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  • 1.
    Addario, Barbara
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Huang, Shenghua
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sauer, Uwe
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Backman, Lars
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Crystallization and preliminary X-ray analysis of the Entamoeba histolytica α-actinin-2 rod domain2011Ingår i: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 67, nr 10, s. 1214-1217Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    -Actinins form antiparallel homodimers that are able to cross-link actin filaments. The protein contains three domains: an N-terminal actin-binding domain followed by a central rod domain and a calmodulin-like EF-hand domain at the C-terminus. Here, crystallization of the rod domain of Entamoeba histolytica -actinin-2 is reported; it crystallized in space group P212121, with unit-cell parameters a = 47.8, b = 79.1, c = 141.8 Å. A Matthews coefficient VM of 2.6 Å3 Da-1 suggests that there are two molecules and 52.5% solvent content in the asymmetric unit. A complete native data set extending to a d-spacing of 2.8 Å was collected on beamline I911-2 at MAX-lab, Sweden.

     

  • 2.
    Bäckström, Stefan
    et al.
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Huang, Shenghua
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Wolf-Watz, Magnus
    Kemi.
    Xie, X Q
    Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Härd, Torleif
    Grundström, Thomas
    Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Sauer, Uwe
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Crystallization and preliminary studies of the DNA-binding runt domain of AML1.2001Ingår i: Acta Crystallogr D Biol Crystallogr, ISSN 0907-4449, Vol. 57, nr Pt 2, s. 269-71Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The acute myeloid leukaemia 1 (AML1) protein belongs to the Runx family of transcription factors and is crucial for haematopoietic development. The genes encoding Runx1 and its associated factor CBF beta are the most frequent targets for chromosomal rearrangements in acute human leukaemias. In addition, point mutations of Runx1 in acute leukaemias and in the familial platelet disorder FPD/AML cluster within the evolutionary conserved runt domain that binds both DNA and CBF beta. Here, the crystallization of the Runx1 runt domain is reported. Crystals belong to space groups C2 and R32 and diffract to 1.7 and 2.0 A resolution, respectively.

  • 3.
    Bäckström, Stefan
    et al.
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Wolf-Watz, Magnus
    Kemi.
    Grundström, Christine
    Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Härd, Torleif
    Grundström, Thomas
    Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Sauer, Uwe
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    The RUNX1 Runt domain at 1.25A resolution: a structural switch and specifically bound chloride ions modulate DNA binding.2002Ingår i: J Mol Biol, ISSN 0022-2836, Vol. 322, nr 2, s. 259-72Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The evolutionarily conserved Runt homology domain is characteristic of the RUNX family of heterodimeric eukaryotic transcription factors, including RUNX1, RUNX2 and RUNX3. The genes for RUNX1, also termed acute myeloid leukemia protein 1, AML1, and its dimerization partner core-binding factor beta, CBFbeta, are essential for hematopoietic development and are together the most common targets for gene rearrangements in acute human leukemias. Here, we describe the crystal structure of the uncomplexed RUNX1 Runt domain at 1.25A resolution and compare its conformation to previously published structures in complex with DNA, CBFbeta or both. We find that complex formation induces significant structural rearrangements in this immunoglobulin (Ig)-like DNA-binding domain. Most pronounced is the movement of loop L11, which changes from a closed conformation in the free Runt structure to an open conformation in the CBFbeta-bound and DNA-bound forms. This transition, which we refer to as the S-switch, and accompanying structural movements that affect other parts of the Runt domain are crucial for sustained DNA binding. The closed to open transition can be induced by CBFbeta alone; suggesting that one role of CBFbeta is to trigger the S-switch and to stabilize the Runt domain in a conformation enhanced for DNA binding.A feature of the Runt domain hitherto unobserved in any Ig-like DNA-binding domain is the presence of two specifically bound chloride ions. One chloride ion is coordinated by amino acid residues that make direct DNA contact. In a series of electrophoretic mobility-shift analyses, we demonstrate a chloride ion concentration-dependent stimulation of the DNA-binding activity of Runt in the physiological range. A comparable DNA-binding stimulation was observed for negatively charged amino acid residues. This suggests a regulatory mechanism of RUNX proteins through acidic amino acid residues provided by activation domains during cooperative interaction with other transcription factors.

  • 4. Carbera Rodrigues, Carlos
    et al.
    Ekström, Fredrik
    Wolf, Jana
    Seibt, Henrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Stier, Gunter
    Grundström, Christin
    Hung, Shen-Hua
    Shingler, Victoria
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Sauer, Uwe H.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    The crystal structure of the DmpR AAA+ domain suggests a novel mode of ATP-dependent transcriptional controlManuskript (preprint) (Övrigt vetenskapligt)
  • 5. Dalhus, Bjørn
    et al.
    Saarinen, Markuu
    Sauer, Uwe
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Eklund, Pär
    Johansson, Kenth
    Karlsson, Andreas
    Ramaswamy, S
    Bjørk, Alexandra
    Synstad, Bjørnar
    Naterstad, Kristine
    Sirevåg, Reidun
    Eklund, Hans
    Structural basis for thermophilic protein stability: structures of thermophilic and mesophilic malate dehydrogenases.2002Ingår i: J Mol Biol, ISSN 0022-2836, Vol. 318, nr 3, s. 707-21Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The three-dimensional structure of four malate dehydrogenases (MDH) from thermophilic and mesophilic phototropic bacteria have been determined by X-ray crystallography and the corresponding structures compared. In contrast to the dimeric quaternary structure of most MDHs, these MDHs are tetramers and are structurally related to tetrameric malate dehydrogenases from Archaea and to lactate dehydrogenases. The tetramers are dimers of dimers, where the structures of each subunit and the dimers are similar to the dimeric malate dehydrogenases. The difference in optimal growth temperature of the corresponding organisms is relatively small, ranging from 32 to 55 degrees C. Nevertheless, on the basis of the four crystal structures, a number of factors that are likely to contribute to the relative thermostability in the present series have been identified. It appears from the results obtained, that the difference in thermostability between MDH from the mesophilic Chlorobium vibrioforme on one hand and from the moderate thermophile Chlorobium tepidum on the other hand is mainly due to the presence of polar residues that form additional hydrogen bonds within each subunit. Furthermore, for the even more thermostable Chloroflexus aurantiacus MDH, the use of charged residues to form additional ionic interactions across the dimer-dimer interface is favored. This enzyme has a favorable intercalation of His-Trp as well as additional aromatic contacts at the monomer-monomer interface in each dimer. A structural alignment of tetrameric and dimeric prokaryotic MDHs reveal that structural elements that differ among dimeric and tetrameric MDHs are located in a few loop regions. (c) 2002 Elsevier Science Ltd.

  • 6.
    Ekström, Fredrik
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Stier, Gunter
    Eaton, Julian T
    Sauer, Uwe
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Crystallization and X-ray analysis of a bacterial non-haem iron-containing phenylalanine hydroxylase from the Gram-negative opportunistic pathogen Pseudomonas aeruginosa.2003Ingår i: Acta Crystallogr D Biol Crystallogr, ISSN 0907-4449, Vol. 59, nr Pt 7, s. 1310-2Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Monooxygenases are frequently involved in the pathways that mediate the pivotal role of microorganisms in recycling carbon from the environment. A structural study of a monooxygenase from Pseudomonas aeruginosa that was identified as a phenylalanine hydroxylase has been initiated. The single-domain monomeric protein harbours a non-haem iron at the active site. The sequence identity to the catalytic domains of tyrosine and tryptophan hydroxylases suggests that the enzyme is not restricted to the substrate phenylalanine alone. Here, the cloning, purification and crystallization of native and SeMet-labelled P. aeruginosa phenylalanine hydroxylase are reported. Crystals grew in space group P6(1), with unit-cell parameters a = b = 210.5, c = 100.7 A, and diffracted to a d spacing of 2.0 A. Crystals of SeMet-labelled protein were used to collect a three-wavelength multiple anomalous dispersion (MAD) data set around the Se K edge.

  • 7.
    Ekström, Fredrik
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Stier, Gunter
    Sauer, Uwe
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Crystallization of the actin-binding domain of human alpha-actinin: analysis of microcrystals of SeMet-labelled protein2003Ingår i: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 59, nr Pt 4, s. 724-726Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Alpha-actinin forms antiparallel homodimers that cross-link actin filaments from adjacent sarcomeres within the Z-discs of striated muscle. The N-terminal actin-binding domain (ABD) is composed of two calponin homology (CH) domains followed by four spectrin-like repeats and a calmodulin-like EF-hand domain at the C-terminus. The ABD of human alpha-actinin crystallizes in space group P2(1), with unit-cell parameters a = 101.9, b = 38.4, c = 154.9 A, beta = 109.2 degrees. A complete native data set from a native crystal was collected extending to 2.0 A resolution and a single-wavelength anomalous dispersion (SAD) data set to 2.9 A resolution was collected from a selenomethionine-labelled microcrystal using the microfocusing beamline ID-13 at the ESRF. Analysis of the anomalous contribution shows a rapid decrease in the sigma(normal)/sigma(anomal) ratio owing to radiation damage.

  • 8.
    Eriksson, Jonas
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Grundström, Christin
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Sauer-Eriksson, A Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Sauer, Uwe H
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Wolf-Watz, Hans
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Elofsson, Mikael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Small Molecule Screening for Inhibitors of the YopH Phosphatase of Yersinia pseudotuberculosis2012Ingår i: Advances in Yersinia Research, New York: Springer, 2012, Vol. 954, s. 357-363Kapitel i bok, del av antologi (Refereegranskat)
    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.

  • 9.
    Good, James A. D.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Andersson, Christopher
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Hansen, Sabine
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Wall, Jessica
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Krishnan, Syam
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Begum, Afshan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Grundström, Christin
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Niemiec, Moritz Sebastian
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Vaitkevicius, Karolis
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Chorell, Erik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Wittung-Stafshede, Pernilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sauer, Uwe H.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Sauer–Eriksson, A. Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Johansson, Jörgen
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Attenuating Listeria monocytogenes virulence by targeting the regulatory protein PrfA2016Ingår i: Cell chemical biology, ISSN 2451-9448, Vol. 23, nr 3, s. 404-414Artikel i tidskrift (Refereegranskat)
    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.

  • 10.
    Hall, Michael
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Grundström, Christin
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Begum, Afshan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Lindberg, Mikael J.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Sauer, Uwe H.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Johansson, Jörgen
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Sauer-Eriksson, A. Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Structural basis for glutathione-mediated activation of the virulence regulatory protein PrfA in Listeria2016Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, nr 51, s. 14733-14738Artikel i tidskrift (Refereegranskat)
    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.

  • 11.
    Hall, Michael
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Kieselbach, Thomas
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sauer, Uwe
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Schröder, Wolfgang P
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Purification, crystallization and preliminary X-ray analysis of PPD6, a PsbP-domain protein from Arabidopsis thaliana2012Ingår i: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 68, nr 3, s. 278-280Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The PsbP protein is an extrinsic component of photosystem II that together with PsbO and PsbQ forms the thylakoid lumenal part of the oxygen-evolving complex in higher plants. In addition to PsbP, the thylakoid lumen contains two PsbP-like proteins (PPLs) and six PsbP-domain proteins (PPDs). While the functions of the PsbP-like proteins PPL1 and PPL2 are currently under investigation, the function of the PsbP-domain proteins still remains completely unknown. PPD6 is unique among the PsbP family of proteins in that it contains a conserved disulfide bond which can be reduced in vitro by thioredoxin. The crystal structure determination of the PPD6 protein has been initiated in order to elucidate its function and to gain deeper insights into redox-regulation pathways in the thylakoid lumen. PPD6 has been expressed, purified and crystallized and preliminary X-ray diffraction data have been collected. The crystals belonged to space group P2(1), with unit-cell parameters a = 47.0, b = 64.3, c = 62.0 Å, β = 94.2°, and diffracted to a maximum d-spacing of 2.1 Å.

  • 12.
    Hall, Michael
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    von Sydow, Lotta
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Storm, Patrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sauer, Uwe
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Kieselbach, Thomas
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Schröder, Wolfgang
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    The lumenal pentapeptide repeat proteins TL15 and TL20.3 are novel chaperone-like proteins in the chloroplast lumen of higher plantsManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    In the thylakoid lumen of Arabidopsis thaliana, three pentapeptide repeat family proteins of unknown function are localized. Pentapeptide repeat proteins (PRP) are comprised of at least eight tandem repeats of five amino acids of the consensus sequence A(D/N)LXX, which fold into a quadrilateral beta helix structure. Here we have solved the crystal structure of the mature form of the lumenal PRP protein TL15 to 1.3 Å resolution. TL15 is comprised of a main pentapeptide domain, consisting of a total of 19 pentapeptide repeats which form five turns of a beta helix, and a C-terminal alpha helix domain consisting of two alpha helices. The alpha helices form a ‘cap’ at the C-terminal end of the beta helix and are connected by a disulphide bond between the conserved cysteine residues C122 and C142. Furthermore we show that the lumenal PRPs TL15 and TL20.3 can assist in refolding of a chemically denatured substrate in vitro, indicating foldase chaperone activity. The three lumenal PRPs have been previously identified as targets of thioredoxin, and interestingly we observed a greatly increased chaperone activity of TL15 and TL20.3 after reduction of their disulphide bonds. Our results provide the high resolution crystal structure of the TL15 protein and our analysis of chaperone activity suggests that TL15 and TL20.3 may constitute a novel type of redox-regulated molecular chaperones in the chloroplast lumen of higher plants.

  • 13.
    Kovermann, Michael
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. University of Konstanz, Department of Chemistry, Constance, Germany.
    Grundström, Christin
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sauer-Eriksson, A. Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sauer, Uwe H.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wolf-Watz, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Structural basis for ligand binding to an enzyme by a conformational selection pathway2017Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, nr 24, s. 6298-6303Artikel i tidskrift (Refereegranskat)
    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.

  • 14.
    Kovermann, Michael
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Grundström, Christin
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sauer-Eriksson, A. Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sauer, Uwe H
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wolf-Watz, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Structural basis for catalytically restrictive dynamics of a high-energy enzyme state2015Ingår i: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 6, artikel-id 7644Artikel i tidskrift (Refereegranskat)
    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.

  • 15.
    Kulén, Martina
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Lindgren, Marie
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Hansen, Sabine
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Cairns, Andrew G.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Grundström, Christin
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Begum, Afshan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    van der Lingen, Ingeborg
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Hall, Michael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Sauer, Uwe H.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Johansson, Jörgen
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Sauer-Eriksson, A. Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Structure-based design of inhibitors targeting PrfA, the master virulence regulator of Listeria monocytogenes2018Ingår i: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 61, nr 9, s. 4165-4175Artikel i tidskrift (Refereegranskat)
    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.

  • 16.
    Lundberg, Erik
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Bäckström, Stefan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Sauer, Uwe
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Sauer-Eriksson, Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    The transthyretin-related protein: structural investigation of a novel protein family2006Ingår i: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 155, nr 3, s. 445-457Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The transthyretin-related protein (TRP) family comprises proteins predicted to be structurally related to the homotetrameric transport protein transthyretin (TTR). The function of TRPs is not yet fully established, but recent data suggest that they are involved in purine catabolism. We have determined the three-dimensional structure of the Escherichia coli TRP in two crystal forms; one at 1.65 A resolution in the presence of zinc, and the other at 2.1 A resolution in the presence of zinc and bromide. The structures revealed five zinc-ion-binding sites per monomer. Of these, the zinc ions bound at sites I and II are coordinated in tetrahedral geometries to the side chains of residues His9, His96, His98, Ser114, and three water molecules at the putative ligand-binding site. Of these four residues, His9, His98, and Ser114 are conserved. His9 and His98 bind the central zinc (site I) together with two water molecules. The side chain of His98 also binds to the zinc ion at site II. Bromide ions bind at site I only, replacing one of the water molecules coordinated to the zinc ion. The C-terminal four amino acid sequence motif Y-[RK]-G-[ST] constitutes the signature sequence of the TRP family. Two Tyr111 residues form direct hydrogen bonds to each other over the tetramer interface at the area, which in TTR constitutes the rear part of its thyroxine-binding channel. The putative substrate/ligand-binding channel of TRP is consequently shallower and broader than its counterpart in TTR.

  • 17.
    Mishra, Yogesh
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Hall, Michael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Chaurasia, Neha
    Rai, Lal Chand
    Jansson, Stefan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Schröder, Wolfgang P
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik.
    Sauer, Uwe H
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Expression, purification, crystallization and preliminary X-ray crystallographic studies of alkyl hydroperoxide reductase (AhpC) from the cyanobacterium Anabaena sp. PCC 71202011Ingår i: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 67, nr 10, s. 1203-1206Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Alkyl hydroperoxide reductase (AhpC) is a key component of a large family of thiol-specific antioxidant (TSA) proteins distributed among prokaryotes and eukaryotes. AhpC is involved in the detoxification of reactive oxygen species (ROS) and reactive sulfur species (RSS). Sequence analysis of AhpC from the cyanobacterium Anabaena sp. PCC 7120 shows that this protein belongs to the 1-Cys class of peroxiredoxins (Prxs). It has recently been reported that enhanced expression of this protein in Escherichia coli offers tolerance to multiple stresses such as heat, salt, copper, cadmium, pesticides and UV-B. However, the structural features and the mechanism behind this process remain unclear. To provide insights into its biochemical function, AhpC was expressed, purified and crystallized by the hanging-drop vapour-diffusion method. Diffraction data were collected to a maximum d-spacing of 2.5 Å using synchrotron radiation. The crystal belonged to space group P212121, with unit-cell parameters a = 80, b = 102, c = 109.6 Å. The structure of AhpC from Anabaena sp. PCC 7120 was determined by molecular-replacement methods using the human Prx enzyme hORF6 (PDB entry1prx) as the template.

     

  • 18.
    Mishra, Yogesh
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Hall, Michael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Locmelis, Roland
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Nam, Kwangho
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, TX, 76019-0065, USA.
    Söderberg, Christopher A. G.
    Storm, Patrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Chaurasia, Neha
    Rai, Lal Chand
    Jansson, Stefan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik.
    Schröder, Wolfgang P.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Sauer, Uwe H.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Active-site plasticity revealed in the asymmetric dimer of AnPrx6 the 1-Cys peroxiredoxin and molecular chaperone from Anabaena sp. PCC 71202017Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, artikel-id 17151Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Peroxiredoxins (Prxs) are vital regulators of intracellular reactive oxygen species levels in all living organisms. Their activity depends on one or two catalytically active cysteine residues, the peroxidatic Cys (C-P) and, if present, the resolving Cys (C-R). A detailed catalytic cycle has been derived for typical 2-Cys Prxs, however, little is known about the catalytic cycle of 1-Cys Prxs. We have characterized Prx6 from the cyanobacterium Anabaena sp. strain PCC7120 (AnPrx6) and found that in addition to the expected peroxidase activity, AnPrx6 can act as a molecular chaperone in its dimeric state, contrary to other Prxs. The AnPrx6 crystal structure at 2.3 angstrom resolution reveals different active site conformations in each monomer of the asymmetric obligate homo-dimer. Molecular dynamic simulations support the observed structural plasticity. A FSH motif, conserved in 1-Cys Prxs, precedes the active site PxxxTxxCp signature and might contribute to the 1-Cys Prx reaction cycle.

  • 19.
    Rogne, Per
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Rosselin, Marie
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Grundström, Christin
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Hedberg, Christian
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    H. Sauer, Uwe
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wolf-Watz, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Molecular mechanism of ATP versus GTP selectivity of adenylate kinase2018Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, nr 12, s. 3012-3017Artikel i tidskrift (Refereegranskat)
    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.

  • 20.
    Seibt, Henrik
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Sauer, Uwe H.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Shingler, Victoria
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    The Y233 gatekeeper of DmpR modulates effector-responsive transcriptional control of δ54-RNA polymerase2019Ingår i: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 21, nr 4, s. 1321-1330Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    DmpR is the obligate transcriptional activator of genes involved in (methyl)phenol catabolism by Pseudomonas putida. DmpR belongs to the AAA+ class of mechano‐transcriptional regulators that employ ATP‐hydrolysis to engage and remodel σ54‐RNA polymerase to allow transcriptional initiation. Previous work has established that binding of phenolic effectors by DmpR is a prerequisite to relieve interdomain repression and allow ATP‐binding to trigger transition to its active multimeric conformation, and further that a structured interdomain linker between the effector‐ and ATP‐binding domains is involved in coupling these processes. Here, we present evidence from ATPase and in vivo and in vitro transcription assays that a tyrosine residue of the interdomain linker (Y233) serves as a gatekeeper to constrain ATP‐hydrolysis and aromatic effector‐responsive transcriptional activation by DmpR. An alanine substitution of Y233A results in both increased ATPase activity and enhanced sensitivity to aromatic effectors. We propose a model in which effector‐binding relocates Y233 to synchronize signal‐reception with multimerisation to provide physiologically appropriate sensitivity of the transcriptional response. Given that Y233 counterparts are present in many ligand‐responsive mechano‐transcriptional regulators, the model is likely to be pertinent for numerous members of this family and has implications for development of enhanced sensitivity of biosensor used to detect pollutants.

  • 21.
    Sellstedt, Magnus
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Dang, Hung The
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Prasad, G. Krishna
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sauer, Uwe
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Four-Component Assembly of Natural-Product-Like Ring-Fused Isoquinuclidines2013Ingår i: European Journal of Organic Chemistry, ISSN 1434-193X, E-ISSN 1099-0690, Vol. 2013, nr 33, s. 7476-7479Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A four-component reaction between formyl-substituted 2-pyridones, aldehydes, amines, and activated alkenes has been developed. The resulting products are ring-fused natural-product-like isoquinuclidines. Three carbon–carbon bonds, two carbon–nitrogen bonds, and four or five stereocentres are formed in the reaction with overall product yields in the range 23–67 %. In most cases a single diastereomer was obtained. An intramolecular version of the reaction yielded analogues of the multi-ring-fused natural product catharanthine in a single step.

  • 22.
    Shaikhali, Jehad
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Norén, Louise
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Barajas-Lopez, Juan de Dios
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Srivastava, Vaibhav
    Koenig, Janine
    Sauer, Uwe H.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wingsle, Gunnar
    Dietz, Karl-Josef
    Strand, Åsa
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Redox-mediated Mechanisms Regulate DNA Binding Activity of the G-group of Basic Region Leucine Zipper (bZIP) Transcription Factors in Arabidopsis2012Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, nr 33, s. 27510-27525Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Plant genes that contain the G-box in their promoters are responsive to a variety of environmental stimuli. Bioinformatics analysis of transcriptome data revealed that the G-box element is significantly enriched in promoters of high light-responsive genes. From nuclear extracts of high light-treated Arabidopsis plants, we identified the AtbZIP16 transcription factor as a component binding to the G-box-containing promoter fragment of light-harvesting chlorophyll a/b-binding protein2.4 (LHCB2.4). AtbZIP16 belongs to the G-group of Arabidopsis basic region leucine zipper (bZIP) type transcription factors. Although AtbZIP16 and its close homologues AtbZIP68 and AtGBF1 bind the G-box, they do not bind the mutated half-sites of the G-box palindrome. In addition, AtbZIP16 interacts with AtbZIP68 and AtGBF1 in the yeast two-hybrid system. A conserved Cys residue was shown to be necessary for redox regulation and enhancement of DNA binding activity in all three proteins. Furthermore, transgenic Arabidopsis lines overexpressing the wild type version of bZIP16 and T-DNA insertion mutants for bZIP68 and GBF1 demonstrated impaired regulation of LHCB2.4 expression. Finally, overexpression lines for the mutated Cys variant of bZIP16 provided support for the biological significance of Cys330 in redox regulation of gene expression. Thus, our results suggest that environmentally induced changes in the redox state regulate the activity of members of the G-group of bZIP transcription factors.

  • 23.
    Singh, Pardeep
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Cairns, Andrew G.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Adolfsson, Dan E.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sauer, Uwe H.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Synthesis of Densely Functionalized N-Alkenyl 2-Pyridones via Benzyne-Induced Ring Opening of Thiazolino-Fused 2-Pyridones2019Ingår i: Organic Letters, ISSN 1523-7060, E-ISSN 1523-7052, Vol. 21, s. 6946-6950Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We report the synthesis of 6-arylthio-substituted-N-alkenyl 2-pyridones by ring opening of bicyclic thiazolino-2-pyridones with arynes. Varied functionalization was used to investigate scope and substituent influences on reactivity. Selected conditions favor thioether ring opening over [4 + 2] cycloaddition and an unusual aryne incorporating ring expansion. Deuterium labeling was used to clarify observed reactivity. Using the knowledge, we produced drug-like molecules with complex substitution patterns and show how thioether ring opening can be used on scaffolds with competing reactivities.

  • 24.
    Tångrot, Jeanette
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för datavetenskap. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Kågström, Bo
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för datavetenskap. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Högpresterande beräkningscentrum norr (HPC2N).
    Sauer, Uwe
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Accurate Domain Identification with Structure-Anchored Hidden Markov Models, saHMMs2009Ingår i: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 76, nr 2, s. 343-352Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The ever increasing speed of DNA sequencing widens the discrepancy between the number of known gene products, and the knowledge of their function and structure. Proper annotation of protein sequences is therefore crucial if the missing information is to be deduced from sequence-based similarity comparisons. These comparisons become exceedingly difficult as the pairwise identities drop to very low values. To improve the accuracy of domain identification, we exploit the fact that the three-dimensional structures of domains are much more conserved than their sequences. Based on structure-anchored multiple sequence alignments of low identity homologues we constructed 850 structure-anchored hidden Markov models (saHMMs), each representing one domain family. Since the saHMMs are highly family specific, they can be used to assign a domain to its correct family and clearly distinguish it from domains belonging to other families, even within the same superfamily. This task is not trivial and becomes particularly difficult if the unknown domain is distantly related to the rest of the domain sequences within the family. In a search with full length protein sequences, harbouring at least one domain as defined by the structural classification of proteins database (SCOP), version 1.71, versus the saHMM database based on SCOP version 1.69, we achieve an accuracy of 99.0%. All of the few hits outside the family fall within the correct superfamily. Compared to Pfam_ls HMMs, the saHMMs obtain about 11% higher coverage. A comparison with BLAST and PSI-BLAST demonstrates that the saHMMs have consistently fewer errors per query at a given coverage. Within our recommended E-value range, the same is true for a comparison with SUPERFAMILY. Furthermore, we are able to annotate 232 proteins with 530 nonoverlapping domains belonging to 102 different domain families among human proteins labelled unknown in the NCBI protein database. Our results demonstrate that the saHMM database represents a versatile and reliable tool for identification of domains in protein sequences. With the aid of saHMMs, homology on the family level can be assigned, even for distantly related sequences. Due to the construction of the saHMMs, the hits they provide are always associated with high quality crystal structures. The saHMM database can be accessed via the FISH server at http://babel.ucmp.umu.se/fish/.

  • 25.
    Tångrot, Jeanette
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för datavetenskap.
    Wang, Lixiao
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Kågström, Bo
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för datavetenskap. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Högpresterande beräkningscentrum norr (HPC2N).
    Sauer, Uwe
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    FISH-Family identification of sequence homologues using structure anchored hidden Markov models2006Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 34, nr Web Server issue, s. W10-W14Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The FISH server is highly accurate in identifying the family membership of domains in a query protein sequence, even in the case of very low sequence identities to known homologues. A performance test using SCOP sequences and an E-value cut-off of 0.1 showed that 99.3% of the top hits are to the correct family saHMM. Matches to a query sequence provide the user not only with an annotation of the identified domains and hence a hint to their function, but also with probable 2D and 3D structures, as well as with pairwise and multiple sequence alignments to homologues with low sequence identity. In addition, the FISH server allows users to upload and search their own protein sequence collection or to quarry public protein sequence data bases with individual saHMMs. The FISH server can be accessed at http://babel.ucmp.umu.se/fish/.

  • 26.
    Tångrot, Jeanette
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för datavetenskap. Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Medicinska fakulteten).
    Wang, Lixiao
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Medicinska fakulteten).
    Kågström, Bo
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för datavetenskap. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Högpresterande beräkningscentrum norr (HPC2N).
    Sauer, Uwe H.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Design, construction and use of the FISH server2007Ingår i: Applied parallel computing: state of the art in scientific computing, Springer Link , 2007, s. 647-657Kapitel i bok, del av antologi (Övrigt vetenskapligt)
    Abstract [en]

    At the core of the FISH (Family Identification with Structure anchored Hidden Markov models, saHMMs) server lies the midnight ASTRAL set. It is a collection of protein domains with low mutual sequence identity within homologous families, according to the structural classification of proteins, SCOP. Here, we evaluate two algorithms for creating the midnight ASTRAL set. The algorithm that limits the number of structural comparisons is about an order of magnitude faster than the all-against-all algorithm. We therefore choose the faster algorithm, although it produces slightly fewer domains in the set. We use the midnight ASTRAL set to construct the structure-anchored Hidden Markov Model data base, saHMM-db, where each saHMM represents one family. Sequence searches using saHMMs provide information about protein function, domain organization, the probable 2D and 3D structure, and can lead to the discovery of homologous domains in remotely related sequences.

  • 27.
    Wang, Lixiao
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sauer, Uwe
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Prediction of protein stability changes due to single amino acid mutationsManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Accurate prediction of the change in protein stability due to single amino acid mutations is important for guiding site-directed mutagenesis and other protein-engineering techniques. Recently, different two state predictors became available aimed at predicting if point mutations stabilize or destabilize a protein. Considering the experimental errors and tolerances of protein with respect to mutations, we realized that the neutral mutations, which only slightly affect the protein’s stability, must be considered as well. Here, we present a new classification scheme for a three-state predictor (destabilizing, neutral and stabilizing mutations) based on multi-class support vector machines (SVM). We have created a refined training dataset of single amino acid mutations and evaluate the predictive ability of models trained on homology clustered and non-clustered training data using two different cross validation procedures. The experimental results reveal the significant difference of prediction accuracy according to different evaluation procedures. Furthermore we demonstrate that, for non-clustered model, the prediction accuracy based on the protein sequence information alone is comparable to the prediction accuracy based on protein structure information. On the other hand, for clustered model, the prediction ability is significantly improved when protein tertiary structure information is included. The comparison of prediction accuracy for the two models reveals that the prediction accuracy of mutation stability on clustered proteins is still a challenging task. Moreover, benchmarking by using previously published datasets, demonstrate that our method has an improved prediction performance over many established methods.

  • 28.
    Wang, Lixiao
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Sauer, Uwe H
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    OnD-CRF: predicting order and disorder in proteins using [corrected] conditional random fields2008Ingår i: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 24, nr 11, s. 1401-1402Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    MOTIVATION: Order and Disorder prediction using Conditional Random Fields (OnD-CRF) is a new method for accurately predicting the transition between structured and mobile or disordered regions in proteins. OnD-CRF applies CRFs relying on features which are generated from the amino acids sequence and from secondary structure prediction. Benchmarking results based on CASP7 targets, and evaluation with respect to several CASP criteria, rank the OnD-CRF model highest among the fully automatic server group. AVAILABILITY: http://babel.ucmp.umu.se/ond-crf/

  • 29.
    Wolf-Watz, Magnus
    et al.
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Kemi.
    Bäckström, Stefan
    Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Grundström, Thomas
    Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Sauer, Uwe
    Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Härd, Torleif
    Chloride binding by the AML1/Runx1 transcription factor studied by NMR2001Ingår i: FEBS Letters, ISSN 0014-5793, Vol. 488, nr 1-2, s. 81-4Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    It is known that the DNA binding Runt domain of the AML1/Runx1 transcription factor coordinates Cl(-) ions. In this paper we have determined Cl(-) binding affinities of AML1 by (35)Cl nuclear magnetic resonance (NMR) linewidth analysis. The Runt domain binds Cl(-) with a dissociation constant (K(d,Cl)) of 34 mM. If CBFbeta is added to form a 1:1 complex, the K(d,Cl) value increases to 56 mM. Homology modeling suggests that a high occupancy Cl(-) binding site overlaps with the DNA binding surface. NMR data show that DNA displaces this Cl(-) ion. Possible biological roles of Cl(-) binding are discussed based on these findings.

  • 30.
    Wolf-Watz, Magnus
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Rogne, Per
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sauer-Eriksson, A. Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sauer, Uwe H.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Hedberg, Christian
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Positive and Negative Substrate Interference Supported by Coinciding Enzyme Residues2019Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 116, nr 3, s. 485A-485AArtikel i tidskrift (Övrigt vetenskapligt)
1 - 30 av 30
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