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  • 1.
    Andersson, Charlotta S.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lundgren, Camilla A. K.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Magnúsdóttir, Auður
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ge, Changrong
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Martinez Molina, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The Mycobacterium tuberculosis Very-Long-Chain Fatty Acyl-CoA Synthetase: Structural Basis for Housing Lipid Substrates Longer than the Enzyme2012In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 20, no 6, p. 1062-1070Article in journal (Refereed)
    Abstract [en]

    The Mycobacterium tuberculosis acid-induced operon MymA encodes the fatty acyl-CoA synthetase FadD13 and is essential for virulence and intracellular growth of the pathogen. Fatty acyl-CoA synthetases activate lipids before entering into the metabolic pathways and are also involved in transmembrane lipid transport. Unlike soluble fatty acyl-CoA synthetases, but like the mammalian integral-membrane very-long-chain acyl-CoA synthetases, FadD13 accepts lipid substrates up to the maximum length tested (C-26). Here, we show that FadD13 is a peripheral membrane protein. The structure and mutational studies reveal an arginine- and aromatic-rich surface patch as the site for membrane interaction. The protein accommodates a hydrophobic tunnel that extends from the active site toward the positive patch and is sealed by an arginine-rich lid-loop at the protein surface. Based on this and previous data, we propose a structural basis for accommodation of lipid substrates longer than the enzyme and transmembrane lipid transport by vectorial CoA-esterification.

  • 2.
    Lundgren, Camilla A. K
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sjöstrand, Dan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Biner, Olivier
    Bennett, Matthew
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rudling, Axel
    Johansson, Ann-Louise
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinsk, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Carlsson, Jens
    von Ballmoos, Christoph
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Scavenging of superoxide by a membrane-bound superoxide oxidase2018In: Nature Chemical Biology, ISSN 1552-4450, E-ISSN 1552-4469, Vol. 14, p. 788-793Article in journal (Refereed)
    Abstract [en]

    Superoxide is a reactive oxygen species produced during aerobic metabolism in mitochondria and prokaryotes. It causes damage to lipids, proteins and DNA and is implicated in cancer, cardiovascular disease, neurodegenerative disorders and aging. As protection, cells express soluble superoxide dismutases, disproportionating superoxide to oxygen and hydrogen peroxide. Here, we describe a membrane-bound enzyme that directly oxidizes superoxide and funnels the sequestered electrons to ubiquinone in a diffusion-limited reaction. Experiments in proteoliposomes and inverted membranes show that the protein is capable of efficiently quenching superoxide generated at the membrane in vitro. The 2.0 Å crystal structure shows an integral membrane di-heme cytochrome b poised for electron transfer from the P-side and proton uptake from the N-side. This suggests that the reaction is electrogenic and contributes to the membrane potential while also conserving energy by reducing the quinone pool. Based on this enzymatic activity, we propose that the enzyme family be denoted superoxide oxidase (SOO).

  • 3.
    Lundgren, Camilla A.K.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structural and Functional Studies of Membrane Proteins: From Characterisation of a Fatty Acyl-CoA Synthetase to the Discovery of Superoxide Oxidase2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis is divided into three parts; the first part describes a method for efficient screening of membrane proteins for crystallography. By utilising the properties of a folding reporter GFP it is possible to quickly and accurately screen the stability of a protein in a range of conditions without full purification. This allows rapid assessment of the suitability of a protein for crystallography and a parallel optimisation of purification conditions for subsequent large-scale protein production.

    The second part describes the discovery of a membrane bound superoxide oxidase (SOO), a novel scavenger of membrane proximal superoxide. SOO is a kinetically perfect enzyme, reacting at rates close to the diffusion limit in a similar fashion to other superoxide scavengers, such as superoxide dismutase. We propose that SOO rescues electrons “lost” to superoxide and recycles them back into the respiratory chain, releasing oxygen. At the same time SOO contributes to the proton motive force by uptake of protons from the cytoplasmic side of the membrane.

    The third part concerns the fatty acyl-CoA synthetase FadD13 from Mycobacterium tuberculosis (M. tuberculosis). It represents a critical node point in M. tuberculosis lipid metabolism and has been suggested to be a vital component of M. tuberculosis survival in host cell macrophages. FadD13 harbours a hydrophobic cavity that is unable to house the very-long-chain substrates the enzyme has preference for. We propose that FadD13 is a peripheral membrane protein, utilising the membrane to house the very-long-chain fatty acid substrates during the activation reaction.

  • 4.
    Lundgren, Camilla
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lerche, Michael
    Norling, Charlotta
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structural and multimerization dynamics of Mycobacterium tuberculosis Fatty acyl CoA synthetase FadD13Manuscript (preprint) (Other academic)
    Abstract [en]

    The very-long-chain fatty acyl-CoA synthetase FadD13 from Mycobacterium tuberculosis activates fatty acids for further use in mycobacterial lipid metabolism.

    FadD13 is a peripheral membrane protein, with both soluble and membrane-bound populations in vivo. The protein displays a prominent positively charged surface patch, suggested to be involved in membrane association. Here we characterize the lipid binding properties of FadD13 and further show that the protein adopts a dimeric arrangement in solution. The dimer interface partly buries the positive patch, seemingly inconsistent with membrane binding. Moreover, the dimer arrangement does not provide an obvious alternative mode of membrane interaction.

    To gain further insight into the membrane binding, two protein variants were created, one where the positive patch was altered to become more negative and one more hydrophobic. The hydrophobic variant displayed an apparent increase in lipid affinity and the negative variant still retained significant lipid binding. Structural analysis showed that dimerization was disrupted in the variant proteins, with both variants being predominantly monomeric in solution, thus exposing the proposed membrane-binding surface. Together, the results suggest that FadD13 membrane interaction is regulated by a dimer-monomer equilibrium.

  • 5.
    Sjöstrand, Dan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Diamanti, Riccardo
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lundgren, Camilla A. K.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wiseman, Benjamin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    A rapid expression and purification condition screening protocol for membrane protein structural biology2017In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 26, no 8, p. 1653-1666Article in journal (Refereed)
    Abstract [en]

    Membrane proteins control a large number of vital biological processes and are often medically important-not least as drug targets. However, membrane proteins are generally more difficult to work with than their globular counterparts, and as a consequence comparatively few high-resolution structures are available. In any membrane protein structure project, a lot of effort is usually spent on obtaining a pure and stable protein preparation. The process commonly involves the expression of several constructs and homologs, followed by extraction in various detergents. This is normally a time-consuming and highly iterative process since only one or a few conditions can be tested at a time. In this article, we describe a rapid screening protocol in a 96-well format that largely mimics standard membrane protein purification procedures, but eliminates the ultracentrifugation and membrane preparation steps. Moreover, we show that the results are robustly translatable to large-scale production of detergent-solubilized protein for structural studies. We have applied this protocol to 60 proteins from an E. coli membrane protein library, in order to find the optimal expression, solubilization and purification conditions for each protein. With guidance from the obtained screening data, we have also performed successful large-scale purifications of several of the proteins. The protocol provides a rapid, low cost solution to one of the major bottlenecks in structural biology, making membrane protein structures attainable even for the small laboratory.

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