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  • 1.
    Benlloch, Reyes
    et al.
    Department of Forest Genetics and Plant Physiology, SLU.
    Shevela, Dmitriy
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hainzl, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Shutova, Tatyana
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Messinger, Johannes
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Samuelsson, Göran
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Crystal structure and functional characterization of Photosystem II-associated carbonic anhydrase CAH3 in Chlamydomonas reinhardtii2015In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 167, no 3, p. 950-962Article in journal (Refereed)
    Abstract [en]

    In oxygenic photosynthesis, light energy is stored in the form of chemical energy by converting CO2 and water into carbohydrates.The light-driven oxidation of water that provides the electrons and protons for the subsequent CO2 fixation takes place inphotosystem II (PSII). Recent studies show that in higher plants, HCO3– increases PSII activity by acting as a mobile acceptor ofthe protons produced by PSII. In the green alga Chlamydomonas reinhardtii, a luminal carbonic anhydrase, CrCAH3, was suggested toimprove proton removal from PSII, possibly by rapid reformation of HCO3– from CO2. In this study, we investigated the interplaybetween PSII and CrCAH3 by membrane inlet mass spectrometry and x-ray crystallography. Membrane inlet mass spectrometrymeasurements showed that CrCAH3 was most active at the slightly acidic pH values prevalent in the thylakoid lumen underillumination. Two crystal structures of CrCAH3 in complex with either acetazolamide or phosphate ions were determined at 2.6- and2.7-Å resolution, respectively. CrCAH3 is a dimer at pH 4.1 that is stabilized by swapping of the N-terminal arms, a feature notpreviously observed in a-type carbonic anhydrases. The structure contains a disulfide bond, and redox titration of CrCAH3 functionwith dithiothreitol suggested a possible redox regulation of the enzyme. The stimulating effect of CrCAH3 and CO2/HCO3– on PSIIactivity was demonstrated by comparing the flash-induced oxygen evolution pattern of wild-type and CrCAH3-less PSIIpreparations. We showed that CrCAH3 has unique structural features that allow this enzyme to maximize PSII activity at lowpH and CO2 concentration.

  • 2.
    Edwin, Aaron
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Öhman, Anders
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Stier, Gunter
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sauer-Eriksson, A Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Domain isolation, expression, purification and proteolytic activity of the metalloprotease PrtV from Vibrio cholerae2014In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 96, p. 39-47Article in journal (Refereed)
    Abstract [en]

    The metalloprotease PrtV from Vibrio cholerae serves an important function for the bacteria's ability to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102kDa) multidomain pre-pro-protein that so far has only been expressed in V. cholerae. Structural studies require high amounts of soluble protein with high purity. Previous attempts for recombinant expression have been hampered by low expression and solubility of protein fragments. Here, we describe results from parallel cloning experiments in Escherichia coli where fusion tagged constructs of PrtV fragments were designed, and protein products tested for expression and solubility. Of more than 100 designed constructs, three produced protein products that expressed well. These include the N-terminal domain (residues 23-103), the PKD1 domain (residues 755-839), and a 25kDa fragment (residues 581-839). The soluble fusion proteins were captured with Ni(2+) affinity chromatography, and subsequently cleaved with tobacco etch virus protease. Purification protocols yielded ∼10-15mg of pure protein from 1L of culture. Proper folding of the shorter domains was confirmed by heteronuclear NMR spectra recorded on (15)N-labeled samples. A modified protocol for the native purification of the secreted 81kDa pro-protein of PrtV is provided. Proteolytic activity measurements suggest that the 37kDa catalytic metalloprotease domain alone is sufficient for activity.

  • 3.
    Eriksson, Jonas
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Sauer-Eriksson, A Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Sauer, Uwe H
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Elofsson, Mikael
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Small Molecule Screening for Inhibitors of the YopH Phosphatase of Yersinia pseudotuberculosis2012In: Advances in Yersinia Research, New York: Springer, 2012, Vol. 954, p. 357-363Chapter in book (Refereed)
    Abstract [en]

    Bacterial virulence systems are attractive targets for development of new antibacterial agents. Yersinia spp. utilize the type III secretion (T3S) system to secrete and translocate Yersinia outer proteins (Yop effectors) into the cytosol of the target cell and thereby overcome host defenses to successfully establish an infection. Thus, the Yop effectors constitute attractive targets for drug development. In the present study we apply small molecule screening to identify inhibitors of one of the secreted proteins YopH, a tyrosine phosphatase required for virulence. Characterization of seven inhibitors indicated that both competitive and noncompetitive inhibitors were identified with IC50 values of 6–20 μM.

  • 4.
    Good, James A. D.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Andersson, Christopher
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Hansen, Sabine
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wall, Jessica
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Krishnan, Syam
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Begum, Afshan
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Niemiec, Moritz Sebastian
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Vaitkevicius, Karolis
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sauer, Uwe H.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Sauer–Eriksson, A. Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Johansson, Jörgen
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Attenuating Listeria monocytogenes virulence by targeting the regulatory protein PrfA2016In: Cell chemical biology, ISSN 2451-9448, Vol. 23, no 3, p. 404-414Article in journal (Refereed)
    Abstract [en]

    The transcriptional activator PrfA, a member of the Crp/Fnr family, controls the expression of some key virulence factors necessary for infection by the human bacterial pathogen Listeria monocytogenes. Phenotypic screening identified ring-fused 2-pyridone molecules that at low micromolar concentrations attenuate L. monocytogenes infectivity by reducing the expression of virulence genes, without compromising bacterial growth. These inhibitors bind the transcriptional regulator PrfA and decrease its affinity for the consensus DNA binding site. Structural characterization of this interaction revealed that one of the ring-fused 2-pyridones, compound 1, binds within a hydrophobic pocket, located between the C- and N-terminal domains of PrfA, and interacts with residues important for PrfA activation. This indicates that these inhibitors maintain the DNA-binding helix-turn-helix motif of PrfA in a disordered state, thereby preventing a PrfA:DNA interaction. Ring-fused 2-pyridones represent a new class of chemical probes for studying virulence in L. monocytogenes.

  • 5.
    Hall, Michael
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Begum, Afshan
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Lindberg, Mikael J.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Sauer, Uwe H.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Johansson, Jörgen
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Sauer-Eriksson, A. Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Structural basis for glutathione-mediated activation of the virulence regulatory protein PrfA in Listeria2016In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, no 51, p. 14733-14738Article in journal (Refereed)
    Abstract [en]

    Infection by the human bacterial pathogen Listeria monocytogenes is mainly controlled by the positive regulatory factor A (PrfA), a member of the Crp/Fnr family of transcriptional activators. Published data suggest that PrfA requires the binding of a cofactor for full activity, and it was recently proposed that glutathione (GSH) could fulfill this function. Here we report the crystal structures of PrfA in complex with GSH and in complex with GSH and its cognate DNA, the hly operator PrfA box motif. These structures reveal the structural basis for a GSH-mediated allosteric mode of activation of PrfA in the cytosol of the host cell. The crystal structure of PrfAWT in complex only with DNA confirms that PrfAWT can adopt a DNA binding-compatible structure without binding the GSH activator molecule. By binding to PrfA in the cytosol of the host cell, GSH induces the correct fold of the HTH motifs, thus priming the PrfA protein for DNA interaction.

  • 6.
    Huang, Shenghua
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hainzl, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Forsman, Cecilia
    Orphan Biovitrum AB, Umeå, Sweden.
    Samuelsson, Göran
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Sauer-Eriksson, A Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Structural studies of β-Carbonic Anhydrase from the Green Alga Coccomyxa: Inhibitor complexes with Anions and Acetazolamide2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 12, p. e28458-Article in journal (Refereed)
    Abstract [en]

    The β-class carbonic anhydrases (β-CAs) are widely distributed among lower eukaryotes, prokaryotes, archaea, and plants. Like all CAs, the β-enzymes catalyze an important physiological reaction, namely the interconversion between carbon dioxide and bicarbonate. In plants the enzyme plays an important role in carbon fixation and metabolism. To further explore the structure-function relationship of β-CA, we have determined the crystal structures of the photoautotroph unicellular green alga Coccomyxa β-CA in complex with five different inhibitors: acetazolamide, thiocyanate, azide, iodide, and phosphate ions. The tetrameric Coccomyxa β-CA structure is similar to other β-CAs but it has a 15 amino acid extension in the C-terminal end, which stabilizes the tetramer by strengthening the interface. Four of the five inhibitors bind in a manner similar to what is found in complexes with α-type CAs. Iodide ions, however, make contact to the zinc ion via a zinc-bound water molecule or hydroxide ion - a type of binding mode not previously observed in any CA. Binding of inhibitors to Coccomyxa β-CA is mediated by side-chain movements of the conserved residue Tyr-88, extending the width of the active site cavity with 1.5-1.8 Å. Structural analysis and comparisons with other α- and β-class members suggest a catalytic mechanism in which the movements of Tyr-88 are important for the CO(2)-HCO(3) (-) interconversion, whereas a structurally conserved water molecule that bridges residues Tyr-88 and Gln-38, seems important for proton transfer, linking water molecules from the zinc-bound water to His-92 and buffer molecules.

  • 7.
    Kovermann, Michael
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. University of Konstanz, Department of Chemistry, Constance, Germany.
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sauer-Eriksson, A. Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sauer, Uwe H.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Structural basis for ligand binding to an enzyme by a conformational selection pathway2017In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 24, p. 6298-6303Article in journal (Refereed)
    Abstract [en]

    Proteins can bind target molecules through either induced fit or conformational selection pathways. In the conformational selection model, a protein samples a scarcely populated high-energy state that resembles a target-bound conformation. In enzymatic catalysis, such high-energy states have been identified as crucial entities for activity and the dynamic interconversion between ground states and high-energy states can constitute the rate-limiting step for catalytic turnover. The transient nature of these states has precluded direct observation of their properties. Here, we present a molecular description of a high-energy enzyme state in a conformational selection pathway by an experimental strategy centered on NMR spectroscopy, protein engineering, and X-ray crystallography. Through the introduction of a disulfide bond, we succeeded in arresting the enzyme adenylate kinase in a closed high-energy conformation that is on-pathway for catalysis. A 1.9-angstrom X-ray structure of the arrested enzyme in complex with a transition state analog shows that catalytic side-chains are properly aligned for catalysis. We discovered that the structural sampling of the substrate free enzyme corresponds to the complete amplitude that is associated with formation of the closed and catalytically active state. In addition, we found that the trapped high-energy state displayed improved ligand binding affinity, compared with the wild-type enzyme, demonstrating that substrate binding to the high-energy state is not occluded by steric hindrance. Finally, we show that quenching of fast time scale motions observed upon ligand binding to adenylate kinase is dominated by enzyme-substrate interactions and not by intramolecular interactions resulting from the conformational change.

  • 8.
    Kovermann, Michael
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sauer-Eriksson, A. Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sauer, Uwe H
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Structural basis for catalytically restrictive dynamics of a high-energy enzyme state2015In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 6, article id 7644Article in journal (Refereed)
    Abstract [en]

    An emerging paradigm in enzymology is that transient high-energy structural states play crucial roles in enzymatic reaction cycles. Generally, these high-energy or ‘invisible’ states cannot be studied directly at atomic resolution using existing structural and spectroscopic techniques owing to their low populations or short residence times. Here we report the direct NMR-based detection of the molecular topology and conformational dynamics of a catalytically indispensable high-energy state of an adenylate kinase variant. On the basis of matching energy barriers for conformational dynamics and catalytic turnover, it was found that the enzyme’s catalytic activity is governed by its dynamic interconversion between the high-energy state and a ground state structure that was determined by X-ray crystallography. Our results show that it is possible to rationally tune enzymes’ conformational dynamics and hence their catalytic power—a key aspect in rational design of enzymes catalysing novel reactions.

  • 9. Krypotou, Emilia
    et al.
    Scortti, Mariela
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Oelker, Melanie
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Luisi, Ben F.
    Sauer-Eriksson, A. Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Vazquez-Boland, Jose
    Control of Bacterial Virulence through the Peptide Signature of the Habitat2019In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 26, no 7, p. 1815-1827Article in journal (Refereed)
    Abstract [en]

    To optimize fitness, pathogens selectively activate their virulence program upon host entry. Here, we report that the facultative intracellular bacterium Listeria monocytogenes exploits exogenous oligopeptides, a ubiquitous organic N source, to sense the environment and control the activity of its virulence transcriptional activator, PrfA. Using a genetic screen in adsorbent- treated ( PrfA-inducing) medium, we found that PrfA is functionally regulated by the balance between activating and inhibitory nutritional peptides scavenged via the Opp transport system. Activating peptides provide essential cysteine precursor for the PrfA-inducing cofactor glutathione ( GSH). Non-cysteine-containing peptides cause promiscuous PrfA inhibition. Biophysical and co-crystallization studies reveal that peptides inhibit PrfA through steric blockade of the GSH binding site, a regulation mechanism directly linking bacterial virulence and metabolism. L. monocytogenes mutant analysis in macrophages and our functional data support a model in which changes in the balance of antagonistic Oppimported oligopeptides promote PrfA induction intra-cellularly and PrfA repression outside the host.

  • 10.
    Kulén, Martina
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Lindgren, Marie
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Hansen, Sabine
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Cairns, Andrew G.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Begum, Afshan
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    van der Lingen, Ingeborg
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hall, Michael
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Sauer, Uwe H.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Johansson, Jörgen
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Sauer-Eriksson, A. Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Structure-based design of inhibitors targeting PrfA, the master virulence regulator of Listeria monocytogenes2018In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 61, no 9, p. 4165-4175Article in journal (Refereed)
    Abstract [en]

    Listeria monocytogenes is a bacterial pathogen that controls much of its virulence through the transcriptional regulator PrfA. In this study, we describe structure guided design and synthesis of a set of PrfA inhibitors based on ring-fused 2-pyridone heterocycles. Our most effective compound decreased virulence factor expression, reduced bacterial uptake into eukaryotic cells, and improved survival of chicken embryos infected with L. monocytogenes compared to previously identified compounds. Crystal structures identified an intraprotein "tunnel" as the main inhibitor binding site (A1), where the compounds participate in an extensive hydrophobic network that restricts the protein's ability to form functional DNA-binding helix−turn−helix (HTH) motifs. Our studies also revealed a hitherto unsuspected structural plasticity of the HTH motif. In conclusion, we have designed 2-pyridone analogues that function as site-A1 selective PrfA inhibitors with potent antivirulence properties.

  • 11.
    Rogne, Per
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Andersson, David
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Linusson, Anna
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Nucleation of an Activating Conformational Change by a Cation−Π Interaction2019In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 58, no 32, p. 3408-3412Article in journal (Refereed)
    Abstract [en]

    As a key molecule in biology, adenosine triphosphate (ATP) has numerous crucial functions in, for instance, energetics, post-translational modifications, nucleotide biosynthesis, and cofactor metabolism. Here, we have discovered an intricate interplay between the enzyme adenylate kinase and its substrate ATP. The side chain of an arginine residue was found to be an efficient sensor of the aromatic moiety of ATP through the formation of a strong cation−π interaction. In addition to recognition, the interaction was found to have dual functionality. First, it nucleates the activating conformational transition of the ATP binding domain and also affects the specificity in the distant AMP binding domain. In light of the functional consequences resulting from the cation−π interaction, it is possible that the mode of ATP recognition may be a useful tool in enzyme design.

  • 12.
    Rogne, Per
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Rosselin, Marie
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hedberg, Christian
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    H. Sauer, Uwe
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Molecular mechanism of ATP versus GTP selectivity of adenylate kinase2018In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 12, p. 3012-3017Article in journal (Refereed)
    Abstract [en]

    Enzymatic substrate selectivity is critical for the precise control of metabolic pathways. In cases where chemically related substrates are present inside cells, robust mechanisms of substrate selectivity are required. Here, we report the mechanism utilized for catalytic ATP versus GTP selectivity during adenylate kinase (Adk) -mediated phosphorylation of AMP. Using NMR spectroscopy we found that while Adk adopts a catalytically competent and closed structural state in complex with ATP, the enzyme is arrested in a catalytically inhibited and open state in complex with GTP. X-ray crystallography experiments revealed that the interaction interfaces supporting ATP and GTP recognition, in part, are mediated by coinciding residues. The mechanism provides an atomic view on how the cellular GTP pool is protected from Adk turnover, which is important because GTP has many specialized cellular functions. In further support of this mechanism, a structure-function analysis enabled by synthesis of ATP analogs suggests that a hydrogen bond between the adenine moiety and the backbone of the enzyme is vital for ATP selectivity. The importance of the hydrogen bond for substrate selectivity is likely general given the conservation of its location and orientation across the family of eukaryotic protein kinases.

  • 13.
    Wikström Hultdin, Ulrika
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lindberg, Stina
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Allgardsson, Anders
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Huang, Shenghua
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Stier, Günter
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Öhman, Anders
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Uhlin, Bernt Eric
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Purification, crystallization and preliminary data analysis of FocB, a transcription factor regulating fimbrial adhesin expression in uropathogenic Escherichia coli2010In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 66, no Pt 3, p. 337-341Article in journal (Refereed)
    Abstract [en]

    The transcription factor FocB belongs to a family of regulators encoded by several different fimbriae gene clusters in uropathogenic Escherichia coli. Recent findings suggest that FocB-family proteins may form different protein-protein complexes and that they may exert both positive and negative effects on the transcription of fimbriae genes. However, little is known about the actual role and mode of action when these proteins interact with the fimbriae operons. The 109-amino-acid FocB transcription factor from the foc gene cluster in E. coli strain J96 has been cloned, expressed and purified. The His6-tagged fusion protein was captured by Ni2+-affinity chromatography, cleaved with tobacco etch virus protease and purified by gel filtration. The purified protein is oligomeric, most likely in the form of dimers. NMR analysis guided the crystallization attempts by showing that probable conformational exchange or oligomerization is reduced at temperatures above 293 K and that removal of the highly flexible His6 tag is advantageous. The protein was crystallized using the hanging-drop vapour-diffusion method at 295 K. A native data set to 2.0 Å resolution was collected at 100 K using synchrotron radiation.

  • 14.
    Wikström Hultdin, Ulrika
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lindberg, Stina
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Huang, Shenghua
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Uhlin, Bernt Eric
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Structure of FocB: a member of a family of transcription factors regulating fimbrial adhesin expression in uropathogenic Escherichia coli2010In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 16, p. 3368-3381Article in journal (Refereed)
    Abstract [en]

    In uropathogenic Escherichia coli, UPEC, different types of fimbriae are expressed in order to mediate interactions with host tissue. FocB belongs to the PapB family of transcription factors involved in the regulation of fimbriae gene clusters. Recent findings suggest that members from this family of proteins may form different protein-protein complexes and that they may exert both positive and negative effects on transcription of fimbriae genes. To elucidate the detailed function of FocB, we have determined its crystal structure at 1.4 Å resolution. FocB is an all alpha helical structure with a helix-turn-helix (HTH) motif. Interestingly, conserved residues important for DNA-binding are not located in the recognition helix of the HTH-motif, but in the preceding helix. Results from protein-DNA binding studies indicated that FocB interacts with minor groove of its cognate DNA, which also points to a DNA-interaction unusual for this motif. Packing interactions in the crystals gave two plausible dimerization interfaces. Conserved residues known to be important for protein oligomerization are present at both interfaces, suggesting that both sites play a role in a functional FocB protein.

  • 15.
    Zetterström, Caroline E.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Uusitalo, Pia
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Qian, Weixing
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Hinch, Shannon
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Caraballo, Remi
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Elofsson, Mikael
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Screening for Inhibitors of Acetaldehyde Dehydrogenase (AdhE) from Enterohemorrhagic Escherichia coli (EHEC)2018In: SLAS Discovery, ISSN 2472-5552, Vol. 23, no 8, p. 815-822Article in journal (Refereed)
    Abstract [en]

    Acetaldehyde dehydrogenase (AdhE) is a bifunctional acetaldehyde-coenzyme A (CoA) dehydrogenase and alcohol dehydrogenase involved in anaerobic metabolism in gram-negative bacteria. This enzyme was recently found to be a key regulator of the type three secretion (T3S) system in Escherichia coli. AdhE inhibitors can be used as tools to study bacterial virulence and a starting point for discovery of novel antibacterial agents. We developed a robust enzymatic assay, based on the acetaldehyde-CoA dehydrogenase activity of AdhE using both absorption and fluorescence detection models (Z' > 0.7). This assay was used to screen similar to 11,000 small molecules in 384-well format that resulted in three hits that were confirmed by resynthesis and validation. All three compounds are noncompetitive with respect to acetaldehyde and display a clear dose-response effect with hill slopes of 1-2. These new inhibitors will be used as chemical tools to study the interplay between metabolism and virulence and the role of AdhE in T3S regulation in gram-negative bacteria, and as starting points for the development of novel antibacterial agents.

  • 16.
    Zhang, Jin
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Begum, Afshan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Iakovleva, Irina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sauer-Eriksson, A. Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Andersson, Patrik L.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Structure-based Virtual Screening Protocol for in silico Identification of Potential Thyroid Disrupting Chemicals Targeting Transthyretin2016In: Environmental Science and Technology, ISSN 0013-936X, E-ISSN 1520-5851, Vol. 50, no 21, p. 11984-11993Article in journal (Refereed)
    Abstract [en]

    Thyroid disruption by xenobiotics is associated with a broad spectrum of severe adverse outcomes. One possible molecular target of thyroid hormone disrupting chemicals (THDCs) is transthyretin (TTR), a thyroid hormone transporter in vertebrates. To better understand the interactions between TTR and THDCs, we determined the crystallographic structures of human TTR in complex with perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), and 2,2',4,4'-tetrahydroxybenzophenone (BP2). The molecular interactions between the ligands and TTR were further characterized using molecular dynamics simulations. A structure-based virtual screening (VS) protocol was developed with the intention of providing an efficient tool for the discovery of novel TTR-binders from the Tox21 inventory. Among the 192 predicted binders, 12 representatives were selected, and their TTR binding affinities were studied with isothermal titration calorimetry, of which seven compounds had binding affinities between 0.26 and 100 mu M. To elucidate structural details in their binding to TTR, crystal structures were determined of TTR in complex with four of the identified compounds including 2,6-dinitro-p-cresol, bisphenol S, clonixin, and triclopyr. The compounds were found to bind in the TTR hormone binding sites as predicted. Our results show that the developed VS protocol is able to successfully identify potential THDCs, and we suggest that it can be used to propose THDCs for future toxicological evaluations.

  • 17.
    Zhang, Jin
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Iakovleva, Irina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lindberg, Mikael J.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Andersson, Patrik L.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sauer-Eriksson, A. Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Interspecies variation between fish and human transthyretins in their binding of thyroid-disrupting chemicals2018In: Environmental Science and Technology, ISSN 0013-936X, E-ISSN 1520-5851, Vol. 52, no 20, p. 11865-11874Article in journal (Refereed)
    Abstract [en]

    Thyroid-disrupting chemicals (TDCs) are xenobiotics that can interfere with the endocrine system and cause adverse effects in organisms and their offspring. TDCs affect both the thyroid gland and regulatory enzymes associated with thyroid hormone homeostasis. Transthyretin (TTR) is found in the serum and cerebrospinal fluid of vertebrates, where it transports thyroid hormones. Here, we explored the interspecies variation in TDC binding to human and fish TTR (exemplified by Gilthead seabream (Sparus aurata)). The in vitro binding experiments showed that TDCs bind with equal or weaker affinity to seabream TTR than to the human TTR, in particular, the polar TDCs (>500-fold lower affinity). Crystal structures of the seabream TTR TDC complexes revealed that all TDCs bound at the thyroid binding sites. However, amino acid substitution of Ser117 in human TTR to Thr117 in seabream prevented polar TDCs from binding deep in the hormone binding cavity, which explains their low affinity to seabream TTR Molecular dynamics and in silico alanine scanning simulation also suggested that the protein backbone of seabream TTR is more rigid than the human one and that Thr117 provides fewer electrostatic contributions than Ser117 to ligand binding. This provides an explanation for the weaker affinities of the ligands that rely on electrostatic interactions with Thr117. The lower affinities of TDCs to fish TTR, in particular the polar ones, could potentially lead to milder thyroid-related effects in fish.

1 - 17 of 17
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