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  • 1.
    Andersson, Charlotta S.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Berthold, Catrine
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    A Dynamic C-terminal Segment in the Mycobacterium tuberculosis Mn/Fe R2lox Protein can Assume a Helical Structure with Possible Functional ConsequencesManuscript (preprint) (Other academic)
  • 2.
    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.

  • 3.
    Andersson, Charlotta Selina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structural studies of R2 and R2–like proteins with a heterodinuclear Mn/Fe cofactor and enzymes involved in Mycobacterium tuberculosis lipid metabolism2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Tuberculosis is a notorious disease responsible for the deaths of 1.4 million people worldwide. A third of the world's population is infected with Mycobacterium tuberculosis, the bacterium causing the disease. The increase of multi drug-resistant strains worsens the situation, and the World Health Organization has declared tuberculosis to be a global emergency. The bacterium envelopes itself with a unique set of very long-chain lipids that play an important role in virulence and drug resistance. Therefore enzymes involved in lipid metabolism are putative drug targets. 

    To allow entry into different metabolic pathways and transmembrane transport, fatty acids have to be activated. This is done primarily by fatty acyl-CoA synthetases (ACSs). We identified an ACS possibly involved in the bacterium’s virulence and solved its structure. Structural interpretation combined with previously reported data gives us insights into the details of its function. This enzyme is known to harbor lipid substrates longer than the enzyme itself, and we now propose how this peripheral membrane protein accommodates its substrates. 

    Some of the most chemically challenging oxidations are performed by dinuclear metalloproteins belonging to the ferritin-like superfamily. We show that the ferritin-like protein, R2lox, from M. tuberculosis contains a new type of heterodinuclear Mn/Fe cofactor. This protein cofactor is capable of performing potent 2-electron oxidations as demonstrated by a novel tyrosine-valine crosslink observed in the protein. 

    Recently a new subclass of ribonucleotide reductase (RNR) R2 proteins, was identified in the intracellular pathogen Chlamydia trachomatis containing the same type of Mn/Fe cofactor mentioned above. The RNR R2 proteins use their metal site to generate a stable radical, essential for the reduction of ribonucleotides to their deoxy forms, the building blocks of DNA. With this work, we were able to characterize the architecture of this metal cofactor.

  • 4. Fourati, Zaineb
    et al.
    Howard, Rebecca J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Heusser, Stephanie A.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Hu, Haidai
    Ruza, Reinis R.
    Sauguet, Ludovic
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Delarue, Marc
    Structural Basis for a Bimodal Allosteric Mechanism of General Anesthetic Modulation in Pentameric Ligand-Gated Ion Channels2018In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 23, no 4, p. 993-1004Article in journal (Refereed)
    Abstract [en]

    Ion channel modulation by general anesthetics is a vital pharmacological process with implications for receptor biophysics and drug development. Functional studies have implicated conserved sites of both potentiation and inhibition in pentameric ligand-gated ion channels, but a detailed structural mechanism for these bimodal effects is lacking[1] . The prokaryotic model protein GLIC recapitulates anesthetic modulation of human ion channels, and is accessible to structure determination in both apparent open and closed states. Here, we report ten X-ray structures and electrophysiological characterization of GLIC variants in the presence and absence of general anesthetics, including the surgical agent propofol. We show that general anesthetics can allosterically favor closed channels by binding in the pore, or favor open channels via various subsites in the transmembrane domain. Our results support an integrated, multi-site mechanism for allosteric modulation, and provide atomic details of both potentiation and inhibition by one of the most common general anesthetics.

  • 5.
    Gustafsson, Robert
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structural and functional studies of proteins of medical relevance: Protein-ligand complexes in cancer and novel structural folds in bacteria2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    X-ray crystallography is a tool for determining the structures of proteins and protein-ligand complexes. In this thesis the method has been employed to study several proteins of medical relevance.

    Cancer is a terrible disease, severely impacting those affected, as well as their family and friends. Current cancer treatments involve a combination of cytostatic drugs, surgery and radiation treatment. Unfortunately many cytostatic drugs also kill healthy cells, which gives rise to serious side-effects. The discovery of treatments which selectively inhibit proteins essential for cancer cell survival but which are non-essential in normal cells, could reduce such side-effects.

    MTH1 is a protein that degrades oxidised nucleotides, which when incorporated into DNA cause mutations and subsequent cell death. Cancer cells have higher levels of reactive oxygen species, which create oxidised nucleotides.  In Paper I it was discovered that cancer cells are dependent on MTH1 for their survival. Crystal structures of MTH1 in complex with small molecules guided their development into potent MTH1 inhibitors, capable of killing cancer cells. Cells with increased amounts of oxidised nucleotides, or with induced hypoxia, were more susceptible to MTH1 inhibition, as shown in Paper II. In Paper III several MTH1 orthologues from organisms often used in pre-clinical studies were tested for MTH1 inhibition. Leucine 116 of mouse MTH1 was determined to be important for the lower inhibition of the developed inhibitors towards this enzyme. A virtual fragment screening study using commercial chemicals resulted in several potent MTH1 inhibitors, as shown in Paper IV. The crystal structures with the fragments or optimised inhibitors did in most cases agree with the docking pose determined from the virtual screening. In addition to the known function of MTH1 in the degradation of oxidised nucleotides, Paper V showed that MTH1 also degrades methylated nucleotides.

    MTHFD2 is responsible for providing one-carbon units for nucleotide synthesis in cancer cells. As MTHFD2 is present in cancer cells but not in healthy cells, targeting the enzyme would make it possible to selectively kill cancer cells. Paper VI presents the first structure of MTHFD2, along with the first inhibitor of the protein. This information provides a starting point for the development of potent and selective MTHFD2 inhibitors.

    The botulinum neurotoxin from the bacterium Clostridium Botulinum is the causative agent of the deadly disease botulism. The action of the botulinum neurotoxin on nerve cells results in paralysis, and is life-threatening if the patient is not helped with breathing support. However, low doses of the neurotoxin are used as a successful treatment for several medical conditions, such as involuntary spasms. In Paper VII the structure of two proteins, P47 and OrfX2, encoded in the gene cluster of a botulinum neurotoxin, were determined. The structures resembled tubular lipid-binding proteins, previously only found in eukaryotes. The proteins were also found to be able to bind lipids. This work gives new insight into the structure and function of this group of proteins, which help the deadly botulinum neurotoxins.

  • 6.
    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.

  • 7.
    Herman Moreno, Maria Dolores
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structural studies of proteins in apoptosis and lipid signaling2008Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Signaling pathways control the fate of the cell. For example, they promote cell survival or commit the cell to death (apoptosis) in response to cell injury or developmental stimuli, decisions, which are vital for the proper development and functioning of metazoan. Tight control of such pathways is essential; dysregulation of apoptosis can disrupt the delicate balance between cell proliferation and cell death ending up in pathological processes, including cancer, autoimmunity diseases, inflammatory diseases, or degenerative disorders. We have used a structural genomic approach to study the structure and function of key proteins involved in apoptosis and lipid signaling: the antiapoptotic Bcl-2 family member Bfl-1 in complex with a Bim peptide, the BIR domains of the Inhibitor of Apoptosis (IAP) family members, cIAP2 and NAIP and the a lipid kinase YegS. The structural analysis of the apoptosis regulatory proteins has revealed important information on the structural determinants for recognition of interacting proteins, which can now assist in the development of therapeutic drugs for human diseases. The structural and complementing biochemical studies of the lipid kinase YegS have reveled the first detailed information on a lipid kinase and explained important aspects of its structure-function relationship.

    Finally, one subject of this work aim to solve what is arguably the most challenging problem in structural projects – to obtain a high production level of proteins suitable for structural studies. We have developed a highthroughput protein solubility screening, the colony filtration (CoFi) blot, which allows soluble clones to be identified from large libraries of protein variants and now constitute a powerful tool for solving difficult protein production problems.

  • 8. 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.

  • 9. 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)
  • 10.
    Magnúsdóttir, Auður
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structural Studies on PP2A and Methods in Protein Production2008Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    PP2A is a major phosphatase in the cell that participates in multiple cell signaling pathways. It is a heterotrimer of a core dimer and variable regulatory subunits. Details of its structure, function and regulation are slowly emerging. Here, the structure of two regulators of PP2A are de-scribed; PTPA and B56γ. PTPA is a highly conserved enzyme that plays a crucial role in PP2A activity but whose biochemical function is still unclear. B56γ is a PP2A regulatory subunit linked to cancer and the structure presented here of B56γ in its free form is particularly valuable in light of the recent structures of the PP2A holoenzyme and core dimer.

    Protein production is a major bottleneck in structural genomic projects. Here, we describe two novel methods for improved protein production. The first is a colony based screening method where any DNA library can be screened for soluble expression of recombinant proteins in E.coli. The second method involves improvements of the well established IMAC purification method. We have seen that a low molecular weight component of E.coli lysate decreases the binding capacity of IMAC columns and by removing the low molecular weight components, recombinant proteins only present at low levels in E.coli lysate can be purified, which has previously been believed to be unfeasible.

  • 11.
    Martinez Molina, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Membrane Protein Tailoring and Structural Studies of Leukotriene C4 Synthase2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Despite a dramatic increase in the number of proteins that have been structurally characterized in recent years, there are still less than 200 unique structures of membrane proteins known today. This is only 1% of the total number of unique protein structures found in structural databases worldwide. There are several reasons for this hindered progress in the structure determination of membrane proteins; it is difficult to generate membrane proteins in recombinant expression systems and it requires the use of appropriate detergents, both for membrane extraction and to keep them stable in solution. This makes isolation and purification problematic. Importantly, once isolated, these proteins are notoriously difficult to crystallize for X-ray structure determination.

     In this thesis, I present two techniques that can be used to increase the likelihood of success in the structural determination of membrane proteins. I started by focusing on problems that occur at an early stage of the process, where I developed a directed-evolution method to overcome problems with low yields during membrane protein production. In addition, I describe a screen for optimal detergent usage when purifying and crystallizing recombinant membrane proteins in eukaryotic hosts.

     The crystal structure of human Leukotriene C4 synthase has been solved. This is the first human membrane protein whose structure has been solved at high resolution. The model provides a structural basis for the formation of potent lipid mediators, which are implicated in the pathophysiology of asthma and chronic inflammation. Furthermore, the structure reveals insight into how specificity can be achieved for lipophilic substrate molecules. In addition, I have determined the crystal structure of a hexahistidine tag and use it to describe the molecular basis of the single most used chromatography method, IMAC.

  • 12.
    Martinez Molina, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structural description of a hexa-His tag chelated with two metal ions - molecular basis of protein poly-His tag IMAC chromatography Manuscript (Other academic)
    Abstract [en]

    In this work we describe the molecular basis for binding of poly histidine tagged protein to IMAC resins such as IDA or NTA and explain early experimental findings on the affinity of various His tags. 

  • 13.
    Martinez Molina, Daniel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Cornvik, Tobias
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Engineering membrane protein overproduction in Escherichia coli2008In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 17, no 4, p. 673-680Article in journal (Refereed)
    Abstract [en]

    Membrane proteins play a fundamental role in human disease and therapy, but suffer from a lack of structural and functional information compared to their soluble counterparts. The paucity of membrane protein structures is primarily due to the unparalleled difficulties in obtaining detergent-solubilized membrane proteins at sufficient levels and quality. We have developed an in vitro evolution strategy for optimizing the levels of detergent-solubilized membrane protein that can be overexpressed and purified from recombinant Escherichia coli. Libraries of random mutants for nine membrane proteins were screened for expression using a novel implementation of the colony filtration blot. In only one cycle of directed evolution were significant improvements of membrane protein yield obtained for five out of nine proteins. In one case, the yield of detergent-solubilized membrane protein was increased 40-fold.

     

  • 14.
    Martinez Molina, Daniel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Niegowski, Damian
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structural basis for synthesis of inflammatory mediators by human leukotriene C4 synthase2007In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 448, no 7153, p. 613-616Article in journal (Refereed)
    Abstract [en]

    Cysteinyl leukotrienes are key mediators in inflammation and have an important role in acute and chronic inflammatory diseases of the cardiovascular and respiratory systems, in particular bronchial asthma. In the biosynthesis of cysteinyl leukotrienes, conversion of arachidonic acid forms the unstable epoxide leukotriene A4 (LTA4). This intermediate is conjugated with glutathione (GSH) to produce leukotriene C4 (LTC4) in a reaction catalysed by LTC4 synthase: this reaction is the key step in cysteinyl leukotriene formation. Here we present the crystal structure of the human LTC4 synthase in its apo and GSH-complexed forms to 2.00 and 2.15 A resolution, respectively. The structure reveals a homotrimer, where each monomer is composed of four transmembrane segments. The structure of the enzyme in complex with substrate reveals that the active site enforces a horseshoe-shaped conformation on GSH, and effectively positions the thiol group for activation by a nearby arginine at the membrane-enzyme interface. In addition, the structure provides a model for how the omega-end of the lipophilic co-substrate is pinned at one end of a hydrophobic cleft, providing a molecular 'ruler' to align the reactive epoxide at the thiol of glutathione. This provides new structural insights into the mechanism of LTC4 formation, and also suggests that the observed binding and activation of GSH might be common for a family of homologous proteins important for inflammatory and detoxification responses.

  • 15.
    Massad, Tariq
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structural Studies of Flexible Biomolecules and a DNA-binding Protein2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The knowledge of the three-dimensional structures of proteins and polypeptides is essential to understand their functions. The work shown in this thesis has two objectives. The first one is to develop a new analytical method based on maximum entropy (ME) theory to analyze NMR experimental data such as NOEs and J-couplings in order to reconstitute φ,ψ Ramachandran plots of flexible biomolecules. Two model systems have been used, the flexible polypeptide motilin and the disaccharide α-D-Mannosep-(1-2)-α-D-Mannosep-O-Me (M2M). The experimental data was defined as constraints that were combined with prior information (priors) which were the φ,ψ distributions obtained from either a coil library, the Protein DataBank or Molecular Dynamics Simulations. ME theory was utilized to formulate φ,ψ distributions (posteriors) that are least committed to the priors and in full agreement with the experimental data. Reparamerization of the Karplus relation was necessary to obtain realistic distributions for the M2M. Clear structural propensities were found in motilin with a nascent α-helix in the central part (residues Y7-E17), a left handed 31 helix in the C-terminus (R18-G21) and an extended conformation in the N-terminus. The contribution of each residue to the thermodynamic entropy (segmental entropy) was calculated from the posteriors and compared favorably to the segmental entropies estimated from 15N-relaxation data. For M2M the dominating conformation of the glycosidic linkage was found to be at φH=-40° ψH=33°, which is governed by the exo-anomeric effect. Another minor conformation with a negative ψH angle was discovered in M2M. The ratio between both populations is about 3:1. The second part of the thesis is a structural study of a DNA-binding protein, the C repressor of the P2 bacteriophage (P2 C). P2 C represses the lytic genes of the P2 bacteriophage, thereby directing the P2 lifecycle toward the lysogenic lifemode. The crystal and solution structures of P2 C have been solved by X-ray crystallography and NMR, respectively. Both structures revealed a homodimeric protein with five rigid α-helices made up by residues 5-66 and a β-strand conformation in residues 69-76 in each monomer. 15N-relaxation data showed that the C-terminus (residues 85-99) is highly flexible and fully unstructured. A model representing the P2 C-DNA complex was built based on the structure and available biochemical data. In the model, P2 C binds DNA cooperatively and two homodimeric P2 C molecules are close enough to interact and bind one direct DNA repeat each.

  • 16.
    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.

  • 17.
    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.

  • 18. 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.

  • 19.
    Shu, Nanjiang
    Stockholm University, Faculty of Science, Department of Physical, Inorganic and Structural Chemistry.
    Prediction of zinc-binding sites in proteins and efficient protein structure description and comparison2008Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    A large number of proteins require certain metals to stabilize their structures or to function properly. About one third of all proteins in the Protein Data Bank (PDB) contain metals and it is estimated that approximately the same proportion of all proteins are metalloproteins.

    Zinc, the second most abundant transition metal found in eukaryotic organisms, plays key roles, mainly structural and catalytic, in many biological functions. Predicting whether a protein binds zinc and even the accurate location of binding sites is important when investigating the function of an experimentally uncharacterized protein.

    Describing and comparing protein structures with both efficiency and accuracy are essential for systematic annotation of functional properties of proteins, be it on an individual or on a genome scale. Dozens of structure comparison methods have been developed in the past decades. In recent years, several research groups have endeavoured in developing methods for fast comparison of protein structures by representing the three-dimensional (3D) protein structures as one-dimensional (1D) geometrical strings based on the shape symbols of clustered regions of φ/ψ torsion angle pairs of the polypeptide backbones. These 1D geometrical strings, shape strings, are as compact as 1D secondary structures but carry more elaborate structural information in loop regions and thus are more suitable for fast structure database searching, classification of loop regions and evaluation of model structures.

    In this thesis, a new method for predicting zinc-binding sites in proteins from amino acid sequences is described. This method predicts zinc-binding Cys, His, Asp and Glu (the four most common zinc-binding residues) with 75% precision (86% for Cys and His only) at 50% recall according to a solid 5-fold cross-validation on a non-redundant set of the PDB chains containing 2727 unique chains, of which 235 bind to zinc. This method predicts zinc-binding Cys and His with about 10% higher precision at different recall levels compared to a previously published method. In addition, different methods for describing and comparing protein structures are reviewed. Some recently developed methods based on 1D geometrical representation of backbone structures are emphasized and analyzed in details.

  • 20.
    Shu, Nanjiang
    et al.
    Stockholm University, Faculty of Science, Department of Physical, Inorganic and Structural Chemistry.
    Hovmöller, Sven
    Stockholm University, Faculty of Science, Department of Physical, Inorganic and Structural Chemistry.
    Zhou, Tuping
    Stockholm University, Faculty of Science, Department of Physical, Inorganic and Structural Chemistry.
    Describing and Comparing Protein Structures Using Shape Strings2008In: Current protein and peptide science, ISSN 1389-2037, E-ISSN 1875-5550, Vol. 9, no 4, p. 310-324Article in journal (Refereed)
    Abstract [en]

    Different methods for describing and comparing the structures of the tens of thousands of proteins that have been determined by X-ray crystallography are reviewed. Such comparisons are important for understanding the structures and functions of proteins and facilitating structure prediction, as well as assessing structure prediction methods. We summarize methods in this field emphasizing ways of representing protein structures as one-dimensional geometrical strings. Such strings are based on the shape symbols of clustered regions of φ/Ψ dihedral angle pairs of the polypeptide backbones as described by the Ramachandran plot. These one-dimensional expressions are as compact as secondary structure description but contain more information in loop regions. They can be used for fast searching for similar structures in databases and for comparing similarities between proteins and between the predicted and native structures.

  • 21.
    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.

  • 22.
    Uzdavinys, Povilas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Establishing the molecular mechanism of sodium/proton exchangers2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Sodium/proton exchangers are ubiquitous secondary active transporters that can be found in all kingdoms of life. These proteins facilitate the transport of protons in exchange for sodium ions to help regulate internal pH, sodium levels, and cell volume. Na+/H+ exchangers belong to the SLC9 family and are involved in many physiological processes including cell proliferation, cell migration and vesicle trafficking. Dysfunction of these proteins has been linked to physiological disorders, such as hypertension, heart failure, epilepsy and diabetes.

    The goal of my thesis is to establish the molecular basis of ion exchange in Na+/H+ exchangers. By establishing how they bind and catalyse the movement of ions across the membrane, we hope we can better understand their role in human physiology.

    In my thesis, I will first present an overview of Na+/H+ exchangers and their molecular mechanism of ion translocation as was currently understood by structural and functional studies when I started my PhD studies. I will outline our important contributions to this field, which were to (i) obtain the first atomic structures of the same Na+/H+ exchanger (NapA) in two major alternating conformations, (ii) show how a transmembrane embedded lysine residue is essential for carrying out electrogenic transport, and (iii) isolate and recorde the first kinetic data of a mammalian Na+/H+ exchanger (NHA2) in an isolated liposome reconstitution system.

  • 23.
    Wetterholm, Anders
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Martinez Molina, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    High-level expression, purification, and crystallization of recombinant rat leukotriene C4 synthase from the yeast Pichia pastoris2008In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 60, no 1, p. 1-6Article in journal (Refereed)
    Abstract [en]

    Leukotriene C(4) synthase (LTC4S) is a member of the MAPEG family of integral membrane proteins and catalyzes the conjugation of leukotriene A(4) with glutathione to form leukotriene C(4), a powerful mediator of allergic inflammation and anaphylaxis. Structural information on this class of proteins would be highly useful for rational drug design. Here, we report the expression, purification, and crystallization of recombinant LTC4S from rat. The enzyme was expressed as an N-terminal hexa-histidine-tagged fusion protein in Pichia pastoris and purified with two steps of affinity chromatography on Ni-Sepharose and S-hexyl-glutathione agarose, followed by gel filtration. From 1l culture, we obtained 0.5-1 mg of apparently homogeneous protein with a specific LTC4S activity ranging between 36 and 49 micromol/mg/min. A small-scale screen identified dodecyl maltoside as a useful detergent for protein extraction and yielded a highly active protein. When tested separately in crystallization trials of the purified LTC4S, six out of seven detergents from all the maltoside family yielded diffracting crystals with the highest resolution at approximately 6 A. Hence, our approach holds promise for solving the structure of rat LTC4S and other members of the MAPEG family of integral membrane proteins.

     

  • 24.
    Zhou, Tuping
    et al.
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Shu, Nanjiang
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Hovmöller, Sven
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    A Novel Method for Accurate One-dimensional Protein Structure Prediction Based on Fragment Matching2010In: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 26, no 4, p. 470-477Article in journal (Refereed)
    Abstract [en]

    Motivation: The precise prediction of one-dimensional (1D) protein structure as represented by the protein secondary structure and 1D string of discrete state of dihedral angles (i.e. Shape Strings) is a prerequisite for the successful prediction of three-dimensional (3D) structure as well as protein-protein interaction. We have developed a novel 1D structure prediction method, called Frag1D, based on a straightforward fragment matching algorithm and demonstrated its success in the prediction of  three sets of 1D structural alphabets, i.e. the classical three-state secondary structure, three-state Shape Strings and eight-state Shape Strings.

    Results: By exploiting the vast protein sequence and protein structure data available, we have brought secondary structure prediction closer to the expected theoretical limit. When tested by a leave-one-out cross validation on a non-redundant set of PDB cutting at 30% sequence identity containing 5860 protein chains, the overall per-residue accuracy for secondary structure prediction, i.e. Q3 is 82.9%. The overall per-residue accuracy for three-state and eight-state Shape Strings are 85.1% and 71.5% respectively. We have also benchmarked our program with the latest version of PSIPRED for secondary structure prediction and our program predicted 0.3% better in Q3 when tested on 2241 chains with the same training set. For Shape Strings, we compared our method with a recently published method with the same dataset and definition as used by that method. Our program predicted at 2.2% better in accuracy for three-state Shape Strings. By quantitatively investigating the effect of data base size on 1D structure prediction we show that the accuracy increases by about 1% with every doubling of the database size.

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