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  • 1.
    Balakrishnan Kumar, Ramakrishnan
    et al.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Department of Biosciences and Nutrition, Karolinska Institutet, Sweden.
    Zhu, Lin
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Department of Biosciences and Nutrition, Karolinska Institutet,.
    Idborg, Helena
    Radmark, Olof
    Jakobsson, Per-Johan
    Rinaldo-Matthis, Agnes
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Department of Biosciences and Nutrition, Karolinska Institutet,.
    Jegerschöld, Caroline
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Department of Biosciences and Nutrition, Karolinska Institutet,.
    Structural and Functional Analysis of Calcium Ion Mediated Binding of 5-Lipoxygenase to Nanodiscs2016In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 3, article id e0152116Article in journal (Refereed)
    Abstract [en]

    An important step in the production of inflammatory mediators of the leukotriene family is the Ca2+ mediated recruitment of 5 Lipoxygenase (5LO) to nuclear membranes. To study this reaction in vitro, the natural membrane mimicking environment of nanodiscs was used. Nanodiscs with 10.5 nm inner diameter were made with the lipid POPC and membrane scaffolding protein MSP1E3D1. Monomeric and dimeric 5LO were investigated. Monomeric 5LO mixed with Ca2+ and nanodiscs are shown to form stable complexes that 1) produce the expected leukotriene products from arachidonic acid and 2) can be, for the first time, visualised by native gel electrophoresis and negative stain transmission electron micros-copy and 3) show a highest ratio of two 5LO per nanodisc. We interpret this as one 5LO on each side of the disc. The dimer of 5LO is visualised by negative stain transmission electron microscopy and is shown to not bind to nanodiscs. This study shows the advantages of nanodiscs to obtain basic structural information as well as functional information of a complex between a monotopic membrane protein and the membrane.

  • 2. Frauenfeld, Jens
    et al.
    Loving, Robin
    Armache, Jean-Paul
    Sonnen, Andreas F-P
    Guettou, Fatma
    Moberg, Per
    Zhu, Lin
    KTH, School of Technology and Health (STH), Health Systems Engineering.
    Jegerschöld, Caroline
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Flayhan, Ali
    Briggs, John A. G.
    Garoff, Henrik
    Low, Christian
    Cheng, Yifan
    Nordlund, Par
    A saposin-lipoprotein nanoparticle system for membrane proteins2016In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 13, no 4, p. 345-351Article in journal (Refereed)
    Abstract [en]

    A limiting factor in membrane protein research is the ability to solubilize and stabilize such proteins. Detergents are used most often for solubilizing membrane proteins, but they are associated with protein instability and poor compatibility with structural and biophysical studies. Here we present a saposin-lipoprotein nanoparticle system, Salipro, which allows for the reconstitution of membrane proteins in a lipid environment that is stabilized by a scaffold of saposin proteins. We demonstrate the applicability of the method on two purified membrane protein complexes as well as by the direct solubilization and nanoparticle incorporation of a viral membrane protein complex from the virus membrane. Our approach facilitated high-resolution structural studies of the bacterial peptide transporter PeptT(S02) by single-particle cryo-electron microscopy (cryo-EM) and allowed us to stabilize the HIV envelope glycoprotein in a functional state.

  • 3. Guettou, Fatma
    et al.
    Quistgaard, Esben M.
    Tresaugues, Lionel
    Moberg, Per
    Jegerschöld, Caroline
    KTH, School of Technology and Health (STH), Structural Biotechnology (Closed 20130701).
    Zhu, Lin
    KTH, School of Technology and Health (STH), Structural Biotechnology (Closed 20130701).
    Jong, Agnes Jin Oi
    Nordlund, Pär
    Löw, Christian
    Structural insights into substrate recognition in proton-dependent oligopeptide transporters2013In: EMBO Reports, ISSN 1469-221X, E-ISSN 1469-3178, Vol. 14, no 9, p. 804-810Article in journal (Refereed)
    Abstract [en]

    Short-chain peptides are transported across membranes through promiscuous proton-dependent oligopeptide transporters (POTs)-a subfamily of the major facilitator superfamily (MFS). The human POTs, PEPT1 and PEPT2, are also involved in the absorption of various drugs in the gut as well as transport to target cells. Here, we present a structure of an oligomeric POT transporter from Shewanella oneidensis (PepT(So2)), which was crystallized in the inward open conformation in complex with the peptidomimetic alafosfalin. All ligand-binding residues are highly conserved and the structural insights presented here are therefore likely to also apply to human POTs.

  • 4.
    Jegerschöld, Caroline
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology (Closed 20130701).
    Paweizik, Sven-Christian
    Purhonen, Pasi
    KTH, School of Technology and Health (STH), Structural Biotechnology (Closed 20130701).
    Bhakat, Priyaranian
    KTH, School of Technology and Health (STH), Structural Biotechnology (Closed 20130701).
    Gheorghe, Karina Roxana
    KTH, School of Technology and Health (STH), Structural Biotechnology (Closed 20130701).
    Gyobu, Nobuhiko
    Mitsuoka, Kaoru
    Morgenstern, Ralf
    Jakobsson, Per-Johan
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology (Closed 20130701).
    Structural basis for induced formation of the inflammatory mediator prostaglandin E-22008In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 32, p. 11110-11115Article in journal (Refereed)
    Abstract [en]

    Prostaglandins (PG) are bioactive lipids produced from arachidonic acid via the action of cyclooxygenases and terminal PG synthases. Microsomal prostaglandin E synthase 1 (MPGES1) constitutes an inducible glutathione-dependent integral membrane protein that catalyzes the oxidoreduction of cyclooxygenase derived PGH(2) into PGE(2). MPGES1 has been implicated in a number of human diseases or pathological conditions, such as rheumatoid arthritis, fever, and pain, and is therefore regarded as a primary target for development of novel antiinflammatory drugs. To provide a structural basis for insight in the catalytic mechanism, we determined the structure of MPGES1 in complex with glutathione by electron crystallography from 2D crystals induced in the presence of phospholipids. Together with results from site-directed mutagenesis and activity measurements, we can thereby demonstrate the role of specific amino acid residues. Glutathione is found to bind in a U-shaped conformation at the interface between subunits in the protein trimer. It is exposed to a site facing the lipid bilayer, which forms the specific environment for the oxidoreduction of PGH(2) to PGE(2) after displacement of the cytoplasmic half of the IN-terminal transmembrane helix. Hence, insight into the dynamic behavior of MPGES1 and homologous membrane proteins in inflammation and detoxification is provided.

  • 5.
    Kuang, Qie
    et al.
    KTH, School of Technology and Health (STH).
    Purhonen, Pasi
    Alander, Johan
    Svensson, Richard
    Hoogland, Veronika
    Winerdal, Jens
    Spahiu, Linda
    Ottosson-Wadlund, Astrid
    Jegerschold, Caroline
    KTH, School of Technology and Health (STH).
    Morgenstern, Ralf
    Hebert, Hans
    KTH, School of Technology and Health (STH), Medical Engineering, Structural Biotechnology.
    Dead-end complex, lipid interactions and catalytic mechanism of microsomal glutathione transferase 1, an electron crystallography and mutagenesis investigation2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 7897Article in journal (Refereed)
    Abstract [en]

    Microsomal glutathione transferase 1 (MGST1) is a detoxification enzyme belonging to the Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism (MAPEG) superfamily. Here we have used electron crystallography of two-dimensional crystals in order to determine an atomic model of rat MGST1 in a lipid environment. The model comprises 123 of the 155 amino acid residues, two structured phospholipid molecules, two aliphatic chains and one glutathione (GSH) molecule. The functional unit is a homotrimer centered on the crystallographic three-fold axes of the unit cell. The GSH substrate binds in an extended conformation at the interface between two subunits of the trimer supported by new in vitro mutagenesis data. Mutation of Arginine 130 to alanine resulted in complete loss of activity consistent with a role for Arginine 130 in stabilizing the strongly nucleophilic GSH thiolate required for catalysis. Based on the new model and an electron diffraction data set from crystals soaked with trinitrobenzene, that forms a dead-end Meisenheimer complex with GSH, a difference map was calculated. The map reveals side chain movements opening a cavity that defines the second substrate site.

  • 6. Kuang, Qie
    et al.
    Purhonen, Pasi
    Jegerschöld, Caroline
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    The projection structure of Kch, a putative potassium channel in Escherichia coli, by electron crystallography2014In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1838, no 1, p. 237-243Article in journal (Refereed)
    Abstract [en]

    The kch gene, the only potassium channel gene in Escherichia coil, has the property to express both full-length Kch and its cytosolic domain (RCK) due to a methionine at position 240. The RCK domains are believed to form an octameric ring structure and regulate the gating of the potassium channels after having bound certain ligands. Several different gating ring structures have been reported for the soluble RCK domains, however, these were studied isolated from their transmembrane parts. We previously reported an octameric structure of Kch in solution by electron microscopy and single particle reconstruction, composed of two tetrameric full-length proteins through RCK interaction. To exclude the effect of the detergent, we have now performed an electron crystallographic study of the full-length Kch in membrane bound form. Well-ordered two-dimensional crystals were grown in a natural phospholipid environment. A projection map merged from the fifteen best images extended to 6 angstrom resolution. The c12 two-sided plane group of the two-dimensional crystals showed that Kch crystallized as two symmetrically related overlapping layers. The arrangement suggests that the two layers of RCK domains are shifted with respect to each other and the RCK octameric gating ring of Kch does not form under the crystallization condition.

  • 7.
    Kuang, Qie
    et al.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sweden.
    Purhonen, Pasi
    Ålander, Johan
    Svensson, Richard
    Hoogland, Veronika
    Winerdal, Jens
    Spahiu, Linda
    Ottosson-Wadlund, Astrid
    Armstrong, Richard
    Jegerschöld, Caroline
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Morgenstern, Ralf
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    A refined atomic model for microsomal glutathione transferase 1 from electron crystallographyManuscript (preprint) (Other academic)
    Abstract [en]

    Microsomal glutathione transferase 1 (MGST1) is a detoxification enzyme belonging to the Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism (MAPEG) superfamily. Here we have used electron crystallography of two-dimensional (2D) crystals in order to determine an atomic model of rat MGST1 in a lipid environment. The 2D crystals were of the p6 two-sided plane group symmetry. For the refinement, information to 3.5 Å resolution from 225 electron diffraction patterns recorded from specimens at tilt angles up to 66° was used. The model comprises 123 of the 155 amino acid residues, two structured phospholipid molecules, two hydrocarbon chains, and one glutathione (GSH) molecule. Interactions between subunits form trimers centered on the crystallographic three-fold axes of the unit cell. The GSH substrate binds in an extended conformation at the interface between two subunits of the trimer. The location of GSH is supported by mutagenesis data in vitro.

  • 8.
    Kumar, Ramakrishnan
    et al.
    Karolinska Inst, Dept Biosci & Nutr, Huddinge, Sweden.
    Hebert, Hans
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH). Karolinska Inst, Dept Biosci & Nutr, Huddinge, Sweden.
    Jegerschöld, Caroline
    KTH, School of Technology and Health (STH). Karolinska Inst, Dept Biosci & Nutr, Huddinge, Sweden.
    Elucidating the Interaction of 5-Lipoxygenase and FLAP2015In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 108, no 2, p. 499A-499AArticle in journal (Other academic)
  • 9. Löw, Christian
    et al.
    Yau, Yin Hoe
    Pardon, Els
    Jegerschöld, Caroline
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Wåhlin, Lisa
    Quistgaard, Esben M.
    Moberg, Per
    Geifman-Shochat, Susana
    Steyaert, Jan
    Nordlund, Pär
    Nanobody Mediated Crystallization of an Archeal Mechanosensitive Channel2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 10, p. e77984-Article in journal (Refereed)
    Abstract [en]

    Mechanosensitive channels (MS) are integral membrane proteins and allow bacteria to survive sudden changes in external osmolarity due to transient opening of their pores. The efflux of cytoplasmic osmolytes reduces the membrane tension and prevents membrane rupture. Therefore these channels serve as emergency valves when experiencing significant environmental stress. The preparation of high quality crystals of integral membrane proteins is a major bottleneck for structure determination by X-ray crystallography. Crystallization chaperones based on various protein scaffolds have emerged as promising tool to increase the crystallization probability of a selected target protein. So far archeal mechanosensitive channels of small conductance have resisted crystallization in our hands. To structurally analyse these channels, we selected nanobodies against an archeal MS channel after immunization of a llama with recombinant expressed, detergent solubilized and purified protein. Here we present the characterization of 23 different binders regarding their interaction with the channel protein using analytical gel filtration, western blotting and surface plasmon resonance. Selected nanobodies bound the target with affinities in the pico- to nanomolar range and some binders had a profound effect on the crystallization of the MS channel. Together with previous data we show that nanobodies are a versatile and valuable tool in structural biology by widening the crystallization space for highly challenging proteins, protein complexes and integral membrane proteins.

  • 10. Pawelzik, Sven-Christian
    et al.
    Uda, Narasimha Rao
    Spahiu, Linda
    Jegerschöld, Caroline
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Stenberg, Patric
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Morgenstern, Ralf
    Jakobsson, Per-Johan
    Identification of Key Residues Determining Species Differences in Inhibitor Binding of Microsomal Prostaglandin E Synthase-12010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 38, p. 29254-29261Article in journal (Refereed)
    Abstract [en]

    Microsomal prostaglandin E synthase-1 (MPGES1) is induced during an inflammatory reaction from low basal levels by pro-inflammatory cytokines and subsequently involved in the production of the important mediator of inflammation, prostaglandin E-2. Nonsteroidal anti-inflammatory drugs prevent prostaglandin E-2 production by inhibiting the upstream enzymes cyclooxygenases 1 and 2. In contrast to these conventional drugs, a new generation of NSAIDs targets the terminal enzyme MPGES1. Some of these compounds potently inhibit human MPGES1 but do not have an effect on the rat orthologue. We investigated this interspecies difference in a rat/human chimeric form of the enzyme as well as in several mutants and identified key residues Thr-131, Leu-135, and Ala-138 in human MPGES1, which play a crucial role as gate keepers for the active site of MPGES1. These residues are situated in transmembrane helix 4, lining the entrance to the cleft between two subunits in the protein trimer, and regulate access of the inhibitor in the rat enzyme. Exchange toward the human residues in rat MPGES1 was accompanied with a gain of inhibitor activity, whereas exchange in human MPGES1 toward the residues found in rat abrogated inhibitor activity. Our data give evidence for the location of the active site at the interface between subunits in the homotrimeric enzyme and suggest a model of how the natural substratePGH(2), or competitive inhibitors of MPGES1, enter the active site via the phospholipid bilayer of the membrane.

  • 11. Petrova, J
    et al.
    Zhu, L
    Axelsson, A
    Mörgelin, M
    Cochran, M
    Monterrubio, A
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Jegerschöld, Caroline
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Voss, CV
    Roberts, LM
    Lagerstedt, JO
    Structural properties of functional HDL and amyloidogenic L178H variant of apo A-12011In: Proceedings of the IX European Symposium of the Protein Society, 2011Conference paper (Refereed)
  • 12.
    Zhu, Lin
    et al.
    KTH, School of Technology and Health (STH), Medical Engineering, Structural Biotechnology. Karolinska Institutet, Sweden.
    Petrlova, J.
    Gysbers, P.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Medical Engineering, Structural Biotechnology. Karolinska Institutet, Sweden.
    Wallin, S.
    Jegerschöld, Caroline
    KTH, School of Technology and Health (STH). Karolinska Institutet, Sweden.
    Lagerstedt, J. O.
    Structures of apolipoprotein A-I in high density lipoprotein generated by electron microscopy and biased simulations2017In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1861, no 11, p. 2726-2738Article in journal (Refereed)
    Abstract [en]

    Background: Apolipoprotein A-I (apoA-I) in high-density lipoprotein (HDL) is a key protein for the transport of cholesterol from the vascular wall to the liver. The formation and structure of nascent HDL, composed of apoA-I and phospholipids, is critical to this process. Methods: The HDL was assembled in vitro from apoA-I, cholesterol and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at a 1:4:50 molar ratio. The structure of HDL was investigated in vitreous samples, frozen at cryogenic temperatures, as well as in negatively stained samples by transmission electron microscopy. Low resolution electron density maps were next used as restraints in biased Monte Carlo simulations of apolipoprotein A-I dimers, with an initial structure derived from atomic resolution X-ray structures. Results: Two final apoA-I structure models for the full-length structure of apoA-I dimer in the lipid bound conformation were generated, showing a nearly circular, flat particle with an uneven particle thickness. Conclusions: The generated structures provide evidence for the discoidal, antiparallel arrangement of apoA-I in nascent HDL, and propose two preferred conformations of the flexible N-termini.

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