Type three secretion is the mechanism of protein secretion found in bacterial flagella and injectisomes. At its centre is the export apparatus (EA), a complex of five membrane proteins through which ...secretion substrates pass the inner membrane. While the complex formed by four of the EA proteins has been well characterised structurally, little is known about the structure of the membrane domain of the largest subunit, FlhA in flagella, SctV in injectisomes. Furthermore, the biologically relevant nonameric assembly of FlhA/SctV has been infrequently observed and differences in conformation of the cytoplasmic portion of FlhA/SctV between open and closed states have been suggested to reflect secretion system specific differences. FlhA has been shown to bind to chaperone-substrate complexes in an open state, but in previous assembled ring structures, SctV is in a closed state. Here, we identify FlhA and SctV homologues that can be recombinantly produced in the oligomeric state and study them using cryo-electron microscopy. The structures of the cytoplasmic domains from both FlhA and SctV are in the open state and we observe a conserved interaction between a short stretch of residues at the N-terminus of the cytoplasmic domain, known as FlhA.sub.L /SctV.sub.L, with a groove on the adjacent protomer's cytoplasmic domain, which stabilises the nonameric ring assembly.
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 β-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.
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.
Export of proteins through type III secretion systems is critical for motility and virulence of many major bacterial pathogens. Three putative integral membrane proteins (FliP, FliQ, FliR) are ...suggested to form the core of an export gate in the inner membrane, but their structure, assembly and location within the final nanomachine remain unclear. Here, we present the cryoelectron microscopy structure of the Salmonella Typhimurium FliP-FliQ-FliR complex at 4.2 Å. None of the subunits adopt canonical integral membrane protein topologies, and common helix-turn-helix structural elements allow them to form a helical assembly with 5:4:1 stoichiometry. Fitting of the structure into reconstructions of intact secretion systems, combined with cross-linking, localize the export gate as a core component of the periplasmic portion of the machinery. This study thereby identifies the export gate as a key element of the secretion channel and implies that it primes the helical architecture of the components assembling downstream.
Centrosomes are important cell organizers. They consist of a pair of centrioles surrounded by pericentriolar material (PCM) that expands dramatically during mitosis—a process termed centrosome ...maturation. How centrosomes mature remains mysterious. Here, we identify a domain in Drosophila Cnn that appears to be phosphorylated by Polo/Plk1 specifically at centrosomes during mitosis. The phosphorylation promotes the assembly of a Cnn scaffold around the centrioles that is in constant flux, with Cnn molecules recruited continuously around the centrioles as the scaffold spreads slowly outward. Mutations that block Cnn phosphorylation strongly inhibit scaffold assembly and centrosome maturation, whereas phosphomimicking mutations allow Cnn to multimerize in vitro and to spontaneously form cytoplasmic scaffolds in vivo that organize microtubules independently of centrosomes. We conclude that Polo/Plk1 initiates the phosphorylation-dependent assembly of a Cnn scaffold around centrioles that is essential for efficient centrosome maturation in flies.
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•Plk1/Polo phosphorylates Cnn in vitro•Phosphorylation appears to occur specifically at centrosomes during mitosis•Phosphorylation allows Cnn to assemble into a dynamic scaffold around centrioles•Cnn scaffold assembly is required for proper centrosome maturation
Centrosomes, important cellular organizers, form when centrioles recruit pericentriolar material (PCM) around themselves. Conduit et al. shed light on PCM spreading/centrosome maturation during mitosis, showing that Polo kinase initiates phosphorylation-dependent assembly of PCM protein Cnn as a scaffold around centrioles. This scaffold then spreads outward to support an expanded PCM.
This review examined evidence for some core predictions of the response styles theory (RST) concerning the relation between response styles and symptoms of depression and gender differences in the ...use of response styles in non-clinical children and adolescents. In summarizing the literature, effect sizes (pooled correlation coefficients) were calculated for cross-sectional and longitudinal studies. Stability of the obtained effect sizes was evaluated by means of a fail-safe N analysis. Results indicated that stable and significant effect sizes were found for rumination being associated with concurrent and future levels of depression. When controlling for baseline levels of depression, effect sizes for rumination and distraction were not stable, indicating that these findings should be interpreted with considerable caution. Finally, significant and stable effect sizes for gender differences in response styles were found only for rumination among adolescents. Taken together, the findings partly support the predictions of the response styles theory examined in this meta-analysis and may implicate that rumination is a cognitive vulnerability factor for depressive symptoms among adolescents.
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.
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•The conserved PReM and CM2 domains of Cnn co-assemble into micron-scale structures•The crystal structure of the PReM-LZ:CM2 complex is solved to 1.82 Å•Mutations that block PReM-LZ:CM2 assembly in vitro block centrosome assembly in vivo•Phosphorylation of PReM by Polo/Plk1 promotes scaffold assembly in vitro and in vivo
Structural and biochemical analyses paint a new picture of how the pericentriolar material forms micron-scale structures that recruit PCM and nucleate microtubules.
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.
Lipid droplets (LDs) are universal lipid storage organelles with a core of neutral lipids, such as triacylglycerols, surrounded by a phospholipid monolayer. This unique architecture is generated ...during LD biogenesis at endoplasmic reticulum (ER) sites marked by Seipin, a conserved membrane protein mutated in lipodystrophy. Here structural, biochemical and molecular dynamics simulation approaches reveal the mechanism of LD formation by the yeast Seipin Sei1 and its membrane partner Ldb16. We show that Sei1 luminal domain assembles a homooligomeric ring, which, in contrast to other Seipins, is unable to concentrate triacylglycerol. Instead, Sei1 positions Ldb16, which concentrates triacylglycerol within the Sei1 ring through critical hydroxyl residues. Triacylglycerol recruitment to the complex is further promoted by Sei1 transmembrane segments, which also control Ldb16 stability. Thus, we propose that LD assembly by the Sei1/Ldb16 complex, and likely other Seipins, requires sequential triacylglycerol-concentrating steps via distinct elements in the ER membrane and lumen.
CD200 is a widely distributed membrane glycoprotein that regulates myeloid cell activity through its interaction with an inhibitory receptor (CD200R). The interaction is of interest as a target for ...treating excessive inflammation and for treating leukemia. There are closely related proteins to CD200R that give activating signals making this a “paired receptor.” We report X-ray crystallography structures for the inhibitory CD200R, the activating receptor CD200RLa, and a complex between CD200R and CD200. Both CD200 and CD200R contain two Ig-like domains and interact through their NH2 terminal domains compatible with immunological synapse-like interactions occurring between myeloid cells and other CD200-expressing cells. The failure of the activating receptor to bind CD200 resides in subtle changes around the interface. CD200 has been acquired by herpes viruses to mimic the host interaction. CD200R has evolved rapidly presumably driven by pathogen pressure but it may also be important in homeostasis through interactions with commensal bacteria.
•Structure of CD200R and in complex with its ligand CD200 complex•Structure of an activating member of the CD200 receptor family•Mutagenesis showing specificity of CD200 paired receptor family
Hatherley et al. present a structural basis explaining why the cell surface protein CD200 binds to its receptor (CD200R) but not to the related activating receptor CD200RLa. The rapid evolution of the receptors was probably driven by pathogens and might play a role in mediating interaction with the microbiome.
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