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Journal articleChayen N, shaffer, govada, et al., 2016,
Controlling crystal nucleation is a crucial step in obtaining high quality protein crystals for structure determination by X-ray crystallography. Carbon nanomaterials (CNMs) including carbon nanotubes, graphene oxide, and carbon black provide a range of surface topographies, porosities and length scales; functionalisation with two different approaches, gas phase radical grafting and liquid phase reductive grafting, provide routes to a range of oligomer functionalised products. These grafted materials, combined with a range of controls, were used in a large-scale assessment of the effectiveness for protein crystal nucleation of 20 different carbon nanomaterials on five proteins. This study has allowed a direct comparison of the key characteristics of carbon-based nucleants: appropriate surface chemistry, porosity and/or roughness are required. The most effective solid system tested in this study, carbon black nanoparticles functionalised with poly(ethylene glycol) methyl ether of mean molecular weight 5000, provides a novel highly effective nucleant, that was able to induce crystal nucleation of four out of the five proteins tested at metastable conditions.
Journal articleRutherford AW, Prell J, MacKellar D, et al., 2016,
Streptomyces thermoautotrophicus UBT1 has been described as a moderately thermophilic chemolithoautotroph with a novel nitrogenase enzyme that is oxygen-insensitive. We have cultured the UBT1 strain, and have isolated two new strains (H1 and P1-2) of very similar phenotypic and genetic characters. These strains show minimal growth on ammonium-free media, and fail to incorporate isotopically labeled N2 gas into biomass in multiple independent assays. The sdn genes previously published as the putative nitrogenase of S. thermoautotrophicus have little similarity to anything found in draft genome sequences, published here, for strains H1 and UBT1, but share >99% nucleotide identity with genes from Hydrogenibacillus schlegelii, a draft genome for which is also presented here. H. schlegelii similarly lacks nitrogenase genes and is a non-diazotroph. We propose reclassification of the species containing strains UBT1, H1, and P1-2 as a non-Streptomycete, non-diazotrophic, facultative chemolithoautotroph and conclude that the existence of the previously proposed oxygen-tolerant nitrogenase is extremely unlikely.
Journal articleTaylor J, Taylor G, Hare S, et al., 2016,
Bacteria have developed a variety of mechanisms for survivingharsh environmental conditions, nutrient stress and overpopulation.Paenibacillus dendritiformis produces a lethal protein (Slf) that is ableto induce cell death in neighboring colonies and a phenotypic switch inmore distant ones. Slf is derived from the secreted precursor protein,DfsB, after proteolytic processing. Here, we present new crystalstructures of DfsB homologues from a variety of bacterial species and asurprising version present in the yeast Saccharomyces cerevisiae.Adopting a four-helix bundle decorated with a further three short heliceswithin intervening loops, DfsB belongs to a non-enzymatic class of theDinB fold. The structure suggests that the biologically-active Slffragment may possess a C-terminal helix rich in basic and aromaticresidues that suggest a functional mechanism akin to that for cationicantimicrobial peptides.
Journal articleLeen EN, Sorgeloos F, Correia S, et al., 2016,
A conserved interaction between a C-terminal motif in norovirus VPg and the HEAT-1 domain of eIF4G is essential for translation initiation, PLOS Pathogens, Vol: 12, ISSN: 1553-7366
Translation initiation is a critical early step in the replication cycle of the positive-sense, single-stranded RNA genome of noroviruses, a major cause of gastroenteritis in humans. Norovirus RNA, which has neither a 5´ m7G cap nor an internal ribosome entry site (IRES), adopts an unusual mechanism to initiate protein synthesis that relies on interactions between the VPg protein covalently attached to the 5´-end of the viral RNA and eukaryotic initiation factors (eIFs) in the host cell. For murine norovirus (MNV) we previously showed that VPg binds to the middle fragment of eIF4G (4GM; residues 652-1132). Here we have used pull-down assays, fluorescence anisotropy, and isothermal titration calorimetry (ITC) to demonstrate that a stretch of ~20 amino acids at the C terminus of MNV VPg mediates direct and specific binding to the HEAT-1 domain within the 4GM fragment of eIF4G. Our analysis further reveals that the MNV C terminus binds to eIF4G HEAT-1 via a motif that is conserved in all known noroviruses. Fine mutagenic mapping suggests that the MNV VPg C terminus may interact with eIF4G in a helical conformation. NMR spectroscopy was used to define the VPg binding site on eIF4G HEAT-1, which was confirmed by mutagenesis and binding assays. We have found that this site is non-overlapping with the binding site for eIF4A on eIF4G HEAT-1 by demonstrating that norovirus VPg can form ternary VPg-eIF4G-eIF4A complexes. The functional significance of the VPg-eIF4G interaction was shown by the ability of fusion proteins containing the C-terminal peptide of MNV VPg to inhibit in vitro translation of norovirus RNA but not cap- or IRES-dependent translation. These observations define important structural details of a functional interaction between norovirus VPg and eIF4G and reveal a binding interface that might be exploited as a target for antiviral therapy.
Book chapterBarretto S, Michoux F, Nixon PJ, 2016,
Temporary Immersion Bioreactors for the Contained Production of Recombinant Proteins in Transplastomic Plants., Recombinant Proteins from Plants: Methods and Protocols, Publisher: Springer, Pages: 149-160, ISBN: 978-1-4939-3288-7
Despite the largely maternal inheritance of plastid genomes, the risk of transgene dissemination from transplastomic plants can limit the scope for field cultivation. There is a need for a cost-effective, scalable process to grow large quantities of transplastomic plant biomass for biosynthesis of biopharmaceuticals and other high-value heterologous proteins. Temporary immersion culture is a means of achieving this under fully contained conditions. This method describes the organogenesis of transplastomic Nicotiana tabacum callus in RITA(®) temporary immersion bioreactors to produce rootless leafy biomass, and subsequent total soluble protein extraction, SDS-PAGE, and Western immunoblot analysis of heterologous protein expression. This method can be used for propagation of plastid or nuclear transformants, though is especially suitable for transplastomic biomass, as organogenesis leads to greater expression and accumulation of transplastomic proteins due to increases in chloroplast number and size.
Journal articleWang Z, Shah UV, Olusanmi D, et al., 2015,
Journal articleWong CT, Xu Y, Gupta A, et al., 2015,
The Neisseriaceae family of bacteria causes a range of diseases including meningitis, septicaemia, gonorrhoea and endocarditis, and extracts haem from haemoglobin as an important iron source within the iron-limited environment of its human host. Herein we report crystal structures of apo- and haemoglobin-bound HpuA, an essential component of this haem import system. The interface involves long loops on the bacterial receptor that present hydrophobic side chains for packing against the surface of haemoglobin. Interestingly, our structural and biochemical analyses of Kingella denitrificans and Neisseria gonorrhoeae HpuA mutants, although validating the interactions observed in the crystal structure, show how Neisseriaceae have the fascinating ability to diversify functional sequences and yet retain the haemoglobin binding function. Our results present the first description of HpuA’s role in direct binding of haemoglobin.
Journal articleTrusch F, Matena A, Vuk M, et al., 2015,
Journal articleLarrouy-Maumus GJ, Abigail Clements, Alain Filloux, et al., 2015,
The purification and characterization of Gram-negative bacterial lipid A is tedious and time-consuming. Herein we report a rapid and sensitive method to identify lipid A directly on intact bacteria without any chemical treatment or purification, using an atypical solvent system to solubilize the matrix combined with MALDI-TOF mass spectrometry.
Journal articleWitcomb LA, Collins JW, McCarthy AJ, et al., 2015,
Bioluminescent Imaging Reveals Novel Patterns of Colonization and Invasion in Systemic Escherichia coli K1 Experimental Infection in the Neonatal Rat, Infection and Immunity, Vol: 83, Pages: 4528-4540, ISSN: 0019-9567
Key features of Escherichia coli K1-mediated neonatal sepsis and meningitis, such as a strong age dependency and development along the gut-mesentery-blood-brain course of infection, can be replicated in the newborn rat. We examined temporal and spatial aspects of E. coli K1 infection following initiation of gastrointestinal colonization in 2-day-old (P2) rats after oral administration of E. coli K1 strain A192PP and a virulent bioluminescent derivative, E. coli A192PP-lux2. A combination of bacterial enumeration in the major organs, two-dimensional bioluminescence imaging, and three-dimensional diffuse light imaging tomography with integrated micro-computed tomography indicated multiple sites of colonization within the alimentary canal; these included the tongue, esophagus, and stomach in addition to the small intestine and colon. After invasion of the blood compartment, the bacteria entered the central nervous system, with restricted colonization of the brain, and also invaded the major organs, in line with increases in the severity of symptoms of infection. Both keratinized and nonkeratinized surfaces of esophagi were colonized to a considerably greater extent in susceptible P2 neonates than in corresponding tissues from infection-resistant 9-day-old rat pups; the bacteria appeared to damage and penetrate the nonkeratinized esophageal epithelium of infection-susceptible P2 animals, suggesting the esophagus represents a portal of entry for E. coli K1 into the systemic circulation. Thus, multimodality imaging of experimental systemic infections in real time indicates complex dynamic patterns of colonization and dissemination that provide new insights into the E. coli K1 infection of the neonatal rat.
Journal articleCurry S, 2015,
Journal articlefilloux A, wood TE, Hachani A, 2015,
Journal articleCarlsson E, Ding JL, Byrne B, 2015,
Toll-like receptors (TLRs) recognise invading pathogens and initiate an innate immune response by recruiting intracellular adaptor proteins via heterotypic Toll/interleukin-1 receptor (TIR) domain interactions. Of the five TIR domain-containing adaptor proteins identified, Sterile α- and armadillo-motif-containing protein (SARM) is functionally unique; suppressing immune signalling instead of promoting it. Here we demonstrate that the recombinantly expressed and purified SARM TIR domain interacts with both the major human TLR adaptors, MyD88 and TRIF. A single glycine residue located in the BB-loop of the SARM TIR domain, G601, was identified as essential for interaction. A short peptide derived from this domain was also found to interact with MyD88 in vitro. SARM expression in HEK-293 cells was found to significantly suppress lipopolysaccharide (LPS)-mediated upregulation of inflammatory cytokines, IL-8 and TNF-α, an effect lost in the G601A mutant. The same result was observed with cytokine activation initiated by MyD88 expression and stimulation of TLR2 with lipoteichoic acid (LTA), suggesting that SARM is capable of suppressing both TRIF- and MyD88- dependent TLR signalling. Our findings indicate that SARM acts on a broader set of target proteins than previously thought, and that the BB-loop motif is functionally important, giving further insight into the endogenous mechanisms used to suppress inflammation in immune cells.
Journal articlePakharukova N, Garnett JA, Tuittila M, et al., 2015,
Structural Insight into Archaic and Alternative Chaperone-Usher Pathways Reveals a Novel Mechanism of Pilus Biogenesis., PLOS Pathogens, Vol: 11, ISSN: 1553-7366
Gram-negative pathogens express fibrous adhesive organelles that mediate targeting to sites of infection. The major class of these organelles is assembled via the classical, alternative and archaic chaperone-usher pathways. Although non-classical systems share a wider phylogenetic distribution and are associated with a range of diseases, little is known about their assembly mechanisms. Here we report atomic-resolution insight into the structure and biogenesis of Acinetobacter baumannii Csu and Escherichia coli ECP biofilm-mediating pili. We show that the two non-classical systems are structurally related, but their assembly mechanism is strikingly different from the classical assembly pathway. Non-classical chaperones, unlike their classical counterparts, maintain subunits in a substantially disordered conformational state, akin to a molten globule. This is achieved by a unique binding mechanism involving the register-shifted donor strand complementation and a different subunit carboxylate anchor. The subunit lacks the classical pre-folded initiation site for donor strand exchange, suggesting that recognition of its exposed hydrophobic core starts the assembly process and provides fresh inspiration for the design of inhibitors targeting chaperone-usher systems.
Journal articlePinzan CF, Sardinha-Silva A, Almeida F, et al., 2015,
Vaccination with Recombinant Microneme Proteins Confers Protection against Experimental Toxoplasmosis in Mice, PLOS One, Vol: 10, ISSN: 1932-6203
Toxoplasmosis, a zoonotic disease caused by Toxoplasma gondii, is an important publichealth problem and veterinary concern. Although there is no vaccine for human toxoplasmosis,many attempts have been made to develop one. Promising vaccine candidates utilizeproteins, or their genes, from microneme organelle of T. gondii that are involved in theinitial stages of host cell invasion by the parasite. In the present study, we used differentrecombinant microneme proteins (TgMIC1, TgMIC4, or TgMIC6) or combinations of theseproteins (TgMIC1-4 and TgMIC1-4-6) to evaluate the immune response and protectionagainst experimental toxoplasmosis in C57BL/6 mice. Vaccination with recombinantTgMIC1, TgMIC4, or TgMIC6 alone conferred partial protection, as demonstrated byreduced brain cyst burden and mortality rates after challenge. Immunization with TgMIC1-4or TgMIC1-4-6 vaccines provided the most effective protection, since 70% and 80% ofmice, respectively, survived to the acute phase of infection. In addition, these vaccinatedmice, in comparison to non-vaccinated ones, showed reduced parasite burden by 59% and68%, respectively. The protective effect was related to the cellular and humoral immuneresponses induced by vaccination and included the release of Th1 cytokines IFN-γ and IL-12, antigen-stimulated spleen cell proliferation, and production of antigen-specific serumantibodies. Our results demonstrate that microneme proteins are potential vaccines againstT. gondii, since their inoculation prevents or decreases the deleterious effects of theinfection.
Journal articleBeeby M, 2015,
The epsilon-proteobacteria are a widespread group of flagellated bacteria frequently associated with either animal digestive tracts or hydrothermal vents, with well-studied examples in the human pathogens of Helicobacter and Campylobacter genera. Flagellated motility is important to both pathogens and hydrothermal vent members, and a number of curious differences between the epsilon-proteobacterial and enteric bacterial motility paradigms make them worthy of further study. The epsilon-proteobacteria have evolved to swim at high speed and through viscous media that immobilize enterics, a phenotype that may be accounted for by the molecular architecture of the unusually large epsilon-proteobacterial flagellar motor. This review summarizes what is known about epsilon-proteobacterial motility and focuses on a number of recent discoveries that rationalize the differences with enteric flagellar motility.
Journal articleBurgess SJ, Hussein T, Yeoman JA, et al., 2015,
Under anoxic conditions the green alga Chlamydomonas reinhardtii activates various 67 fermentation pathways leading to the creation of formate, acetate, ethanol and small 68 amounts of other metabolites including D-lactate and hydrogen. Progress has been 69 made in identifying the enzymes involved in these pathways and their sub-cellular 70 locations; however, the identity of the enzyme involved in reducing pyruvate to D-71 lactate has remained unclear. Based on sequence comparisons, enzyme activity 72 measurements, X-ray crystallography, biochemical fractionation and analysis of 73 knock-down mutants we conclude that pyruvate reduction in the chloroplast is 74 catalysed by a tetrameric NAD⁺-dependent D-lactate dehydrogenase encoded by 75 Cre07.g324550. Its expression during aerobic growth supports a possible function as a 76 ‘lactate valve’ for the export of lactate to the mitochondrion for oxidation by 77 cytochrome-dependent D-lactate dehydrogenases and by glycolate dehydrogenase. 78 We also present a revised spatial model of fermentation based on our 79 immunochemical detection of the likely pyruvate decarboxylase, PDC3, in the 80 cytoplasm.
Journal articleShah UV, Wang Z, Olusanmi D, et al., 2015,
Journal articleKrynická V, Shao S, Nixon PJ, et al., 2015,
The oxygen-evolving photosystem II (PSII) complex located inchloroplasts and cyanobacteria is sensitive to light-induceddamage1 that unless repaired causes reduction in photosyntheticcapacity and growth. Although a potential target forcrop improvement, the mechanism of PSII repair remainsunclear. The D1 reaction center protein is the main target forphotodamage2, with repair involving the selective degradationof the damaged protein by FtsH protease3. How a singledamaged PSII subunit is recognized for replacement isunknown. Here, we have tested the dark stability of PSII subunitsin strains of the cyanobacterium Synechocystis PCC6803 blocked at specific stages of assembly. We have foundthat when D1, which is normally shielded by the CP43subunit, becomes exposed in a photochemically active PSIIcomplex lacking CP43, it is selectively degraded by FtsH evenin the dark. Removal of the CP47 subunit, which increasesaccessibility of FtsH to the D2 subunit, induced dark degradationof D2 at a faster rate than that of D1. In contrast,CP47 and CP43 are resistant to degradation in the dark. Ourresults indicate that protease accessibility induced by PSII disassemblyis an important determinant in the selection of the D1and D2 subunits to be degraded by FtsH.
Journal articleChaban B, Hughes HV, Beeby M, 2015,
The bacterial flagellum is an amazingly complex molecular machine with a diversity of roles in pathogenesis including reaching the optimal host site, colonization or invasion, maintenance at the infection site, and post-infection dispersal. Multi-megadalton flagellar motors self-assemble across the cell wall to form a reversible rotary motor that spins a helical propeller - the flagellum itself - to drive the motility of diverse bacterial pathogens. The flagellar motor responds to the chemoreceptor system to redirect swimming toward beneficial environments, thus enabling flagellated pathogens to seek out their site of infection. At their target site, additional roles of surface swimming and mechanosensing are mediated by flagella to trigger pathogenesis. Yet while these motility-related functions have long been recognized as virulence factors in bacteria, many bacteria have capitalized upon flagellar structure and function by adapting it to roles in other stages of the infection process. Once at their target site, the flagellum can assist adherence to surfaces, differentiation into biofilms, secretion of effector molecules, further penetration through tissue structures, or in activating phagocytosis to gain entry into eukaryotic cells. Next, upon onset of infection, flagellar expression must be adapted to deal with the host's immune system defenses, either by reduced or altered expression or by flagellar structural modification. Finally, after a successful growth phase on or inside a host, dispersal to new infection sites is often flagellar motility-mediated. Examining examples of all these processes from different bacterial pathogens, it quickly becomes clear that the flagellum is involved in bacterial pathogenesis for motility and a whole lot more.
Journal articlevan Wilderen L, Silkstone G, Mason M, et al., 2015,
Kinetic studies on the oxidation of semiquinone and hydroquinone forms of Arabidopsis cryptochrome by molecular oxygen, FEBS Open Bio, Vol: 5, Pages: 885-892, ISSN: 2211-5463
Cryptochromes (crys) are flavoprotein photoreceptors present throughout the biological kingdom that play important roles in plant development and entrainment of the circadian clock in several organisms. Crys non‐covalently bind flavin adenine dinucleotide (FAD) which undergoes photoreduction from the oxidised state to a radical form suggested to be active in signalling in vivo. Although the photoreduction reactions have been well characterised by a number of approaches, little is known of the oxidation reactions of crys and their mechanisms. In this work, a stopped‐flow kinetics approach is used to investigate the mechanism of cry oxidation in the presence and absence of an external electron donor. This in vitro study extends earlier investigations of the oxidation of Arabidopsis cryptochrome1 by molecular oxygen and demonstrates that, under some conditions, a more complex model for oxidation of the flavin than was previously proposed is required to accommodate the spectral evidence (see P. Müller and M. Ahmad (2011) J. Biol. Chem. 286, 21033–21040 ). In the absence of an electron donor, photoreduction leads predominantly to the formation of the radical FADH. Dark recovery most likely forms flavin hydroperoxide (FADHOOH) requiring superoxide. In the presence of reductant (DTT), illumination yields the fully reduced flavin species (FADH−). Reaction of this with dioxygen leads to transient radical (FADH) and simultaneous accumulation of oxidised species (FAD), possibly governed by interplay between different cryptochrome molecules or cooperativity effects within the cry homodimer.
Journal articleHohenester E, Paracuellos P, Briggs DC, et al., 2015,
Insights into collagen uptake by C-type mannose receptors from the crystal structure of Endo180 domains 1-4, Structure, Vol: 23, Pages: 2133-2142, ISSN: 1878-4186
The C-type mannose receptor and its homologEndo180 (or uPARAP, for urokinase plasminogenactivator receptor-associated protein) mediate theendocytic uptake of collagen by macrophages and fi-broblasts. This process is required for normal tissueremodeling, but also facilitates the growth anddissemination of tumors. We have determined thecrystal structure at 2.5 A˚ resolution of the N-terminalregion of Endo180, consisting of a ricin-like domain,a fibronectin type II (FN2) domain, and two C-typelectin (CTL) domains. The L-shaped arrangement ofthese domains creates a shallow trench spanningthe FN2 and CTL1 domains, which was shown bymutagenesis to bind triple-helical and denaturedcollagen. Small-angle X-ray scattering showed thatthe L-shaped structure is maintained in solution atneutral and acidic pH, irrespective of calcium ionloading. Collagen binding was equally unaffectedby acidic pH, suggesting that collagen release in endosomesis not regulated by changes within theEndo180 N-terminal region.
Journal articleSaito K, Rutherford AW, Ishikita H, 2015,
In photosystem II (PSII), the Mn4CaO5 cluster catalyses the water splitting reaction. The crystal structure of PSII shows the presence of a hydrogen-bonded water molecule directly linked to O4. Here we show the detailed properties of the H-bonds associated with the Mn4CaO5 cluster using a quantum mechanical/molecular mechanical approach. When O4 is taken as a μ-hydroxo bridge acting as a hydrogen-bond donor to water539 (W539), the S0 redox state best describes the unusually short O4–OW539 distance (2.5 Å) seen in the crystal structure. We find that in S1, O4 easily releases the proton into a chain of eight strongly hydrogen-bonded water molecules. The corresponding hydrogen-bond network is absent for O5 in S1. The present study suggests that the O4-water chain could facilitate the initial deprotonation event in PSII. This unexpected insight is likely to be of real relevance to mechanistic models for water oxidation.
Journal articleCota E, Hoyer LL, 2015,
The Candida albicans agglutinin-like sequence family of adhesins: functional insights gained from structural analysis, Future Microbiology, Vol: 10, Pages: 1635-1648, ISSN: 1746-0921
Candida albicans colonizes many host sites suggesting its interaction with diverse ligands.Candida albicans adhesion is mediated by a number of proteins including those in the Als(agglutinin-like sequence) family, which have been studied intensively. The recent solutionof the Als binding domain structure ended years of speculation regarding the molecularmechanism for Als adhesive function. Als adhesins bind flexible C termini from a broadcollection of proteins, providing the basis for adhesion to various cell types and perhaps forC. albicans broad tissue tropism. Understanding adhesive functions at the molecular level willreveal the sequence of events in C. albicans pathogenesis, from host recognition to complexinteractions such as development of polymicrobial biofilms or disseminated disease.
Journal articleWilliams DR, Quigley A, 2015,
The second virial coefficient as a predictor of protein aggregation propensity: a self-interaction chromatography study, European Journal of Pharmaceutics and Biopharmaceutics, Vol: 96, Pages: 282-290, ISSN: 0939-6411
The second osmotic virial coefficients (b2) of four proteins – lysozyme, recombinant human lactoferrin, concanavalin A and catalase were measured by self-interaction chromatography (SIC) in solutions of varying salt type, concentration and pH. Protein aggregate sizes based on the initial hydrodynamic radius of the protein solution species present were measured using dynamic light scattering, and the relationship between b2 and protein aggregate size was studied. A linear correlation was established between b2 values and protein aggregate hydrodynamic size for all proteins, and for almost all solution conditions. Aggregate sizes of <∼10 nm, indicative of non-aggregated protein systems, were consistently observed to have b2 values >0. The observed b2 trends as a function of solution conditions were very much protein dependent, with notable trends including the existence of attractive interactions (negative b2 values) at low ionic strengths for catalase and concanavalin A, and the highly positive b2 values observed for lactoferrin over a wide range of solution conditions, reflecting lactoferrin’s innately high stability. It is concluded that the quantification of protein–protein interactions using SIC based b2 data is a potentially valuable screening tool for predicting protein aggregation propensity.
Journal articleSchroeder GN, Frankel G, Tate EW, et al., 2015,
Legionella pneumophila is a bacterial pathogen that thrives in alveolar macrophages, causing a severe pneumonia. The virulence of L. pneumophila depends on its Dot/Icm type IV secretion system (T4SS), which delivers more than 300 effector proteins into the host, where they rewire cellular signaling to establish a replication-permissive niche, the Legionella-containing vacuole (LCV). Biogenesis of the LCV requires substantial redirection of vesicle trafficking and remodeling of intracellular membranes. In order to achieve this, several T4SS effectors target regulators of membrane trafficking, while others resemble lipases. Here, we characterized LpdA, a phospholipase D effector, which was previously proposed to modulate the lipid composition of the LCV. We found that ectopically expressed LpdA was targeted to the plasma membrane and Rab4- and Rab14-containing vesicles. Subcellular targeting of LpdA required a C-terminal motif, which is posttranslationally modified by S-palmitoylation. Substrate specificity assays showed that LpdA hydrolyzed phosphatidylinositol, -inositol-3- and -4-phosphate, and phosphatidylglycerol to phosphatidic acid (PA) in vitro. In HeLa cells, LpdA generated PA at vesicles and the plasma membrane. Imaging of different phosphatidylinositol phosphate (PIP) and organelle markers revealed that while LpdA did not impact on membrane association of various PIP probes, it triggered fragmentation of the Golgi apparatus. Importantly, although LpdA is translocated inefficiently into cultured cells, an L. pneumophila ΔlpdA mutant displayed reduced replication in murine lungs, suggesting that it is a virulence factor contributing to L. pneumophila infection in vivo.
Journal articleBeis K, 2015,
Journal articleGu R-X, Corradi V, Singh G, et al., 2015,
Journal articleCotton CAR, Kabasakal BV, Miah N, et al., 2015,
Structure of the dual-function fructose-1,6/sedoheptulose-1,7-bisphosphatase from Thermosynechococcus elongatus bound with sedoheptulose-7-phosphate, Acta Crystallographica Section F, Vol: F71, Pages: 1341-1345, ISSN: 2053-230X
The dual-function fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) in cyanobacteria carries out two activities in the Calvin cycle. Structures of this enzyme from the cyanobacterium Synechocystis sp. PCC 6803 exist, but only with adenosine monophosphate (AMP) or fructose-1,6-bisphosphate and AMP bound. The mechanisms which control both selectivity between the two sugars and the structural mechanisms for redox control are still unresolved. Here, the structure of the dual-function FBP/SBPase from the thermophilic cyanobacterium Thermosynechococcus elongatus is presented with sedoheptulose-7-phosphate bound and in the absence of AMP. The structure is globally very similar to the Synechocystis sp. PCC 6803 enzyme, but highlights features of selectivity at the active site and loop ordering at the AMP-binding site. Understanding the selectivity and control of this enzyme is critical for understanding the Calvin cycle in cyanobacteria and for possible biotechnological application in plants.
Journal articleZhang N, Schaefer J, Sharma A, et al., 2015,
Mutations in RNA Polymerase Bridge Helix and Switch Regions Affect Active-Site Networks and Transcript-Assisted Hydrolysis, Journal of Molecular Biology, Vol: 427, Pages: 3516-3526, ISSN: 1089-8638
In bacterial RNA polymerase (RNAP), the bridge helix and switch regions form an intricate network with the catalytic active centre and the main channel. These interactions are important for catalysis, hydrolysis and clamp domain movement. By targeting conserved residues in Escherichia coli RNAP, we are able to show that functions of these regions are differentially required during σ70-dependent and the contrasting σ54-dependent transcription activations and thus potentially underlie the key mechanistic differences between the two transcription paradigms. We further demonstrate that the transcription factor DksA directly regulates σ54-dependent activation both positively and negatively. This finding is consistent with the observed impacts of DksA on σ70-dependent promoters. DksA does not seem to significantly affect RNAP binding to a pre-melted promoter DNA but affects extensively activity at the stage of initial RNA synthesis on σ54-regulated promoters. Strikingly, removal of the σ54 Region I is sufficient to invert the action of DksA (from stimulation to inhibition or vice versa) at two test promoters. The RNAP mutants we generated also show a strong propensity to backtrack. These mutants increase the rate of transcript-hydrolysis cleavage to a level comparable to that seen in the Thermus aquaticus RNAP even in the absence of a non-complementary nucleotide. These novel phenotypes imply an important function of the bridge helix and switch regions as an anti-backtracking ratchet and an RNA hydrolysis regulator.
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