Structural Basis for Mitotic Centrosome Assembly in Flies.

In flies, Centrosomin (Cnn) forms a phosphorylation-dependent scaffold that recruits proteins to the mitotic centrosome, but how Cnn assembles into a scaffold is unclear. We show that scaffold assembly requires conserved leucine zipper (LZ) and Cnn-motif 2 (CM2) domains that co-assemble into a 2:2 complex in vitro. We solve the crystal structure of the LZ:CM2 complex, revealing that both proteins form helical dimers that assemble into an unusual tetramer. A slightly longer version of the LZ can form micron-scale structures with CM2, whose assembly is stimulated by Plk1 phosphorylation in vitro. Mutating individual residues that perturb LZ:CM2 tetramer assembly perturbs the formation of these micron-scale assemblies in vitro and Cnn-scaffold assembly in vivo. Thus, Cnn molecules have an intrinsic ability to form large, LZ:CM2-interaction-dependent assemblies that are critical for mitotic centrosome assembly. These studies provide the first atomic insight into a molecular interaction required for mitotic centrosome assembly.

Structural basis of antifolate recognition and transport by PCFT.

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-adenylsmethionine1,2. Folate deficiency is associated with a number of developmental, immune and neurological disorders3-5. Mammals cannot synthesize folates de novo; several systems have therefore evolved to take up folates from the diet and distribute them within the body3,6. The proton-coupled folate transporter (PCFT) (also known as SLC46A1) mediates folate uptake across the intestinal brush border membrane and the choroid plexus4,7, and is an important route for the delivery of antifolate drugs in cancer chemotherapy8-10. 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.

Capturing heterogeneous conformers of cobalamin riboswitch by cryo-EM.

RNA conformational heterogeneity often hampers its high-resolution structure determination, especially for large and flexible RNAs devoid of stabilizing proteins or ligands. The adenosylcobalamin riboswitch exhibits heterogeneous conformations under 1 mM Mg2+ concentration and ligand binding reduces conformational flexibility. Among all conformers, we determined one apo (5.3 Å) and four holo cryo-electron microscopy structures (overall 3.0-3.5 Å, binding pocket 2.9-3.2 Å). The holo dimers exhibit global motions of helical twisting and bending around the dimer interface. A backbone comparison of the apo and holo states reveals a large structural difference in the P6 extension position. The central strand of the binding pocket, junction 6/3, changes from an 'S'- to a 'U'-shaped conformation to accommodate ligand. Furthermore, the binding pocket can partially form under 1 mM Mg2+ and fully form under 10 mM Mg2+ within the bound-like structure in the absence of ligand. Our results not only demonstrate the stabilizing ligand-induced conformational changes in and around the binding pocket but may also provide further insight into the role of the P6 extension in ligand binding and selectivity.

The CD46-Jagged1 interaction is critical for human TH1 immunity.

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 (T(H)1) 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.

Type 9 secretion system structures reveal a new protein transport mechanism.

The type 9 secretion system (T9SS) is the protein export pathway of bacteria of the Gram-negative Fibrobacteres-Chlorobi-Bacteroidetes superphylum and is an essential determinant of pathogenicity in severe periodontal disease. The central element of the T9SS is a so-far uncharacterized protein-conducting translocon located in the bacterial outer membrane. Here, using cryo-electron microscopy, we provide structural evidence that the translocon is the T9SS protein SprA. SprA forms an extremely large (36-strand) single polypeptide transmembrane ß-barrel. The barrel pore is capped on the extracellular end, but has a lateral opening to the external membrane surface. Structures of SprA bound to different components of the T9SS show that partner proteins control access to the lateral opening and to the periplasmic end of the pore. Our results identify a protein transporter with a distinctive architecture that uses an alternating access mechanism in which the two ends of the protein-conducting channel are open at different times.

The conserved C2 phospholipid-binding domain in Delta contributes to robust Notch signalling.

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 ß1-2 loop that is involved in phospholipid binding. Mutations in the ß1-2 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 ß1-2 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.

An inhibitor of complement C5 provides structural insights into activation.

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.

Maintenance of the Shigella sonnei Virulence Plasmid Is Dependent on Its Repertoire and Amino Acid Sequence of Toxin-Antitoxin Systems.

Shigella sonnei is a major cause of bacillary dysentery and an increasing concern due to the spread of multidrug resistance. S. sonnei harbors pINV, an ∼210 kb plasmid that encodes a type III secretion system (T3SS), which is essential for virulence. During growth in the laboratory, avirulence arises spontaneously in S. sonnei at high frequency, hampering studies on and vaccine development against this important pathogen. Here, we investigated the molecular basis for the emergence of avirulence in S. sonnei and showed that avirulence mainly results from pINV loss, which is consistent with previous findings. Ancestral deletions have led to the loss from S. sonnei pINV of two toxin-antitoxin (TA) systems involved in plasmid maintenance, CcdAB and GmvAT, which are found on pINV in Shigella flexneri. We showed that the introduction of these TA systems into S. sonnei pINV reduced but did not eliminate pINV loss, while the single amino acid polymorphisms found in the S. sonnei VapBC TA system compared with S. flexneri VapBC also contributed to pINV loss. Avirulence also resulted from deletions of T3SS-associated genes in pINV through recombination between insertion sequences (ISs) on the plasmid. These events differed from those observed in S. flexneri due to the different distribution and repertoire of ISs. Our findings demonstrated that TA systems and ISs influenced plasmid dynamics and loss in S. sonnei and could be exploited for the design and evaluation of vaccines. IMPORTANCE Shigella sonnei is the major cause of shigellosis in high-income and industrializing countries and is an emerging, multidrug-resistant pathogen. A significant challenge when studying this bacterium is that it spontaneously becomes avirulent during growth in the laboratory through loss of its virulence plasmid (pINV). Here, we deciphered the mechanisms leading to avirulence in S. sonnei and how the limited repertoire and amino acid sequences of plasmid-encoded toxin-antitoxin (TA) systems make the maintenance of pINV in this bacterium less efficient compared with Shigella flexneri. Our findings highlighted how subtle differences in plasmids in closely related species have marked effects and could be exploited to reduce plasmid loss in S. sonnei. This should facilitate research on this bacterium and vaccine development.

The DNA transporter ComEC has metal-dependent nuclease activity that is important for natural transformation.

In the process of natural transformation bacteria import extracellular DNA molecules for integration into their genome. One strand of the incoming DNA molecule is degraded, whereas the remaining strand is transported across the cytoplasmic membrane. The DNA transport channel is provided by the protein ComEC. Many ComEC proteins have an extracellular C-terminal domain (CTD) with homology to the metallo-ß-lactamase fold. Here we show that this CTD binds Mn2+ ions and exhibits Mn2+ -dependent phosphodiesterase and nuclease activities. Inactivation of the enzymatic activity of the CTD severely inhibits natural transformation in Bacillus subtilis. These data suggest that the ComEC CTD is a nuclease responsible for degrading the nontransforming DNA strand during natural transformation and that this process is important for efficient DNA import.

Structural and functional dissection of the interplay between lipid and Notch binding by human Notch ligands.

Recent data have expanded our understanding of Notch signalling by identifying a C2 domain at the N-terminus of Notch ligands, which has both lipid- and receptor-binding properties. We present novel structures of human ligands Jagged2 and Delta-like4 and human Notch2, together with functional assays, which suggest that ligand-mediated coupling of membrane recognition and Notch binding is likely to be critical in establishing the optimal context for Notch signalling. Comparisons between the Jagged and Delta family show a huge diversity in the structures of the loops at the apex of the C2 domain implicated in membrane recognition and Jagged1 missense mutations, which affect these loops and are associated with extrahepatic biliary atresia, lead to a loss of membrane recognition, but do not alter Notch binding. Taken together, these data suggest that C2 domain binding to membranes is an important element in tuning ligand-dependent Notch signalling in different physiological contexts.

Structure of and influence of a tick complement inhibitor on human complement component 5.

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.

A signal sequence suppressor mutant that stabilizes an assembled state of the twin arginine translocase.

The twin-arginine protein translocation (Tat) system mediates transport of folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. The Tat system of Escherichia coli is made up of TatA, TatB, and TatC components. TatBC comprise the substrate receptor complex, and active Tat translocases are formed by the substrate-induced association of TatA oligomers with this receptor. Proteins are targeted to TatBC by signal peptides containing an essential pair of arginine residues. We isolated substitutions, locating to the transmembrane helix of TatB that restored transport activity to Tat signal peptides with inactivating twin arginine substitutions. A subset of these variants also suppressed inactivating substitutions in the signal peptide binding site on TatC. The suppressors did not function by restoring detectable signal peptide binding to the TatBC complex. Instead, site-specific cross-linking experiments indicate that the suppressor substitutions induce conformational change in the complex and movement of the TatB subunit. The TatB F13Y substitution was associated with the strongest suppressing activity, even allowing transport of a Tat substrate lacking a signal peptide. In vivo analysis using a TatA-YFP fusion showed that the TatB F13Y substitution resulted in signal peptide-independent assembly of the Tat translocase. We conclude that Tat signal peptides play roles in substrate targeting and in triggering assembly of the active translocase.

FHR-1 Binds to C-Reactive Protein and Enhances Rather than Inhibits Complement Activation.

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.

Multi-professional IAPT CBT training: clinical competence and patient outcomes.

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.

Structural and Functional Characterization of the Bacterial Type III Secretion Export Apparatus.

Bacterial type III protein secretion systems inject effector proteins into eukaryotic host cells in order to promote survival and colonization of Gram-negative pathogens and symbionts. Secretion across the bacterial cell envelope and injection into host cells is facilitated by a so-called injectisome. Its small hydrophobic export apparatus components SpaP and SpaR were shown to nucleate assembly of the needle complex and to form the central "cup" substructure of a Salmonella Typhimurium secretion system. However, the in vivo placement of these components in the needle complex and their function during the secretion process remained poorly defined. Here we present evidence that a SpaP pentamer forms a 15 Å wide pore and provide a detailed map of SpaP interactions with the export apparatus components SpaQ, SpaR, and SpaS. We further refine the current view of export apparatus assembly, consolidate transmembrane topology models for SpaP and SpaR, and present intimate interactions of the periplasmic domains of SpaP and SpaR with the inner rod protein PrgJ, indicating how export apparatus and needle filament are connected to create a continuous conduit for substrate translocation.

Structure of the TatC core of the twin-arginine protein transport system.

The twin-arginine translocation (Tat) pathway is one of two general protein transport systems found in the prokaryotic cytoplasmic membrane and is conserved in the thylakoid membrane of plant chloroplasts. The defining, and highly unusual, property of the Tat pathway is that it transports folded proteins, a task that must be achieved without allowing appreciable ion leakage across the membrane. The integral membrane TatC protein is the central component of the Tat pathway. TatC captures substrate proteins by binding their signal peptides. TatC then recruits TatA family proteins to form the active translocation complex. Here we report the crystal structure of TatC from the hyperthermophilic bacterium Aquifex aeolicus. This structure provides a molecular description of the core of the Tat translocation system and a framework for understanding the unique Tat transport mechanism.

Structural Insights into Notch Receptor-Ligand Interactions.

Pioneering cell aggregation experiments from the Artavanis-Tsakonas group in the late 1980's localized the core ligand recognition sequence in the Drosophila Notch receptor to epidermal growth factor-like (EGF) domains 11 and 12. Since then, advances in protein expression, structure determination methods and functional assays have enabled us to define the molecular basis of the core receptor/ligand interaction and given new insights into the architecture of the Notch complex at the cell surface. We now know that Notch EGF11 and 12 interact with the Delta/Serrate/LAG-2 (DSL) and C2 domains of ligand and that membrane-binding, together with additional protein-protein interactions outside the core recognition domains, are likely to fine-tune generation of the Notch signal. Furthermore, structure determination of O-glycosylated variants of Notch alone or in complex with receptor fragments, has shown that these sugars contribute directly to the binding interface, as well as to stabilizing intra-molecular domain structure, providing some mechanistic insights into the observed modulatory effects of O-glycosylation on Notch activity.Future challenges lie in determining the complete extracellular architecture of ligand and receptor in order to understand (i) how Notch/ligand complexes may form at the cell surface in response to physiological cues, (ii) the role of lipid binding in stabilizing the Notch/ligand complex, (iii) the impact of O-glycosylation on binding and signalling and (iv) to dissect the different pathologies that arise as a consequence of mutations that affect proteins involved in the Notch pathway.

Structural basis for specificity and promiscuity in a carrier protein/enzyme system from the sulfur cycle.

The bacterial Sox (sulfur oxidation) pathway is an important route for the oxidation of inorganic sulfur compounds. Intermediates in the Sox pathway are covalently attached to the heterodimeric carrier protein SoxYZ through conjugation to a cysteine on a protein swinging arm. We have investigated how the carrier protein shuttles intermediates between the enzymes of the Sox pathway using the interaction between SoxYZ and the enzyme SoxB as our model. The carrier protein and enzyme interact only weakly, but we have trapped their complex by using a "suicide enzyme" strategy in which an engineered cysteine in the SoxB active site forms a disulfide bond with the incoming carrier arm cysteine. The structure of this trapped complex, together with calorimetric data, identifies sites of protein-protein interaction both at the entrance to the enzyme active site tunnel and at a second, distal, site. We find that the enzyme distinguishes between the substrate and product forms of the carrier protein through differences in their interaction kinetics and deduce that this behavior arises from substrate-specific stabilization of a conformational change in the enzyme active site. Our analysis also suggests how the carrier arm-bound substrate group is able to outcompete the adjacent C-terminal carboxylate of the carrier arm for binding to the active site metal ions. We infer that similar principles underlie carrier protein interactions with other enzymes of the Sox pathway.

Characterisation of Shigella Spa33 and Thermotoga FliM/N reveals a new model for C-ring assembly in T3SS.

Flagellar type III secretion systems (T3SS) contain an essential cytoplasmic-ring (C-ring) largely composed of two proteins FliM and FliN, whereas an analogous substructure for the closely related non-flagellar (NF) T3SS has not been observed in situ. We show that the spa33 gene encoding the putative NF-T3SS C-ring component in Shigella flexneri is alternatively translated to produce both full-length (Spa33-FL) and a short variant (Spa33-C), with both required for secretion. They associate in a 1:2 complex (Spa33-FL/C2) that further oligomerises into elongated arrays in vitro. The structure of Spa33-C2 and identification of an unexpected intramolecular pseudodimer in Spa33-FL reveal a molecular model for their higher order assembly within NF-T3SS. Spa33-FL and Spa33-C are identified as functional counterparts of a FliM-FliN fusion and free FliN respectively. Furthermore, we show that Thermotoga maritima FliM and FliN form a 1:3 complex structurally equivalent to Spa33-FL/C2 , allowing us to propose a unified model for C-ring assembly by NF-T3SS and flagellar-T3SS.

Costs of the police service and mental healthcare pathways experienced by individuals with enduring mental health needs.

BACKGROUND: Substantial policy, communication and operational gaps exist between mental health services and the police for individuals with enduring mental health needs. AIMS: To map and cost pathways through mental health and police services, and to model the cost impact of implementing key policy recommendations. METHOD: Within 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. RESULTS: Under 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%. CONCLUSIONS: Recommended enhancements to care pathways only marginally increase individual-level costs.