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  • 1.
    Andersson, Charlotta S.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Öhrström, Maria
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Popović-Bijelić, Ana
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The manganese ion of the heterodinuclear Mn/Fe cofactor in Chlamydia trachomatis ribonucleotide reductase R2c is located at metal position 1.2012In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 134, no 1, p. 123-125Article in journal (Refereed)
    Abstract [en]

    The essential catalytic radical of Class-I ribonucleotide reductase is generated and delivered by protein R2, carrying a dinuclear metal cofactor. A new R2 subclass, R2c, prototyped by the Chlamydia trachomatis protein was recently discovered. This protein carries an oxygen-activating heterodinuclear Mn(II)/Fe(II) metal cofactor and generates a radical-equivalent Mn(IV)/Fe(III) oxidation state of the metal site, as opposed to the tyrosyl radical generated by other R2 subclasses. The metal arrangement of the heterodinuclear cofactor remains unknown. Is the metal positioning specific, and if so, where is which ion located? Here we use X-ray crystallography with anomalous scattering to show that the metal arrangement of this cofactor is specific with the manganese ion occupying metal position 1. This is the position proximal to the tyrosyl radical site in other R2 proteins and consistent with the assumption that the high-valent Mn(IV) species functions as a direct substitute for the tyrosyl radical.

  • 2.
    Berntsson, Ronnie P. -A.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Odegrip, Richard
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Sehlén, Wilhelmina
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Skaar, Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Svensson, Linda M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Massad, Tariq
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Haggard-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structural insight into DNA binding and oligomerization of the multifunctional Cox protein of bacteriophage P22014In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 4, p. 2725-2735Article in journal (Refereed)
    Abstract [en]

    The Cox protein from bacteriophage P2 is a small multifunctional DNA-binding protein. It is involved in site-specific recombination leading to P2 prophage excision and functions as a transcriptional repressor of the P2 Pc promoter. Furthermore, it transcriptionally activates the unrelated, defective prophage P4 that depends on phage P2 late gene products for lytic growth. In this article, we have investigated the structural determinants to understand how P2 Cox performs these different functions. We have solved the structure of P2 Cox to 2.4 angstrom resolution. Interestingly, P2 Cox crystallized in a continuous oligomeric spiral with its DNA-binding helix and wing positioned outwards. The extended C-terminal part of P2 Cox is largely responsible for the oligomerization in the structure. The spacing between the repeating DNA-binding elements along the helical P2 Cox filament is consistent with DNA binding along the filament. Functional analyses of alanine mutants in P2 Cox argue for the importance of key residues for protein function. We here present the first structure from the Cox protein family and, together with previous biochemical observations, propose that P2 Cox achieves its various functions by specific binding of DNA while wrapping the DNA around its helical oligomer.

  • 3.
    Berntsson, Ronnie P. A.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Peng, Lisheng
    Dong, Min
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structure of dual receptor binding to botulinum neurotoxin B2013In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 4, p. 2058-Article in journal (Refereed)
    Abstract [en]

    Botulinum neurotoxins are highly toxic, and bind two receptors to achieve their high affinity and specificity for neurons. Here we present the first structure of a botulinum neurotoxin bound to both its receptors. We determine the 2.3-angstrom structure of a ternary complex of botulinum neurotoxin type B bound to both its protein receptor synaptotagmin II and its ganglioside receptor GD1a. We show that there is no direct contact between the two receptors, and that the binding affinity towards synaptotagmin II is not influenced by the presence of GD1a. The interactions of botulinum neurotoxin type B with the sialic acid 5 moiety of GD1a are important for the ganglioside selectivity. The structure demonstrates that the protein receptor and the ganglioside receptor occupy nearby but separate binding sites, thus providing two independent anchoring points.

  • 4.
    Berntsson, Ronnie P.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Peng, Lisheng
    Svensson, Linda M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Dong, Min
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Crystal Structures of Botulinum Neurotoxin DC in Complex with Its Protein Receptors Synaptotagmin I and II2013In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 21, no 9, p. 1602-1611Article in journal (Refereed)
    Abstract [en]

    Botulinum neurotoxins (BoNTs) can cause paralysis at exceptionally low concentrations and include seven serotypes (BoNT/A-G). The chimeric BoNT/DC toxin has a receptor binding domain similar to the same region in BoNT/C. However, BoNT/DC does not share protein receptor with BoNT/C. Instead, it shares synaptotagmin (Syt) I and II as receptors with BoNT/B, despite their low sequence similarity. Here, we present the crystal structures of the binding domain of BoNT/DC in complex with the recognition domains of its protein receptors, Syt-I and Syt-II. The structures reveal that BoNT/DC possesses a Syt binding site, distinct from the established Syt-II binding site in BoNT/B. Structure-based mutagenesis further shows that hydrophobic interactions play a key role in Syt binding. The structures suggest that the BoNT/DC ganglioside binding sites are independent of the protein receptor binding site. Our results reveal the remarkable versatility in the receptor recognition of the BoNTs.

  • 5. Bräutigam, Lars
    et al.
    Pudelko, Linda
    Jemth, Ann-Sofie
    Gad, Helge
    Narwal, Mohit
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gustafsson, Robert
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Karsten, Stella
    Carreras Puigvert, Jordi
    Homan, Evert
    Berndt, Carsten
    Warpman Berglund, Ulrika
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Helleday, Thomas
    Hypoxic Signaling and the Cellular Redox Tumor Environment Determine Sensitivity to MTH1 Inhibition2016In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 76, no 8, p. 2366-2375Article in journal (Refereed)
    Abstract [en]

    Cancer cells are commonly in a state of redox imbalance that drives their growth and survival. To compensate for oxidative stress induced by the tumor redox environment, cancer cells upregulate specific nononcogenic addiction enzymes, such as MTH1 (NUDT1), which detoxifies oxidized nucleotides. Here, we show that increasing oxidative stress in nonmalignant cells induced their sensitization to the effects of MTH1 inhibition, whereas decreasing oxidative pressure in cancer cells protected against inhibition. Furthermore, we purified zebrafish MTH1 and solved the crystal structure of MTH1 bound to its inhibitor, highlighting the zebrafish as a relevant tool to study MTH1 biology. Delivery of 8-oxo-dGTP and 2-OH-dATP to zebrafish embryos was highly toxic in the absence of MTH1 activity. Moreover, chemically or genetically mimicking activated hypoxia signaling in zebrafish revealed that pathologic upregulation of the HIF1 alpha response, often observed in cancer and linked to poor prognosis, sensitized embryos to MTH1 inhibition. Using a transgenic zebrafish line, in which the cellular redox status can be monitored in vivo, we detected an increase in oxidative pressure upon activation of hypoxic signaling. Pretreatment with the antioxidant N-acetyl-L-cysteine protected embryos with activated hypoxia signaling against MTH1 inhibition, suggesting that the aberrant redox environment likely causes sensitization. In summary, MTH1 inhibition may offer a general approach to treat cancers characterized by deregulated hypoxia signaling or redox imbalance.

  • 6. Carreras-Puigvert, Jordi
    et al.
    Zitnik, Marinka
    Jemth, Ann-Sofie
    Carter, Megan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Unterlass, Judith E.
    Hallström, Björn
    Loseva, Olga
    Karem, Zhir
    Calderón-Montaño, José Manuel
    Lindskog, Cecilia
    Edqvist, Per-Henrik
    Matuszewski, Damian J.
    Blal, Hammou Ait
    Berntsson, Ronnie P. A.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Häggblad, Maria
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Martens, Ulf
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Studham, Matthew
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Lundgren, Bo
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Wählby, Carolina
    Sonnhammer, Erik L. L.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Lundberg, Emma
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Zupan, Blaz
    Helleday, Thomas
    A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family2017In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 8, article id 1541Article in journal (Refereed)
    Abstract [en]

    The NUDIX enzymes are involved in cellular metabolism and homeostasis, as well as mRNA processing. Although highly conserved throughout all organisms, their biological roles and biochemical redundancies remain largely unclear. To address this, we globally resolve their individual properties and inter-relationships. We purify 18 of the human NUDIX proteins and screen 52 substrates, providing a substrate redundancy map. Using crystal structures, we generate sequence alignment analyses revealing four major structural classes. To a certain extent, their substrate preference redundancies correlate with structural classes, thus linking structure and activity relationships. To elucidate interdependence among the NUDIX hydrolases, we pairwise deplete them generating an epistatic interaction map, evaluate cell cycle perturbations upon knockdown in normal and cancer cells, and analyse their protein and mRNA expression in normal and cancer tissues. Using a novel FUSION algorithm, we integrate all data creating a comprehensive NUDIX enzyme profile map, which will prove fundamental to understanding their biological functionality.

  • 7.
    Carter, Megan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jemth, Ann-Sofie
    Carreras-Puigvert, Jordi
    Herr, Patrick
    Martínez Carranza, Markel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Vallin, Karl S. A.
    Throup, Adam
    Helleday, Thomas
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Human NUDT22 Is a UDP-Glucose/Galactose Hydrolase Exhibiting a Unique Structural Fold2018In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 26, no 2, p. 295-303Article in journal (Refereed)
    Abstract [en]

    Human NUDT22 belongs to the diverse NUDIX family of proteins, but has, until now, remained uncharacterized. Here we show that human NUDT22 is a Mg2+-dependent UDP-glucose and UDP-galactose hydrolase, producing UMP and glucose 1-phosphate or galactose 1-phosphate. We present the structure of human NUDT22 alone and in a complex with the substrate UDP-glucose. These structures reveal a partially conserved NUDIX fold domain preceded by a unique N-terminal domain responsible for UDP moiety binding and recognition. The NUDIX domain of NUDT22 contains a modified NUDIX box identified using structural analysis and confirmed through functional analysis of mutants. Human NUDT22's distinct structure and function as a UDP-carbohydrate hydrolase establish a unique NUDIX protein subfamily.

  • 8.
    Carter, Megan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jemth, Ann-Sofie
    Hagenkort, Anna
    Page, Brent D. G.
    Gustafsson, Robert
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Griese, Julia J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gad, Helge
    Valerie, Nicholas C. K.
    Desroses, Matthieu
    Boström, Johan
    Berglund, Ulrika Warpman
    Helleday, Thomas
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Crystal structure, biochemical and cellular activities demonstrate separate functions of MTH1 and MTH22015In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 6, article id 7871Article in journal (Refereed)
    Abstract [en]

    Deregulated redox metabolism in cancer leads to oxidative damage to cellular components including deoxyribonucleoside triphosphates (dNTPs). Targeting dNTP pool sanitizing enzymes, such as MTH1, is a highly promising anticancer strategy. The MTH2 protein, known as NUDT15, is described as the second human homologue of bacterial MutT with 8-oxo-dGTPase activity. We present the first NUDT15 crystal structure and demonstrate that NUDT15 prefers other nucleotide substrates over 8-oxo-dGTP. Key structural features are identified that explain different substrate preferences for NUDT15 and MTH1. We find that depletion of NUDT15 has no effect on incorporation of 8-oxo-dGTP into DNA and does not impact cancer cell survival in cell lines tested. NUDT17 and NUDT18 were also profiled and found to have far less activity than MTH1 against oxidized nucleotides. We show that NUDT15 is not a biologically relevant 8-oxo-dGTPase, and that MTH1 is the most prominent sanitizer of the cellular dNTP pool known to date.

  • 9. Egeblad, Louise
    et al.
    Welin, Martin
    Johansson, Andreas
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wang, Liya
    Flodin, Susanne
    Nyman, Tomas
    Trésaugues, Lionel
    Kotenyova, Tetyana
    Johansson, Ida
    Eriksson, Staffan
    Eklund, Hans
    Nordlund, Pär
    Structural and functional studies of the human phosphoribosyltransferase domain containing protein 12010In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 23, p. 4920-30Article in journal (Refereed)
    Abstract [en]

    Human hypoxanthine-guanine phosphoribosyltransferase (HPRT) (EC 2.4.2.8) catalyzes the conversion of hypoxanthine and guanine to their respective nucleoside monophosphates. Human HPRT deficiency as a result of genetic mutations is linked to both Lesch-Nyhan disease and gout. In the present study, we have characterized phosphoribosyltransferase domain containing protein 1 (PRTFDC1), a human HPRT homolog of unknown function. The PRTFDC1 structure has been determined at 1.7 Å resolution with bound GMP. The overall structure and GMP binding mode are very similar to that observed for HPRT. Using a thermal-melt assay, a nucleotide metabolome library was screened against PRTFDC1 and revealed that hypoxanthine and guanine specifically interacted with the enzyme. It was subsequently confirmed that PRTFDC1 could convert these two bases into their corresponding nucleoside monophosphate. However, the catalytic efficiency (k(cat)/K(m)) of PRTFDC1 towards hypoxanthine and guanine was only 0.26% and 0.09%, respectively, of that of HPRT. This low activity could be explained by the fact that PRTFDC1 has a Gly in the position of the proposed catalytic Asp of HPRT. In PRTFDC1, a water molecule at the position of the aspartic acid side chain position in HPRT might be responsible for the low activity observed by acting as a weak base. The data obtained in the present study indicate that PRTFDC1 does not have a direct catalytic role in the nucleotide salvage pathway.

  • 10. Frykholm, Karolin
    et al.
    Berntsson, Ronnie Per-Arne
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Claesson, Magnus
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    de Battice, Laura
    Odegrip, Richard
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Westerlund, Fredrik
    DNA compaction by the bacteriophage protein Cox studied on the single DNA molecule level using nanofluidic channels2016In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 15, p. 7219-7227Article in journal (Refereed)
    Abstract [en]

    The Cox protein from bacteriophage P2 forms oligomeric filaments and it has been proposed that DNA can be wound up around these filaments, similar to how histones condense DNA. We here use fluorescence microscopy to study single DNA-Cox complexes in nanofluidic channels and compare how the Cox homologs from phages P2 and W Phi affect DNA. By measuring the extension of nanoconfined DNA in absence and presence of Cox we show that the protein compacts DNA and that the binding is highly cooperative, in agreement with the model of a Cox filament around which DNA is wrapped. Furthermore, comparing microscopy images for the wild-type P2 Cox protein and two mutants allows us to discriminate between compaction due to filament formation and compaction by monomeric Cox. P2 and W Phi Cox have similar effects on the physical properties of DNA and the subtle, but significant, differences in DNA binding are due to differences in binding affinity rather than binding mode. The presented work highlights the use of single DNA molecule studies to confirm structural predictions from X-ray crystallography. It also shows how a small protein by oligomerization can have great impact on the organization of DNA and thereby fulfill multiple regulatory functions.

  • 11. Gad, Helge
    et al.
    Svensson, Linda M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Saleh, Aljona
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    Berntsson, Ronnie P.-A.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gustafsson, Robert
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Djureinovic, Tatjana
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Häggblad, Maria
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Martens, Ulf
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Lundgren, Bo
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Granelli, Ingrid
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Helleday, Thomas
    MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool2014In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 508, no 7495, p. 215-221Article in journal (Refereed)
    Abstract [en]

    Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bind in the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.

  • 12.
    Gustafsson, Robert
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Berntsson, Ronnie P-A
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Umeå University, Sweden.
    Martínez-Carranza, Markel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    El Tekle, Geniver
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Odegrip, Richard
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Johnson, Eric A.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Crystal structures of OrfX2 and P47 from a Botulinum neurotoxin OrfX-type gene cluster2017In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 591, no 22, p. 3781-3792Article in journal (Refereed)
    Abstract [en]

    Botulinum neurotoxins are highly toxic substances and are all encoded together with one of two alternative gene clusters, the HA or the OrfX gene cluster. Very little is known about the function and structure of the proteins encoded in the OrfX gene cluster, which in addition to the toxin contains five proteins (OrfX1, OrfX2, OrfX3, P47, and NTNH). We here present the structures of OrfX2 and P47, solved to 2.1 and 1.8 Å, respectively. We show that they belong to the TULIP protein superfamily, which are often involved in lipid binding. OrfX1 and OrfX2 were both found to bind phosphatidylinositol lipids.

  • 13.
    Gustafsson, Robert
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jemth, Ann-Sofie
    Gustafsson, Nina M. S.
    Färnegårdh, Katarina
    Stockholm University, Faculty of Science, Department of Organic Chemistry. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Loseva, Olga
    Wiita, Elisée
    Bonagas, Nadilly
    Dahllund, Leif
    Llona-Minguez, Sabin
    Häggblad, Maria
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Henriksson, Martin
    Andersson, Yasmin
    Homan, Evert
    Helleday, Thomas
    Stenmark, Pal
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Crystal Structure of the Emerging Cancer Target MTHFD2 in Complex with a Substrate-Based Inhibitor2017In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 77, no 4, p. 937-948Article in journal (Refereed)
    Abstract [en]

    To sustain their proliferation, cancer cells become dependent on one-carbon metabolism to support purine and thymidylate synthesis. Indeed, one of the most highly upregulated enzymes during neoplastic transformation is MTHFD2, a mitochondrial methylenetetrahydrofolate dehydrogenase and cyclohydrolase involved in one-carbon metabolism. Because MTHFD2 is expressed normally only during embryonic development, it offers a disease-selective therapeutic target for eradicating cancer cells while sparing healthy cells. Here we report the synthesis and preclinical characterization of the first inhibitor of human MTHFD2. We also disclose the first crystal structure of MTHFD2 in complex with a substrate-based inhibitor and the enzyme cofactors NAD(+) and inorganic phosphate. Our work provides a rationale for continued development of a structural framework for the generation of potent and selective MTHFD2 inhibitors for cancer treatment.

  • 14.
    Hamark, Christoffer
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Berntsson, Ronnie P. -A.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Masuyer, Geoffrey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Henriksson, Linda M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gustafsson, Robert
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Glycans Confer Specificity to the Recognition of Ganglioside Receptors by Botulinum Neurotoxin A2017In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 139, no 1, p. 218-230Article in journal (Refereed)
    Abstract [en]

    The highly poisonous botulinum neurotoxins, produced by the bacterium Clostridium botulinum, act on their hosts by a high-affinity association to two receptors on neuronal cell surfaces as the first step of invasion. The glycan motifs of gangliosides serve as initial coreceptors for these protein complexes, whereby a membrane protein receptor is bound. Herein we set out to characterize the carbohydrate minimal binding epitope of the botulinum neurotoxin serotype A. By means of ligand-based NMR spectroscopy, X-ray crystallography, computer simulations, and isothermal titration calorimetry, a screening of ganglioside analogues together with a detailed characterization of various carbohydrate ligand complexes with the toxin were accomplished. We show that the representation of the glycan epitope to the protein affects the details of binding. Notably, both branches of the oligosaccharide GD la can associate to botulinum neurotoxin serotype A when expressed as individual trisaccharides. It is, however, the terminal branch of GD1a as well as this trisaccharide motif alone, corresponding to the sialyl-Thomsen-Friedenreich antigen, that represents the active ligand epitope, and these compounds bind to the neurotoxin with a high degree of predisposition but with low affinities. This finding does not correlate with the oligosaccharide moieties having a strong contribution to the total affinity, which was expected to be the case. We here propose that the glycan part of the ganglioside receptors mainly provides abundance and specificity, whereas the interaction with the membrane itself and protein receptor brings about the strong total binding of the toxin to the neuronal membrane.

  • 15.
    Hamark, Christoffer
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Berntsson, Ronnie Per-Arne
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gustafsson, Robert
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Svensson, Linda M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Recognition of Ganglioside Receptors by Botulinum Neurotoxin AManuscript (preprint) (Other academic)
  • 16. Jacobson, Mark J.
    et al.
    Lin, Guangyun
    Tepp, William
    Dupuy, Jerome
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Stevens, Raymond C.
    Johnson, Eric A.
    Purification, Modeling, and Analysis of Botulinum Neurotoxin Subtype A5 (BoNT/A5) from Clostridium botulinum Strain A6612222011In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 77, no 12, p. 4217-4222Article in journal (Refereed)
    Abstract [en]

    A Clostridium botulinum type A strain (A661222) in our culture collection was found to produce the botulinum neurotoxin subtype A5 (BoNT/A5). Its neurotoxin gene was sequenced to determine its degree of similarity to available sequences of BoNT/A5 and the well-studied BoNT/A1. Thirty-six amino acid differences were observed between BoNT/A5 and BoNT/A1, with the predominant number being located in the heavy chain. The amino acid chain of the BoNT/A from the A661222 strain was superimposed over the crystal structure of the known structure of BoNT/A1 to assess the potential significance of these differences-specifically how they would affect antibody neutralization. The BoNT/A5 neurotoxin was purified to homogeneity and evaluated for certain properties, including specific toxicity and antibody neutralization. This study reports the first purification of BoNTA5 and describes distinct differences in properties between BoNT/A5 and BoNT/A1.

  • 17. Jemth, Ann-Sofie
    et al.
    Gustafsson, Robert
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bräutigam, Lars
    Henriksson, Linda
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Desroses, Matthieu
    Carreras Puigvert, Jordi
    Homan, Evert
    Warpman Berglund, Ulrika
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Helleday, Thomas
    MutT homologue 1 (MTH1) catalyses the hydrolysis of mutagenic O6-methyl-dGTPManuscript (preprint) (Other academic)
  • 18.
    Kmiec, Beata
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Teixeira, Pedro F.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Berntsson, Ronnie P. -A.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Murcha, Monika W.
    Branca, Rui M. M.
    Radomiljac, Jordan D.
    Regberg, Jakob
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Svensson, Linda M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bakali, Amin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lehtio, Janne
    Whelan, James
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Organellar oligopeptidase (OOP) provides a complementary pathway for targeting peptide degradation in mitochondria and chloroplasts2013In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, no 40, p. E3761-E3769Article in journal (Refereed)
    Abstract [en]

    Both mitochondria and chloroplasts contain distinct proteolytic systems for precursor protein processing catalyzed by the mitochondrial and stromal processing peptidases and for the degradation of targeting peptides catalyzed by presequence protease. Here, we have identified and characterized a component of the organellar proteolytic systems in Arabidopsis thaliana, the organellar oligopeptidase, OOP (At5g65620). OOP belongs to the M3A family of peptide-degrading metalloproteases. Using two independent in vivo methods, we show that the protease is dually localized to mitochondria and chloroplasts. Furthermore, we localized the OPP homolog At5g10540 to the cytosol. Analysis of peptide degradation by OOP revealed substrate size restriction from 8 to 23 aa residues. Short mitochondrial targeting peptides (presequence of the ribosomal protein L29 and presequence of 1-aminocyclopropane-1-carboxylic acid deaminase 1) and N- and C-terminal fragments derived from the presequence of the ATPase beta subunit ranging in size from 11 to 20 aa could be degraded. MS analysis showed that OOP does not exhibit a strict cleavage pattern but shows a weak preference for hydrophobic residues (F/L) at the P1 position. The crystal structures of OOP, at 1.8-1.9 angstrom, exhibit an ellipsoidal shape consisting of two major domains enclosing the catalytic cavity of 3,000 angstrom(3). The structural and biochemical data suggest that the protein undergoes conformational changes to allow peptide binding and proteolysis. Our results demonstrate the complementary role of OOP in targeting-peptide degradation in mitochondria and chloroplasts.

  • 19. Llona-Minguez, Sabin
    et al.
    Desroses, Matthieu
    Ghassemian, Artin
    Jacques, Sylvain A.
    Eriksson, Lars
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Isacksson, Rebecka
    Koolmeister, Tobias
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Scobie, Martin
    Helleday, Thomas
    Vinylic MIDA Boronates: New Building Blocks for the Synthesis of Aza-Heterocycles2015In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 21, no 20, p. 7394-7398Article in journal (Refereed)
    Abstract [en]

    A two-step synthesis of structurally diverse pyrrole-containing bicyclic systems is reported. ortho-Nitro-haloarenes coupled with vinylic N-methyliminodiacetic acid (MIDA) boronates generate ortho-vinyl-nitroarenes, which undergo a metal-free nitrene insertion, resulting in a new pyrrole ring. This novel synthetic approach has a wide substrate tolerance and it is applicable in the preparation of more complex drug-like molecules. Interestingly, an ortho-nitro-allylarene derivative furnished a cyclic beta-aminophosphonate motif.

  • 20. Llona-Minguez, Sabin
    et al.
    Höglund, Andreas
    Jacques, Sylvain A.
    Johanson, Lars
    Calderon-Montano, Jose Manuel
    Claesson, Magnus
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Loseva, Olga
    Valerie, Nicholas C. K.
    Lundbäck, Thomas
    Piedrafita, Javier
    Maga, Giovanni
    Crespan, Emmanuele
    Meijer, Laurent
    Burgos Moron, Estefania
    Baranczewski, Pawel
    Hagbjörk, Ann-Louise
    Svensson, Richard
    Wiita, Elisee
    Almlöf, Ingrid
    Visnes, Torkild
    Jeppsson, Fredrik
    Sigmundsson, Kristmundur
    Jenmalm Jensen, Annika
    Artursson, Per
    Jemth, Ann-Sofie
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Warpman Berglund, Ulrika
    Scobie, Martin
    Helleday, Thomas
    Discovery of the First Potent and Selective Inhibitors of Human dCTP Pyrophosphatase 12016In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 59, no 3, p. 1140-1148Article in journal (Refereed)
    Abstract [en]

    The dCTPase pyrophosphatase 1 (dCTPase) regulates the intracellular nucleotide pool through hydrolytic degradation of canonical and noncanonical nucleotide triphosphates (dNTPs). dCTPase is highly expressed in multiple carcinomas and is associated with cancer cell sternness. Here we report on the development of the first potent and selective dCTPase inhibitors that enhance the cytotoxic effect of cytidine analogues in leukemia cells. Boronate 30 displays a promising in vitro ADME profile, including plasma and mouse microsomal half-lives, aqueous solubility, cell permeability and CYP inhibition, deeming it a suitable compound for in vivo studies.

  • 21. Llona-Minguez, Sabin
    et al.
    Throup, Adam
    Steiner, Emilie
    Lightowler, Molly
    Van der Haegen, Sandra
    Homan, Evert
    Eriksson, Lars
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jenmalm-Jensen, Annika
    Helleday, Thomas
    Novel spirocyclic systems via multicomponent aza-Diels-Alder reaction2017In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 15, no 37, p. 7758-7764Article in journal (Refereed)
    Abstract [en]

    Here we present a two-step diastereoselective methodology building on a multicomponent aza-Diels-Alder reaction. Using previously unexplored cyclic ketones, heterocyclic amines and cyclopentadiene derivatives, we obtained novel spiro-heterocyclic frameworks at the interphase between drug-like molecules and natural products.

  • 22.
    Magnusdottir, Audur
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Stenmark, Pål
    Flodin, Susanne
    Nyman, Tomas
    Kotenyova, Tatyana
    Gräslund, Susanne
    Ogg, Derek
    Nordlund, Pär
    The structure of the PP2A regulatory subunit B56gamma: The remaining piece of the PP2A jigsaw puzzle2009In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 74, no 1, p. 212-221Article in journal (Refereed)
    Abstract [en]

    The PP2A serine/threonine phosphatase regulates a plethora of cellular processes. In the cell the predominant form of the enzyme is a heterotrimer, formed by a core dimer composed of a catalytic and a scaffolding subunit, which assemble together with one of a range of different regulatory B subunits. Here, we present the first structure of a free non-complexed B subunit, B56. Comparison with the recent structures of a heterotrimeric complex and the core dimer reveals several significant conformational changes in the interface region between the B56 and the core dimer. These allow for an assembly scheme of the PP2A holoenzyme to be put forth where B56 first complexes with the scaffolding subunit and subsequently binds to the catalytic subunit and this induces the formation of a binding site for the invariant C-terminus of the catalytic subunit that locks in the complex as a last step of assembly.

  • 23.
    Massad, Tariq
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Papadopolos, Evangelos
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Harvard Medical School, USA.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Damberg, Peter
    The C repressor of the P2 bacteriophage2016In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 64, no 2, p. 175-180Article in journal (Refereed)
  • 24.
    Massad, Tariq
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Papadopoulos, Evangelos
    Beth Israel Deaconess Med. Center Harvard Institute of Medicin.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Damberg, Peter
    Department of Neurobiology, Care Sciences and Society, Karolinska Institutet .
    NMR Structure Note: The C Repressor of the P2 BacteriophageManuscript (preprint) (Other academic)
  • 25.
    Massad, Tariq
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Skaar, Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, Hanna
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Damberg, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Henriksson-Peltola, Petri
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Crystal structure of the P2 C-repressor: a binder of nonpalindromic direct DNA repeats2010In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, no 21, p. 7778-7790Article in journal (Refereed)
    Abstract [en]

    As opposed to the vast majority of prokaryoticrepressors, the immunity repressor of temperateEscherichia coli phage P2 (C) recognizes nonpalindromicdirect repeats of DNA rather thaninverted repeats. We have determined the crystalstructure of P2 C at 1.8A ° . This constitutes the firststructure solved from the family of C proteins fromP2-like bacteriophages. The structure reveals thatthe P2 C protein forms a symmetric dimer orientedto bind the major groove of two consecutive turns ofthe DNA. Surprisingly, P2 C has great similarities tobinders of palindromic sequences. Nevertheless, thetwo identical DNA-binding helixes of the symmetricP2 C dimer have to bind different DNA sequences.Helix 3 is identified as the DNA-recognition motif inP2 C by alanine scanning and the importance for theindividual residues in DNA recognition is defined.A truncation mutant shows that the disorderedC-terminus is dispensable for repressor function.The short distance between the DNA-bindinghelices together with a possible interaction betweentwo P2 C dimers are proposed to be responsible forextensive bending of the DNA. The structure providesinsight into the mechanisms behind the mutants ofP2 C causing dimer disruption, temperature sensitivityand insensitivity to the P4 antirepressor.

  • 26.
    Masuyer, Geoffrey
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Conrad, Julian
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The structure of the tetanus toxin reveals pH-mediated domain dynamics2017In: EMBO Reports, ISSN 1469-221X, E-ISSN 1469-3178, Vol. 18, no 8, p. 1306-1317Article in journal (Refereed)
    Abstract [en]

    The tetanus neurotoxin (TeNT) is a highly potent toxin produced by Clostridium tetani that inhibits neurotransmission of inhibitory interneurons, causing spastic paralysis in the tetanus disease. TeNT differs from the other clostridial neurotoxins by its unique ability to target the central nervous system by retrograde axonal transport. The crystal structure of the tetanus toxin reveals a closed domain arrangement stabilised by two disulphide bridges, and the molecular details of the toxin's interaction with its polysaccharide receptor. An integrative analysis combining X-ray crystallography, solution scattering and single particle electron cryo-microscopy reveals pH-mediated domain rearrangements that may give TeNT the ability to adapt to the multiple environments encountered during intoxication, and facilitate binding to distinct receptors.

  • 27.
    Narwal, Mohit
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jemth, Ann-Sofie
    Gustafsson, Robert
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Almlöf, Ingrid
    Warpman Berglund, Ulrika
    Helleday, Thomas
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Crystal Structures and Inhibitor Interactions of Mouse and Dog MTH1 Reveal Species-Specific Differences in Affinity2018In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 57, no 5, p. 593-603Article in journal (Refereed)
    Abstract [en]

    MTH1 hydrolyzes oxidized nucleoside triphosphates, thereby sanitizing the nucleotide pool from oxidative damage. This prevents incorporation of damaged nucleotides into DNA, which otherwise would lead to mutations and cell death. The high level of reactive oxygen species in cancer cells leads to a higher level of oxidized nucleotides in cancer cells compared to non-malignant cells making cancer cells more dependent on MTH1 for survival. The possibility to specifically target cancer cells by inhibiting MTH1 has highlighted MTH1 as a promising cancer target. Progression of MTH1 inhibitors into the clinic requires animal studies and knowledge about species differences in potency of inhibitors are of vital importance. We here show that the human MTH1 inhibitor TH588 is approximately twenty fold less potent for inhibition of mouse MTH1 compared to human, rat, pig, and dog MTH1. We present the crystal structures of mouse MTH1 in complex with TH588 and dog MTH1and elucidate the structural and sequence basis for the observed difference in affinity for TH588. We identify amino acid residue 116 in MTH1 as an important determinant for TH588 affinity. Furthermore, we present the structure of mouse MTH1 in complex with the substrate 8-oxo-dGTP. The crystal structures provide insight into the high degree of structural conservation between MTH1 from different organisms and provide a detailed view of interactions between MTH1 and the inhibitor, revealing that minute structural differences can have a large impact on affinity and specificity.

  • 28. Page, Brent D. G.
    et al.
    Valerie, Nicholas C. K.
    Wright, Roni H. G.
    Wallner, Olov
    Isaksson, Rebecka
    Carter, Megan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rudd, Sean G.
    Loseva, Olga
    Jemth, Ann-Sofie
    Almlöf, Ingrid
    Font-Mateu, Jofre
    Llona-Minguez, Sabin
    Baranczewski, Pawel
    Jeppsson, Fredrik
    Homan, Evert
    Almqvist, Helena
    Axelsson, Hanna
    Regmi, Shruti
    Gustavsson, Anna-Lena
    Lundback, Thomas
    Scobie, Martin
    Strömberg, Kia
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Beato, Miguel
    Helleday, Thomas
    Targeted NUDT5 inhibitors block hormone signaling in breast cancer cells2018In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, article id 250Article in journal (Refereed)
    Abstract [en]

    With a diverse network of substrates, NUDIX hydrolases have emerged as a key family of nucleotide-metabolizing enzymes. NUDT5 (also called NUDIX5) has been implicated in ADPribose and 8-oxo-guanine metabolism and was recently identified as a rheostat of hormone-dependent gene regulation and proliferation in breast cancer cells. Here, we further elucidate the physiological relevance of known NUDT5 substrates and underscore the biological requirement for NUDT5 in gene regulation and proliferation of breast cancer cells. We confirm the involvement of NUDT5 in ADP-ribose metabolism and dissociate a relationship to oxidized nucleotide sanitation. Furthermore, we identify potent NUDT5 inhibitors, which are optimized to promote maximal NUDT5 cellular target engagement by CETSA. Lead compound, TH5427, blocks progestin-dependent, PAR-derived nuclear ATP synthesis and subsequent chromatin remodeling, gene regulation and proliferation in breast cancer cells. We herein present TH5427 as a promising, targeted inhibitor that can be used to further study NUDT5 activity and ADP-ribose metabolism.

  • 29. Patton, Gregory C.
    et al.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gollapalli, Deviprasad R.
    Sevastik, Robin
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Kursula, Petri
    Flodin, Susanne
    Schuler, Herwig
    Swales, Colin T.
    Eklund, Hans
    Himo, Fahmi
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Nordlund, Par
    Hedstrom, Lizbeth
    Cofactor mobility determines reaction outcome in the IMPDH and GMPR (beta-alpha)(8) barrel enzymes2011In: Nature Chemical Biology, ISSN 1552-4450, E-ISSN 1552-4469, Vol. 7, no 12, p. 950-958Article in journal (Refereed)
    Abstract [en]

    Inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate reductase (GMPR) belong to the same structural family, share a common set of catalytic residues and bind the same ligands. The structural and mechanistic features that determine reaction outcome in the IMPDH and GMPR family have not been identified. Here we show that the GMPR reaction uses the same intermediate E-XMP(star) as IMPDH, but in this reaction the intermediate reacts with ammonia instead of water. A single crystal structure of human GMPR type 2 with IMP and NADPH fortuitously captures three different states, each of which mimics a distinct step in the catalytic cycle of GMPR. The cofactor is found in two conformations: an 'in' conformation poised for hydride transfer and an 'out' conformation in which the cofactor is 6 angstrom from IMP. Mutagenesis along with substrate and cofactor analog experiments demonstrate that the out conformation is required for the deamination of GMP. Remarkably, the cofactor is part of the catalytic machinery that activates ammonia.

  • 30. Peng, Lisheng
    et al.
    Berntsson, Ronnie P.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tepp, William H.
    Pitkin, Rose M.
    Johnson, Eric A.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Dong, Min
    Botulinum neurotoxin D-C uses synaptotagmin I and II as receptors, and human synaptotagmin II is not an effective receptor for type B, D-C and G toxins2012In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 125, no 13, p. 3233-3242Article in journal (Refereed)
    Abstract [en]

    Botulinum neurotoxins (BoNTs) are classified into seven types (A-G), but multiple subtype and mosaic toxins exist. These subtype and mosaic toxins share a high sequence identity, and presumably the same receptors and substrates with their parental toxins. Here, we report that a mosaic toxin, type D-C (BoNT/D-C), uses different receptors from its parental toxin BoNT/C. BoNT/D-C, but not BoNT/C, binds directly to the luminal domains of synaptic vesicle proteins synaptotagmin (Syt) I and II, and requires expression of SytI/II to enter neurons. The SytII luminal fragment containing the toxin-binding site can block the entry of BoNT/D-C into neurons and reduce its toxicity in vivo in mice. We also found that gangliosides increase binding of BoNT/D-C to SytI/II and enhance the ability of the SytII luminal fragment to block BoNT/D-C entry into neurons. These data establish SytI/II, in conjunction with gangliosides, as the receptors for BoNT/D-C, and indicate that BoNT/D-C is functionally distinct from BoNT/C. We further found that BoNT/D-C recognizes the same binding site on SytI/II where BoNT/B and G also bind, but utilizes a receptor-binding interface that is distinct from BoNT/B and G. Finally, we also report that human and chimpanzee SytII has diminished binding and function as the receptor for BoNT/B, D-C and G owing to a single residue change from rodent SytII within the toxin binding site, potentially reducing the potency of these BoNTs in humans and chimpanzees.

  • 31.
    Rudling, Axel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gustafsson, Robert
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Almlöf, Ingrid
    Homan, Evert
    Scobie, Martin
    Warpman Berglund, Ulrika
    Helleday, Thomas
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Carlsson, Jens
    Fragment-Based Discovery and Optimization of Enzyme Inhibitors by Docking of Commercial Chemical Space2017In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 60, no 19, p. 8160-8169Article in journal (Refereed)
    Abstract [en]

    Fragment-based lead discovery has emerged as a leading drug development strategy for novel therapeutic targets. Although fragment-based drug discovery benefits immensely from access to atomic-resolution information, structure-based virtual screening has rarely been used to drive fragment discovery and optimization. Here, molecular docking of 0.3 million fragments to a crystal structure of cancer target MTH1 was performed. Twenty-two predicted fragment ligands, for which analogs could be acquired commercially, were experimentally evaluated. Five fragments inhibited MTH1 with IC50 values ranging from 6 to 79 mu M. Structure-based optimization guided by predicted binding modes and analogs from commercial chemical libraries yielded nanomolar inhibitors. Subsequently solved crystal structures confirmed binding modes predicted by docking for three scaffolds. Structure-guided exploration of commercial chemical space using molecular docking gives access to fragment libraries that are several orders of magnitude larger than those screened experimentally and can enable efficient optimization of hits to potent leads.

  • 32. Ryan, Hannah
    et al.
    Carter, Megan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. University of Colorado, USA.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Stewart, James J. P.
    Braun-Sand, Sonja B.
    A comparison of X-ray and calculated structures of the enzyme MTH12016In: Journal of Molecular Modeling, ISSN 1610-2940, E-ISSN 0948-5023, Vol. 22, no 7, article id 168Article in journal (Refereed)
    Abstract [en]

    Modern computational chemistry methods provide a powerful tool for use in refining the geometry of proteins determined by X-ray crystallography. Specifically, computational methods can be used to correctly place hydrogen atoms unresolved by this experimental method and improve bond geometry accuracy. Using the semiempirical method PM7, the structure of the nucleotide-sanitizing enzyme MTH1, complete with hydrolyzed substrate 8-oxo-dGMP, was optimized and the resulting geometry compared with the original X-ray structure of MTH1. After determining hydrogen atom placement and the identification of ionized sites, the charge distribution in the binding site was explored. Where comparison was possible, all the theoretical predictions were in good agreement with experimental observations. However, when these were combined with additional predictions for which experimental observations were not available, the result was a new and alternative description of the substrate-binding site interaction. An estimate was made of the strengths and weaknesses of the PM7 method for modeling proteins on varying scales, ranging from overall structure to individual interatomic distances. An attempt to correct a known fault in PM7, the under-estimation of steric repulsion, is also described. This work sheds light on the specificity of the enzyme MTH1 toward the substrate 8-oxo-dGTP; information that would facilitate drug development involving MTH1.

  • 33.
    Skaar, Karin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Claesson, Magnus
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Odegrip, Richard
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Magnus
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Crystal structure of the bacteriophage P2 integrase catalytic domain2015In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 589, no 23, p. 3556-3563Article in journal (Refereed)
    Abstract [en]

    Bacteriophage P2 is a temperate phage capable of integrating its DNA into the host genome by site-specific recombination upon lysogenization. Integration and excision of the phage genome requires P2 integrase, which performs recognition, cleavage and joining of DNA during these processes. This work presents the high-resolution crystal structure of the catalytic domain of P2 integrase, and analysis of several non-functional P2 integrase mutants. The DNA binding area is characterized by a large positively charged patch, harbouring key residues. The structure reveals potential for large dimer flexibility, likely essential for rearrangement of DNA strands upon integration and excision.

  • 34.
    Stenmark, Pål
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Dong, Min
    Dupuy, Jerome
    Chapman, Edwin R.
    Stevens, Raymond C.
    Crystal Structure of the Botulinum Neurotoxin Type G Binding Domain: Insight into Cell Surface Binding2010In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 397, no 5, p. 1287-1297Article in journal (Refereed)
    Abstract [en]

    Botulinum neurotoxins (BoNTs) typically bind the neuronal cell surface via dual interactions with both protein receptors and gangliosides. We present here the 1.9-angstrom X-ray structure of the BoNT serotype G (BoNT/G) receptor binding domain (residues 868-1297) and a detailed view of protein receptor and ganglioside binding regions. The ganglioside binding motif (SxWY) has a conserved structure compared to the corresponding regions in BoNT serotype A and BoNT serotype B (BoNT/B), but several features of interactions with the hydrophilic face of the ganglioside are absent at the opposite side of the motif in the BoNT/G ganglioside binding cleft. This may significantly reduce the affinity between BoNT/G and gangliosides. BoNT/G and BoNT/B share the protein receptor synaptotagmin (Syt) I/II. The Syt binding site has a conserved hydrophobic plateau located centrally in the proposed protein receptor binding interface (Tyr1189, Phe1202, Ala1204, Pro1205, and Phe1212). Interestingly, only 5 of 14 residues that are important for binding between Syt-II and BoNT/B are conserved in BoNT/G, suggesting that the means by which BoNT/G and BoNT/B bind Syt diverges more than previously appreciated. Indeed, substitution of Syt-II Phe47 and Phe55 with alanine residues had little effect on the binding of BoNT/G, but strongly reduced the binding of BoNT/B. Furthermore, an extended solvent-exposed hydrophobic loop, located between the Syt binding site and the ganglioside binding cleft, may serve as a third membrane association and binding element to contribute to high-affinity binding to the neuronal membrane. While BoNT/G and BoNT/B are homologous to each other and both utilize Syt-I/Syt-II as their protein receptor, the precise means by which these two toxin serotypes bind to Syt appears surprisingly divergent.

  • 35.
    Stenmark, Pål
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gurmu, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordlund, Pär
    Crystal Structure of CaiB, a Type-III CoA Transferase in Carnitine Metabolism2004In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 44, p. 13996-14003Article in journal (Refereed)
    Abstract [en]

    Carnitine is an important molecule in human metabolism, mainly because of its role in the transport of long-chain fatty acids across the inner mitochondrial membrane. Escherichia coli uses carnitine as a terminal electron acceptor during anaerobic metabolism. Bacteria present in our large intestine break down carnitine that is not absorbed in the small intestine. One part of this catabolic pathway is reversible and can be utilized for bioproduction of large amounts of stereochemically pure l-carnitine, which is used medically for the treatment of a variety of human diseases. Here, we present the crystal structure of the E. coli protein CaiB, which is a member of the recently identified type-III coenzyme A (CoA) transferase family and catalyzes the transfer of the CoA moiety between γ-butyrobetaine−CoA and carnitine forming carnityl-CoA and γ-butyrobetaine. This is the first protein from the carnitine metabolic pathway to be structurally characterized. The structure of CaiB reveals a spectacular fold where two monomers are interlaced to form an interlocked dimer. A molecule of the crystallization buffer bis-(2-hydroxyethyl)imino-tris(hydroxymethyl)methane (bis-tris) is bound in a large pocket located primarily in the small domain, and we propose that this pocket constitutes the binding site for both substrate moieties participating in the CaiB transfer reaction. The binding of CoA to CaiB induces a domain movement that closes the active site of the protein. This is the first observation of a domain movement in the type-III CoA transferase family and can play an important role in coupling substrate binding to initiation of the catalytic reaction.

  • 36.
    Stenmark, Pål
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Moche, Martin
    Gurmu, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordlund, Pär
    The Crystal Structure of the Bifunctional Deaminase/Reductase RibD of the Riboflavin Biosynthetic Pathway in Escherichia coli: Implications for the Reductive Mechanism2007In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 373, no 1, p. 48-64Article in journal (Refereed)
    Abstract [en]

    We have determined the crystal structure of the bi-functional deaminase/reductase enzyme from Escherichia coli (EcRibD) that catalyzes two consecutive reactions during riboflavin biosynthesis. The polypeptide chain of EcRibD is folded into two domains where the 3D structure of the N-terminal domain (1–145) is similar to cytosine deaminase and the C-terminal domain (146–367) is similar to dihydrofolate reductase. We showed that EcRibD is dimeric and compared our structure to tetrameric RibG, an ortholog from Bacillus subtilis (BsRibG). We have also determined the structure of EcRibD in two binary complexes with the oxidized cofactor (NADP+) and with the substrate analogue ribose-5-phosphate (RP5) and superposed these two in order to mimic the ternary complex. Based on this superposition we propose that the invariant Asp200 initiates the reductive reaction by abstracting a proton from the bound substrate and that the pro-R proton from C4 of the cofactor is transferred to C1 of the substrate. A highly flexible loop is found in the reductase active site (159–173) that appears to control cofactor and substrate binding to the reductase active site and was therefore compared to the corresponding Met20 loop of E. coli dihydrofolate reductase (EcDHFR). Lys152, identified by comparing substrate analogue (RP5) coordination in the reductase active site of EcRibD with the homologous reductase from Methanocaldococcus jannaschii (MjaRED), is invariant among bacterial RibD enzymes and could contribute to the various pathways taken during riboflavin biosynthesis in bacteria and yeast.

  • 37.
    Svensson, Linda M.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jemth, Ann-Sofie
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Desroses, Matthieu
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Loseva, Olga
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Hoegbom, Martin
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Crystal structure of human MTH1 and the 8-oxo-dGMP product complex2011In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 585, no 16, p. 2617-2621Article in journal (Refereed)
    Abstract [en]

    MTH1 hydrolyzes oxidized nucleotide triphosphates, thereby preventing them from being incorporated into DNA. We here present the structures of human MTH1 (1.9 angstrom) and its complex with the product 8-oxo-dGMP (1.8 angstrom). Unexpectedly MTH1 binds the nucleotide in the anti conformation with no direct interaction between the 8-oxo group and the protein. We suggest that the specificity depends on the stabilization of an enol tautomer of the 8-oxo form of dGTP. The binding of the product induces no major structural changes. The structures reveal the mode of nucleotide binding in MTH1 and provide the structural basis for inhibitor design.

  • 38. Tao, Liang
    et al.
    Peng, Lisheng
    Berntsson, Ronnie P. -A.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Umeå University, Sweden.
    Liu, Sai Man
    Park, SunHyun
    Yu, Feifan
    Boone, Christopher
    Palan, Shilpa
    Beard, Matthew
    Chabrier, Pierre-Etienne
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Krupp, Johannes
    Dong, Min
    Engineered botulinum neurotoxin B with improved efficacy for targeting human receptors2017In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 8, article id 53Article in journal (Refereed)
    Abstract [en]

    Botulinum neurotoxin B is a Food and Drug Administration-approved therapeutic toxin. However, it has lower binding affinity toward the human version of its major receptor, synaptotagmin II (h-Syt II), compared to mouse Syt II, because of a residue difference. Increasing the binding affinity to h-Syt II may improve botulinum neurotoxin B's therapeutic efficacy and reduce adverse effects. Here we utilized the bacterial adenylate cyclase two-hybrid method and carried out a saturation mutagenesis screen in the Syt II-binding pocket of botulinum neurotoxin B. The screen identifies E1191 as a key residue: replacing it with M/C/V/Q enhances botulinum neurotoxin B binding to human synaptotagmin II. Adding S1199Y/W or W1178Q as a secondary mutation further increases binding affinity. Mutant botulinum neurotoxin B containing E1191M/S1199Y exhibits similar to 11-fold higher efficacy in blocking neurotransmission than wild-type botulinum neurotoxin B in neurons expressing human synaptotagmin II, demonstrating that enhancing receptor binding increases the overall efficacy at functional levels. The engineered botulinum neurotoxin B provides a platform to develop therapeutic toxins with improved efficacy.

  • 39.
    Teixeira, Pedro F.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Masuyer, Geoffrey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pinho, Catarina M.
    Branca, Rui M. M.
    Kmiec, Beata
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wallin, Cecilia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wärmländer, Sebastian K. T. S.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Berntsson, Ronnie P. -A.
    Ankarcrona, Maria
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lehtiö, Janne
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mechanism of Peptide Binding and Cleavage by the Human Mitochondrial Peptidase Neurolysin2018In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 430, no 3, p. 348-362Article in journal (Refereed)
    Abstract [en]

    Proteolysis plays an important role in mitochondria! biogenesis, from the processing of newly imported precursor proteins to the degradation of mitochondrial targeting peptides. Disruption of peptide degradation activity in yeast, plant and mammalian mitochondria is known to have deleterious consequences for organism physiology, highlighting the important role of mitochondrial peptidases. In the present work, we show that the human mitochondrial peptidase neurolysin (hNLN) can degrade mitochondrial presequence peptides as well as other fragments up to 19 amino acids long. The crystal structure of hNLN(E475Q) in complex with the products of neurotensin cleavage at 2.7 angstrom revealed a closed conformation with an internal cavity that restricts substrate length and highlighted the mechanism of enzyme opening/closing that is necessary for substrate binding and catalytic activity. Analysis of peptide degradation in vitro showed that hNLN cooperates with presequence protease (PreP or PITRM1) in the degradation of long targeting peptides and amyloid-beta peptide, A beta 1-40, associated with Alzheimer disease, particularly cleaving the hydrophobic fragment A beta 35-40. These findings suggest that a network of proteases may be required for complete degradation of peptides localized in mitochondria.

  • 40. Valerie, Nicholas C. K.
    et al.
    Hagenkort, Anna
    Page, Brent D. G.
    Masuyer, Geoffrey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rehling, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Carter, Megan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bevc, Luka
    Herr, Patrick
    Homan, Evert
    Sheppard, Nina G.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jemth, Ann-Sofie
    Helleday, Thomas
    NUDT15 Hydrolyzes 6-Thio-DeoxyGTP to Mediate the Anticancer Efficacy of 6-Thioguanine2016In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 76, no 18, p. 5501-5511Article in journal (Refereed)
    Abstract [en]

    Thiopurines are a standard treatment for childhood leukemia, but like all chemotherapeutics, their use is limited by inherent or acquired resistance in patients. Recently, the nucleoside diphosphate hydrolase NUDT15 has received attention on the basis of its ability to hydrolyze the thiopurine effector metabolites 6-thio-deoxyGTP (6-thio-dGTP) and 6-thio-GTP, thereby limiting the efficacy of thiopurines. In particular, increasing evidence suggests an association between the NUDT15 missense variant, R139C, and thiopurine sensitivity. In this study, we elucidated the role of NUDT15 and NUDT15 R139C in thiopurine metabolism. In vitro and cellular results argued that 6-thio-dGTP and 6-thio-GTP are favored substrates for NUDT15, a finding supported by a crystallographic determination of NUDT15 in complex with 6-thio-GMP. We found that NUDT15 R139C mutation did not affect enzymatic activity but instead negatively influenced protein stability, likely due to a loss of supportive intramolecular bonds that caused rapid proteasomal degradation in cells. Mechanistic investigations in cells indicated that NUDT15 ablation potentiated induction of the DNA damage checkpoint and cancer cell death by 6-thioguanine. Taken together, our results defined how NUDT15 limits thiopurine efficacy and how genetic ablation via the R139C missense mutation confers sensitivity to thiopurine treatment in patients.

  • 41. Welin, Martin
    et al.
    Grossmann, Jörg Günter
    Flodin, Susanne
    Nyman, Tomas
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Trésaugues, Lionel
    Kotenyova, Tetyana
    Johansson, Ida
    Nordlund, Pär
    Lehtiö, Lari
    Structural studies of tri-functional human GART2010In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, no 20, p. 7308-19Article in journal (Refereed)
    Abstract [en]

    Human purine de novo synthesis pathway contains several multi-functional enzymes, one of which, tri-functional GART, contains three enzymatic activities in a single polypeptide chain. We have solved structures of two domains bearing separate catalytic functions: glycinamide ribonucleotide synthetase and aminoimidazole ribonucleotide synthetase. Structures are compared with those of homologous enzymes from prokaryotes and analyzed in terms of the catalytic mechanism. We also report small angle X-ray scattering models for the full-length protein. These models are consistent with the enzyme forming a dimer through the middle domain. The protein has an approximate seesaw geometry where terminal enzyme units display high mobility owing to flexible linker segments. This resilient seesaw shape may facilitate internal substrate/product transfer or forwarding to other enzymes in the pathway.

  • 42. Zhang, Sicai
    et al.
    Berntsson, Ronnie P. -A.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Umeå University, Sweden.
    Tepp, William H.
    Tao, Liang
    Johnson, Eric A.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Dong, Min
    Structural basis for the unique ganglioside and cell membrane recognition mechanism of botulinum neurotoxin DC2017In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 8, article id 1637Article in journal (Refereed)
    Abstract [en]

    Botulinum neurotoxins (BoNTs), the most potent toxins known, are potential bioterrorism agents. It is well established that all seven serotypes of BoNTs (BoNT/A-G) require complex gangliosides as co-receptors. Here, we report that BoNT/DC, a presumed mosaic toxin between BoNT/D and BoNT/C1, binds and enters efficiently into neurons lacking complex gangliosides and shows no reduction in toxicity in mice deficient in complex gangliosides. The co-crystal structure of BoNT/DC with sialyl-Thomsen-Friedenreich antigen (Sialyl-T) suggests that BoNT/DC recognizes only the sialic acid, but not other moieties in gangliosides. Using liposome flotation assays, we demonstrate that an extended loop in BoNT/DC directly interacts with lipid membranes, and the co-occurring sialic acid binding and loop-membrane interactions mediate the recognition of gangliosides in membranes by BoNT/DC. These findings reveal a unique mechanism for cell membrane recognition and demonstrate that BoNT/DC can use a broad range of sialic acid-containing moieties as co-receptors.

  • 43. Zhang, Sicai
    et al.
    Lebreton, Francois
    Mansfield, Michael J.
    Miyashita, Shin-Ichiro
    Zhang, Jie
    Schwartzman, Julia A.
    Tao, Liang
    Masuyer, Geoffrey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Martínez-Carranza, Markel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gilmore, Michael S.
    Doxey, Andrew C.
    Dong, Min
    Identification of a Botulinum Neurotoxin-like Toxin in a Commensal Strain of Enterococcus faecium2018In: Cell Host and Microbe, ISSN 1931-3128, E-ISSN 1934-6069, Vol. 23, no 2, p. 169-+Article in journal (Refereed)
    Abstract [en]

    Botulinumneurotoxins (BoNTs), produced by various Clostridium strains, are a family of potent bacterial toxins and potential bioterrorism agents. Here we report that an Enterococcus faecium strain isolated from cow feces carries a BoNT-like toxin, designated BoNT/En. It cleaves both VAMP2 and SNAP-25, proteins that mediate synaptic vesicle exocytosis in neurons, at sites distinct from known BoNT cleavage sites on these two proteins. Comparative genomic analysis determines that the E. faecium strain carrying BoNT/En is a commensal type and that the BoNT/En gene is located within a typical BoNT gene cluster on a 206 kb putatively conjugative plasmid. Although the host species targeted by BoNT/En remains to be determined, these findings establish an extended member of BoNTs and demonstrate the capability of E. faecium, a commensal organism ubiquitous in humans and animals and a leading cause of hospital-acquired multi-drug-resistant (MDR) infections, to horizontally acquire, and possibly disseminate, a unique BoNT gene cluster.

  • 44. Zhang, Sicai
    et al.
    Masuyer, Geoffrey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Zhang, Jie
    Shen, Yi
    Lundin, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Henriksson, Linda
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Miyashita, Shin-Ichiro
    Martinez-Carranza, Markel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Dong, Min
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Identification and characterization of a novel botulinum neurotoxin2017In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 8, article id 14130Article in journal (Refereed)
    Abstract [en]

    Botulinum neurotoxins are known to have seven serotypes (BoNT/A-G). Here we report a new BoNT serotype, tentatively named BoNT/X, which has the lowest sequence identity with other BoNTs and is not recognized by antisera against known BoNTs. Similar to BoNT/B/D/F/G, BoNT/X cleaves vesicle-associated membrane proteins (VAMP) 1, 2 and 3, but at a novel site (Arg66-Ala67 in VAMP2). Remarkably, BoNT/X is the only toxin that also cleaves non-canonical substrates VAMP4, VAMP5 and Ykt6. To validate its activity, a small amount of full-length BoNT/X was assembled by linking two non-toxic fragments using a transpeptidase (sortase). Assembled BoNT/X cleaves VAMP2 and VAMP4 in cultured neurons and causes flaccid paralysis in mice. Thus, BoNT/X is a novel BoNT with a unique substrate profile. Its discovery posts a challenge to develop effective countermeasures, provides a novel tool for studying intracellular membrane trafficking, and presents a new potential therapeutic toxin for modulating secretions in cells.

  • 45.
    Öhrström, Maria
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Popovic-Bijelic, Ana
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oligopeptide inhibition of class Ic ribonucleotide reductase from Chlamydia trachomatisManuscript (preprint) (Other (popular science, discussion, etc.))
  • 46.
    Öhrström, Maria
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Popović-Bijelić, Ana
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Luo, Jinghui
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Inhibition of chlamydial class Ic ribonucleotide reductase by C-terminal peptides from protein R22011In: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 17, no 11, p. 756-762Article in journal (Refereed)
    Abstract [en]

    Chlamydia trachomatis ribonucleotide reductase (RNR) is a class Ic RNR. It has two homodimeric subunits: proteins R1 and R2. Class Ic protein R2 in its most active form has a manganese-iron metal cofactor, which functions in catalysis like the tyrosyl radical in classical class Ia and Ib RNRs. Oligopeptides with the same sequence as the C-terminus of C. trachomatis protein R2 inhibit the catalytic activity of C. trachomatis RNR, showing that the class Ic enzyme shares a similar highly specific inhibition mechanism with the previously studied radical-containing class Ia and Ib RNRs. The results indicate that the catalytic mechanism of this class of RNRs with a manganese-iron cofactor is similar to that of the tyrosyl-radical-containing RNRs, involving reversible long-range radical transfer between proteins R1 and R2. The competitive binding of the inhibitory R2-derived oligopeptide blocks the transfer pathway. We have constructed three-dimensional structure models of C. trachomatis protein R1, based on homologous R1 crystal structures, and used them to discuss possible binding modes of the peptide to protein R1. Typical half maximal inhibitory concentration values for C. trachomatis RNR are about 200 µ m for a 20-mer peptide, indicating a less efficient inhibition compared with those for an equally long peptide in the Escherichia coli class Ia RNR. A possible explanation is that the C. trachomatis R1/R2 complex has other important interactions, in addition to the binding mediated by the R1 interaction with the C-terminus of protein R2.

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