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  • Journal article
    Wobbe L, Nixon PJ, 2013,

    The mTERF protein MOC1 terminates mitochondrial DNA transcription in the unicellular green alga Chlamydomonas reinhardtii

    , Nucleic Acids Research, Vol: 41, Pages: 6553-6567, ISSN: 1362-4962

    The molecular function of mTERFs (mitochondrialtranscription termination factors) has so far onlybeen described for metazoan members of theprotein family and in animals they control mitochondrialreplication, transcription and translation. Cells ofphotosynthetic eukaryotes harbour chloroplasts andmitochondria, which are in an intense cross-talk thatis vital for photosynthesis. Chlamydomonasreinhardtii is a unicellular green alga widely used asa model organism for photosynthesis research andgreen biotechnology. Among the six nuclear C.reinhardtii mTERF genes is mTERF-like gene ofChlamydomonas (MOC1), whose inactivation altersmitorespiration and interestingly also light-acclimationprocesses in the chloroplast that favour theenhanced production of biohydrogen. We show herefrom in vitro studies that MOC1 binds specifically to asequence within the mitochondrial rRNA-codingmodule S3, and that a knockout of MOC1 in themutant stm6 increases read-through transcription atthis site, indicating that MOC1 acts as a transcriptionterminator in vivo. Whereas the level of certainantisense RNA species is higher in stm6, the amountof unprocessed mitochondrial sense transcripts isstrongly reduced, demonstrating that a loss of MOC1causes perturbed mitochondrial DNA (mtDNA) expression.Overall, we provide evidence for the existenceof mitochondrial antisense RNAs in C. reinhardtiiand show that mTERF-mediated transcription terminationis an evolutionary-conserved mechanismoccurring in phototrophic protists and metazoans.
  • Journal article
    Emerson M, Solomon A, Smyth E, Vydyanath A, Luther P, Tetley TDet al., 2013,

    Role of platelets in driving the thrombotic risk and protective processes associated with exposure to diesel exhaust particles

    , JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Vol: 11, Pages: 643-644, ISSN: 1538-7933
  • Journal article
    Harding CR, Stoneham CA, Schuelein R, Newton H, Oates CV, Hartland EL, Schroeder GN, Frankel Get al., 2013,

    The Dot/Icm Effector SdhA Is Necessary for Virulence of <i>Legionella pneumophila</i> in <i>Galleria mellonella</i> and A/J Mice

    , INFECTION AND IMMUNITY, Vol: 81, Pages: 2598-2605, ISSN: 0019-9567
  • Journal article
    Balasco N, Esposito L, De Simone A, Vitagliano Let al., 2013,

    Role of loops connecting secondary structure elements in the stabilization of proteins isolated from thermophilic organisms

    , PROTEIN SCIENCE, Vol: 22, Pages: 1016-1023, ISSN: 0961-8368
  • Journal article
    Smyth E, Solomon A, Vydyanath A, Luther P, Pitchford S, Tetley TD, Emerson Met al., 2013,

    The potencies and mechanisms by which engineered nanoparticles induce platelet aggregation are dependent upon their precise physicochemistry

    , JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Vol: 11, Pages: 895-896, ISSN: 1538-7933
  • Conference paper
    Horejs C, Bertazzo S, Hohenester E, Stevens Met al., 2013,

    The cleavage of Laminin-111 by MMP-2 affects early differentiation of murine ESCs and iPS cells

    , 38th Congress of the Federation-of-European-Biochemical-Societies (FEBS), Publisher: WILEY-BLACKWELL, Pages: 446-446, ISSN: 1742-464X
  • Journal article
    Bertheleme N, Singh S, Dowell SJ, Hubbard J, Byrne Bet al., 2013,

    Loss of constitutive activity is correlated with increased thermostability of the human adenosine A2A receptor

    , BRITISH JOURNAL OF PHARMACOLOGY, Vol: 169, Pages: 988-998, ISSN: 0007-1188
  • Journal article
    Suzuki H, Yu J, Kobayashi T, Nakanishi H, Nixon PJ, Noguchi Tet al., 2013,

    Functional Roles of D2-Lys317 and the Interacting Chloride Ion in the Water Oxidation Reaction of Photosystem II As Revealed by Fourier Transform Infrared Analysis

    , BIOCHEMISTRY, Vol: 52, Pages: 4748-4757, ISSN: 0006-2960

    Photosynthetic water oxidation in plants andcyanobacteria is catalyzed by a Mn4CaO5 cluster within thephotosystem II (PSII) protein complex. Two Cl− ions boundnear the Mn4CaO5 cluster act as indispensable cofactors, buttheir functional roles remain to be clarified. We haveinvestigated the role of the Cl− ion interacting with D2-K317 (designated Cl-1) by Fourier transform infraredspectroscopy (FTIR) analysis of the D2-K317R mutant ofSynechocystis sp. PCC 6803 in combination with Cl−/NO3−replacement. The D2-K317R mutation perturbed the bands inthe regions of the COO− stretching and backbone amidevibrations in the FTIR difference spectrum upon the S1 → S2 transition. In addition, this mutation altered the 15N isotope-editedNO3− bands in the spectrum of NO3−-treated PSII. These results provide the first experimental evidence that the Cl-1 site iscoupled with the Mn4CaO5 cluster and its interaction is affected by the S1 → S2 transition. It was also shown that a negative bandat 1748 cm−1 arising from COOH group(s) was altered to a positive intensity by the D2-K317R mutation as well as by NO3−treatment, suggesting that the Cl-1 site affects the pKa of COOH/COO− group(s) near the Mn4CaO5 cluster in a commonhydrogen bond network. Together with the observation that the efficiency of the S3 → S0 transition significantly decreased in thecore complexes of D2-K317R upon moderate dehydration, it is suggested that D2-K317 and Cl-1 are involved in a protontransfer pathway from the Mn4CaO5 cluster to the lumen, which functions in the S3 → S0 transition.
  • Journal article
    MacDonald JT, Kelley LA, Freemont PS, 2013,

    Validating a Coarse-Grained Potential Energy Function through Protein Loop Modelling

    , PLOS One, Vol: 8, ISSN: 1932-6203

    Coarse-grained (CG) methods for sampling protein conformational space have the potential to increase computational efficiency by reducing the degrees of freedom. The gain in computational efficiency of CG methods often comes at the expense of non-protein like local conformational features. This could cause problems when transitioning to full atom models in a hierarchical framework. Here, a CG potential energy function was validated by applying it to the problem of loop prediction. A novel method to sample the conformational space of backbone atoms was benchmarked using a standard test set consisting of 351 distinct loops. This method used a sequence-independent CG potential energy function representing the protein using -carbon positions only and sampling conformations with a Monte Carlo simulated annealing based protocol. Backbone atoms were added using a method previously described and then gradient minimised in the Rosetta force field. Despite the CG potential energy function being sequence-independent, the method performed similarly to methods that explicitly use either fragments of known protein backbones with similar sequences or residue-specific /-maps to restrict the search space. The method was also able to predict with sub-Angstrom accuracy two out of seven loops from recently solved crystal structures of proteins with low sequence and structure similarity to previously deposited structures in the PDB. The ability to sample realistic loop conformations directly from a potential energy function enables the incorporation of additional geometric restraints and the use of more advanced sampling methods in a way that is not possible to do easily with fragment replacement methods and also enable multi-scale simulations for protein design and protein structure prediction. These restraints could be derived from experimental data or could be design restraints in the case of computational protein design. C++ source code is available for download from http://www.sbg.
  • Journal article
    Squeglia F, Romano M, Ruggiero A, Vitagliano L, De Simone A, Berisio Ret al., 2013,

    Carbohydrate Recognition by RpfB from Mycobacterium tuberculosis Unveiled by Crystallographic and Molecular Dynamics Analyses.

    , Biophys J, Vol: 104, Pages: 2530-2539

    Resuscitation of Mtb is crucial to the etiology of Tuberculosis, because latent tuberculosis is estimated to affect one-third of the world population. The resuscitation-promoting factor RpfB is mainly responsible for Mtb resuscitation from dormancy. Given the impact of latent Tuberculosis, RpfB represents an interesting target for tuberculosis drug discovery. However, no molecular models of substrate binding and catalysis are hitherto available for this enzyme. Here, we identified key interactions involved in substrate binding to RpfB by combining x-ray diffraction studies and computational approaches. The crystal structure of RpfB catalytic domain in complex with N,N',N″-triacetyl-chitotriose, as described here, provides the first, to our knowledge, atomic representation of ligand recognition by RpfB and demonstrates that the strongest interactions are established by the N-acetylglucosamine moiety in the central region of the enzyme binding cleft. Molecular dynamics analyses provided information on the dynamic behavior of protein-substrate interactions and on the role played by the solvent in RpfB function. These data combined with sequence conservation analysis suggest that Glu-292 is the sole residue crucial for catalysis, implying that RpfB acts via the formation of an oxocarbenium ion rather than a covalent intermediate. Present data represent a solid base for the design of effective drug inhibitors of RpfB. Moreover, homology models were generated for the catalytic domains of all members of the Mtb Rpf family (RpfA-E). The analysis of these models unveiled analogies and differences among the different members of the Rpf protein family.
  • Journal article
    Tabib-Salazar A, Liu B, Doughty P, Lewis RA, Ghosh S, Parsy M-L, Simpson PJ, O'Dwyer K, Matthews SJ, Paget MSet al., 2013,

    The actinobacterial transcription factor RbpA binds to the principal sigma subunit of RNA polymerase

    , NUCLEIC ACIDS RESEARCH, Vol: 41, Pages: 5679-5691, ISSN: 0305-1048
  • Journal article
    Corrigan RM, Campeotto I, Jeganathan T, Roelofs KG, Lee VT, Gruendling Aet al., 2013,

    Systematic identification of conserved bacterial c-di-AMP receptor proteins

    , PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 110, Pages: 9084-9089, ISSN: 0027-8424
  • Journal article
    Saito K, Rutherford AW, Ishikita H, 2013,

    Mechanism of tyrosine D oxidation in Photosystem II

    , PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 110, Pages: 7690-7695, ISSN: 0027-8424
  • Journal article
    Saraiva N, Prole DL, Carrara G, de Motes CM, Johnson BF, Byrne B, Taylor CW, Smith GLet al., 2013,

    Human and Viral Golgi Anti-apoptotic Proteins (GAAPs) Oligomerize via Different Mechanisms and Monomeric GAAP Inhibits Apoptosis and Modulates Calcium

    , JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 288, Pages: 13057-13067
  • Journal article
    Beeby M, Gumbart JC, Roux B, Jensen GJet al., 2013,

    Architecture and assembly of the Gram-positive cell wall

    , Molecular Microbiology, Vol: 88, Pages: 664-672, ISSN: 0950-382X
  • Journal article
    Nederlof I, Li YW, van Heel M, Abrahams JPet al., 2013,

    Imaging protein three-dimensional nanocrystals with cryo-EM

    , ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY, Vol: 69, Pages: 852-859, ISSN: 2059-7983
  • Journal article
    Leen EN, Kwok KYR, Birtley JR, Simpson PJ, Subba-Reddy CV, Chaudhry Y, Sosnovtsev SV, Green KY, Prater SN, Tong M, Young JC, Chung LMW, Marchant J, Roberts LO, Kao CC, Matthews S, Goodfellow IG, Curry Set al., 2013,

    Structures of the Compact Helical Core Domains of Feline Calicivirus and Murine Norovirus VPg Proteins

    , JOURNAL OF VIROLOGY, Vol: 87, Pages: 5318-5330, ISSN: 0022-538X
  • Journal article
    Aurass P, Schlegel M, Metwally O, Harding CR, Schroeder GN, Frankel G, Flieger Aet al., 2013,

    The <i>Legionella pneumophila</i> Dot/Icm-secreted Effector PlcC/CegC1 Together with PlcA and PlcB Promotes Virulence and Belongs to a Novel Zinc Metallophospholipase C Family Present in Bacteria and Fungi

    , JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 288, Pages: 11080-11092
  • Journal article
    Figueira R, Watson KG, Holden DW, Helaine Set al., 2013,

    Identification of salmonella pathogenicity island-2 type III secretion system effectors involved in intramacrophage replication of S. enterica serovar typhimurium: implications for rational vaccine design

    , mBio, Vol: 4, ISSN: 2161-2129

    Salmonella enterica serovars cause severe diseases in humans, such as gastroenteritis and typhoid fever. The development of systemic disease is dependent on a type III secretion system (T3SS) encoded by Salmonella pathogenicity island-2 (SPI-2). Translocation of effector proteins across the Salmonella-containing vacuole, via the SPI-2 T3SS, enables bacterial replication within host cells, including macrophages. Here, we investigated the contribution of these effectors to intramacrophage replication of Salmonella enterica serovar Typhimurium using Fluorescence Dilution, a dual-fluorescence tool which allows direct measurement of bacterial replication. Of 32 strains, each carrying single mutations in genes encoding effectors, 10 (lacking sifA, sseJ, sopD2, sseG, sseF, srfH, sseL, spvD, cigR, or steD) were attenuated in replication in mouse bone marrow-derived macrophages. The replication profiles of strains combining deletions in effector genes were also investigated: a strain lacking the genes sseG, sopD2, and srfH showed an increased replication defect compared to single-mutation strains and was very similar to SPI-2 T3SS-deficient bacteria with respect to its replication defect. This strain was substantially attenuated in virulence in vivo and yet retained intracellular vacuole integrity and a functional SPI-2 T3SS. Moreover, this strain was capable of SPI-2 T3SS-mediated delivery of a model antigen for major histocompatibility complex (MHC) class I-dependent T-cell activation. This work establishes a basis for the use of a poly-effector mutant strain as an attenuated vaccine carrier for delivery of heterologous antigens directly into the cytoplasm of host cells.IMPORTANCE Live attenuated strains of Salmonella enterica serotype Typhi have generated much interest in the search for improved vaccines against typhoid fever and as vaccine vectors for the delivery of heterologous antigens. A promising vaccine candidate is the ΔaroC ΔssaV S. Typhi strain, whic
  • Journal article
    Mercadante D, Melton LD, Jameson GB, Williams MAK, De Simone Aet al., 2013,

    Substrate Dynamics in Enzyme Action: Rotations of Monosaccharide Subunits in the Binding Groove are Essential for Pectin Methylesterase Processivity

    , BIOPHYSICAL JOURNAL, Vol: 104, Pages: 1731-1739, ISSN: 0006-3495
  • Conference paper
    Jensen G, Briegel A, Beeby M, 2013,

    Visualizing large macromolecular assemblies in vivo with electron cryotomography

    , 245th National Spring Meeting of the American-Chemical-Society (ACS), Publisher: AMER CHEMICAL SOC, ISSN: 0065-7727
  • Journal article
    Manzine LR, Balasco Serrao VH, Trambaioli da Rocha e Lima LM, de Souza MM, Bettini J, Portugal RV, van Heel M, Thiemann OHet al., 2013,

    Assembly stoichiometry of bacterial selenocysteine synthase and SelC (tRNA<SUP>sec</SUP>)

    , FEBS LETTERS, Vol: 587, Pages: 906-911, ISSN: 0014-5793
  • Journal article
    Fitzpatrick AWP, Debelouchina GT, Bayro MJ, Clare DK, Caporini MA, Bajaj VS, Jaroniec CP, Wang L, Ladizhansky V, Mueller SA, MacPhee CE, Waudby CA, Mott HR, De Simone A, Knowles TPJ, Saibil HR, Vendruscolo M, Orlova EV, Griffin RG, Dobson CMet al., 2013,

    Atomic structure and hierarchical assembly of a cross-β amyloid fibril

    , PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 110, Pages: 5468-5473, ISSN: 0027-8424
  • Journal article
    Sheppard C, James E, Barton G, Matthews S, Severinov K, Wigneshweraraj Set al., 2013,

    A non-bacterial transcription factor inhibits bacterial transcription by a multipronged mechanism

    , RNA BIOLOGY, Vol: 10, Pages: 495-501, ISSN: 1547-6286
  • Journal article
    Lansky S, Alalouf O, Solomon V, Alhassid A, Govada L, Chayan NE, Belrhali H, Shoham Y, Shoham Get al., 2013,

    Crystallization and preliminary crystallographic analysis of Axe2, an acetylxylan esterase from <i>Geobacillus stearothermophilus</i>

    , ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS, Vol: 69, Pages: 430-434
  • Journal article
    Helaine S, Holden DW, 2013,

    Heterogeneity of intracellular replication of bacterial pathogens

    , CURRENT OPINION IN MICROBIOLOGY, Vol: 16, Pages: 184-191, ISSN: 1369-5274
  • Journal article
    Faunce T, Styring S, Wasielewski MR, Brudvig GW, Rutherford AW, Messinger J, Lee AF, Hill CL, deGroot H, Fontecave M, MacFarlane DR, Hankamer B, Nocera DG, Tiede DM, Dau H, Hillier W, Wang L, Amal Ret al., 2013,

    Artificial photosynthesis as a frontier technology for energy sustainability

    , ENERGY & ENVIRONMENTAL SCIENCE, Vol: 6, Pages: 1074-1076, ISSN: 1754-5692
  • Journal article
    Carrara M, Prischi F, Ali MMU, 2013,

    UPR Signal Activation by Luminal Sensor Domains

    , Int. j. Mol. Sci., Vol: 14
  • Journal article
    Hachani A, Lossi NS, Filloux A, 2013,

    A visual assay to monitor T6SS-mediated bacterial competition

    , Jove-Journal of Visualized Experiments, ISSN: 1940-087X

    Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.The data available emphasized a numerical approach to the bacterial competition assay
  • Journal article
    Canning P, Cooper CDO, Krojer T, Murray JW, Pike ACW, Chaikuad A, Keates T, Thangaratnarajah C, Hojzan V, Marsden BD, Gileadi O, Knapp S, von Delft F, Bullock ANet al., 2013,

    Structural Basis for Cul3 Protein Assembly with the BTB-Kelch Family of E3 Ubiquitin Ligases

    , JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 288, Pages: 7803-7814

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