we determined the structural basis of the low level of interaction between fHbp and murine fH; in addition to changes in amino acids in the fHbp binding site, murine fH has a distinct conformation compared with the human protein that would sterically inhibit binding to fHbp. Non-functional V1 fHbps were further characterised by binding and structural studies, and shown in non-transgenic and transgenic mice (expressing chimeric fH that binds fHbp and precisely regulates complement system) to retain their immunogenicity. Our findings provide a catalogue of non-functional fHbps from all variant groups that can be included in new generation meningococcal vaccines, and establish proof-in-principle for clinical studies to compare their efficacy with wild-type fHbps., Author Summary Vaccines are currently available against several serogroups of Neisseria meningitidis. However broadly effective serogroup B vaccines are still required as capsule-based approaches cannot be implemented with this serogroup because of the risks of auto-immunity. As a result, vaccines based on proteins in the bacterial outer membrane are being developed. Factor H binding protein (fHbp) is an important meningococcal immunogen which is able to bind the human complement regulator factor H (fH) at high affinity; this interaction could impair the efficacy of fHbp-based vaccines. Here we perform structure:function analyses to define non-functional fHbps and to explain the basis for the host specificity of the fHbp:fH interaction. The vaccine candidacy of non-functional fHbps was compared with wild-type proteins in a relevant transgenic model. These findings should allow the design and evaluation of future fHbp vaccines against this important human pathogen.

CD46 is a complement regulator with important roles related to the immune response. CD46 functions as a pathogen receptor and is a potent costimulator for the induction of interferon-γ (IFN-γ)-secreting effector T helper type 1 (TH1) cells and their subsequent switch into interleukin 10 (IL-10)-producing regulatory T cells. Here we identified the Notch family member Jagged1 as a physiological ligand for CD46. Furthermore, we found that CD46 regulated the expression of Notch receptors and ligands during T cell activation and that disturbance of the CD46-Notch crosstalk impeded induction of IFN-γ and switching to IL-10. Notably, CD4+ T cells from CD46-deficient patients and patients with hypomorphic mutations in the gene encoding Jagged1 (Alagille syndrome) failed to mount appropriate T H 1 responses in vitro and in vivo, which suggested that CD46-Jagged1 crosstalk is responsible for the recurrent infections in subpopulations of these patients. © 2012 Nature America, Inc. All rights reserved.