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  • 51. Lendzian, F.
    et al.
    Voevodskaya, Nina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Galander, M.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The high-valent Fe(III)Fe(IV) center in class Ic ribonucleotide reductase of chlamydia trachomatis: EPR and ENDOR studies2007Conference paper (Other (popular science, discussion, etc.))
  • 52.
    Lerche, Michael
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sandhu, Hena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Flöckner, Lukas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rapp, Mikaela
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structure and Cooperativity of the Cytosolic Domain of the CorA Mg2+ Channel from Escherichia coli2017In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 25, no 8, p. 1175-1186.e4Article in journal (Refereed)
    Abstract [en]

    Structures of the Mg2+ bound (closed) and apo (open) states of CorA suggests that channel gating is accomplished by rigid-body motions between symmetric and asymmetric assemblies of the cytosolic portions of the five subunits in response to ligand (Mg2+) binding/unbinding at interfacial sites. Here, we structurally and biochemically characterize the isolated cytosolic domain from Escherichia coli CorA. The data reveal an Mg2+-ligand binding site located in a novel position between each of the five subunits and two Mg2+ ions trapped inside the pore. Soaking experiments show that cobalt hexammine outcompetes Mg2+ at the pore site closest to the membrane. This represents the first structural information of how an analog of hexa-hydrated Mg2+ (and competitive inhibitor of CorA) associates to the CorA pore. Biochemical data on the isolated cytoplasmic domain and full-length protein suggests that gating of the CorA channel is governed cooperatively.

  • 53.
    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).

  • 54.
    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.

  • 55.
    Lundin, Daniel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Poole, Anthony M.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Use of Structural Phylogenetic Networks for Classification of the Ferritin-like Superfamily2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 24, p. 20565-20575Article in journal (Refereed)
    Abstract [en]

    In the postgenomic era, bioinformatic analysis of sequence similarity is an immensely powerful tool to gain insight into evolution and protein function. Over long evolutionary distances, however, sequence-based methods fail as the similarities become too low for phylogenetic analysis. Macromolecular structure generally appears better conserved than sequence, but clear models for how structure evolves over time are lacking. The exponential growth of three-dimensional structural information may allow novel structure-based methods to drastically extend the evolutionary time scales amenable to phylogenetics and functional classification of proteins. To this end, we analyzed 80 structures from the functionally diverse ferritin-like superfamily. Using evolutionary networks, we demonstrate that structural comparisons can delineate and discover groups of proteins beyond the twilight zone where sequence similarity does not allow evolutionary analysis, suggesting that considerable and useful evolutionary signal is preserved in three-dimensional structures.

  • 56.
    Lundin, Daniel
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Poole, Anthony
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The functional diversity and evolutionary relationships of ferritin-like proteinsManuscript (preprint) (Other academic)
    Abstract [en]

    BackgroundThe ferritin-like proteins are evolutionarily related, as evidenced by the topology oftheir signature metal-binding four-helix bundle. They perform diverse functions suchas iron/oxygen detoxification, iron storage, DNA protection and substrate oxidation.Though evolutionarily related, sequence similarity between families and often withinfamilies is low. To analyse the evolutionary relationships between individual familiesand their functional roles systematically, we turned to 3D structural alignment andphylogenetic methods.ResultsOur phylogenetic network recovers all characterised functional groups of ferritin-likeproteins and suggests evolutionary relationships between them. The evolutionaryhypotheses that are suggested by the phylogeny are tested against availableindependent evidence such as dimerisation geometries, qualitative comparison ofstructures and sequence based partial phylogenies. Generally our hypotheses stand upagainst the evidence, but in a few cases verification have to await further data.ConclusionsTwo large evolutionary groups of ferritin-like proteins are identified from structuralphylogeny: ferritins, bacterioferritins and Dps proteins on one hand, and substrateoxidising proteins, bacterial multicomponent monooxygenases, fatty acid desaturasesand class I ribonucleotide reductase radical generating subunits, on the other. Themethod we present provides a robust way of evolutionarily classifying a functionallydiverse group of distantly related proteins, as well as examining the possible functionsof poorly-characterised proteins.

  • 57.
    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.

  • 58. Maugeri, Pearson T.
    et al.
    Griese, Julia J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Branca, Rui M.
    Miller, Effie K.
    Smith, Zachary R.
    Eirich, Jürgen
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Shafaat, Hannah S.
    Driving Protein Conformational Changes with Light: Photoinduced Structural Rearrangement in a Heterobimetallic Oxidase2018In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 140, no 4, p. 1471-1480Article in journal (Refereed)
    Abstract [en]

    The heterobimetallic R2lox protein binds both manganese and iron ions in a site-selective fashion and activates oxygen, ultimately performing C-H bond oxidation to generate a tyrosine-valine crosslink near the active site. In this work, we demonstrate that, following assembly, R2lox undergoes photoinduced changes to the active site geometry and metal coordination motif. Through spectroscopic, structural, and mass spectrometric characterization, the photoconverted species is found to consist of a tyrosinate-bound iron center following light-induced decarboxylation of a coordinating glutamate residue and cleavage of the tyrosine-valine cross-link. This process occurs with high quantum efficiencies (Phi = 3%) using violet and near-ultraviolet light, suggesting that the photodecarboxylation is initiated via ligandto-metal charge transfer excitation. Site-directed mutagenesis and structural analysis suggest that the cross-linked tyrosine-162 is the coordinating residue. One primary product is observed following irradiation, indicating potential use of this class of proteins, which contains a putative substrate channel, for controlled photoinduced decarboxylation processes, with relevance for in vivo functionality of R2lox as well as application in environmental remediation.

  • 59. Moure, Vivian R.
    et al.
    Siöberg, Catrine L. B.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Valdameri, Glaucio
    Nji, Emmanuel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oliveira, Marco Aurelio S.
    Gerdhardt, Edileusa C. M.
    Pedrosa, Fabio O.
    Mitchell, David A.
    Seefeldt, Lance C.
    Huergo, Luciano F.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordlund, Stefan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Souza, Emanuel M.
    The ammonium transporter AmtB and the PII signal transduction protein GlnZ are required to inhibit DraG in Azospirillum brasilense2019In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 286, no 6, p. 1214-1229Article in journal (Refereed)
    Abstract [en]

    The ammonium-dependent posttranslational regulation of nitrogenase activity in Azospirillum brasilense requires dinitrogenase reductase ADPribosyl transferase (DraT) and dinitrogenase reductase ADP-glycohydrolase (DraG). These enzymes are reciprocally regulated by interaction with the PII proteins, GlnB and GlnZ. In this study, purified ADP-ribosylated Fe-protein was used as substrate to study the mechanism involved in the regulation of A. brasilense DraG in vitro. The data show that DraG is partially inhibited by GlnZ and that DraG inhibition is further enhanced by the simultaneous presence of GlnZ and AmtB. These results are the first to demonstrate experimentally that DraG inactivation requires the formation of a ternary DraG-GlnZ-AmtB complex in vitro. Previous structural data have revealed that when the DraG-GlnZ complex associates with AmtB, the flexible T-loops of the trimeric GlnZ bind to AmtB and become rigid; these molecular events stabilize the DraG-GlnZ complex, resulting in DraG inactivation. To determine whether restraining the flexibility of the GlnZ T-loops is a limiting factor in DraG inhibition, we used a GlnZ variant that carries a partial deletion of the T-loop (GlnZD42-54). However, although the GlnZD42-54 variant was more effective in inhibiting DraG in vitro, it bound to DraG with a slightly lower affinity than does wild-type GlnZ and was not competent to completely inhibit DraG activity either in vitro or in vivo. We, therefore, conclude that the formation of a ternary complex between DraG-GlnZ-AmtB is necessary for the inactivation of DraG.

  • 60.
    Nitharwal, Ram Gopal
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Schäfer, Jacob
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wiseman, Benjamin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sjöstrand, Dan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kuang, Qie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Biochemical and structural characterization of a superoxide dismutase-containing respiratory supercomplex from Mycobacterium smegmatisManuscript (preprint) (Other academic)
  • 61.
    Nordlund, Stefan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    ADP-ribosylation, a mechanism regulating nitrogenase activity2013In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 280, no 15, p. 3484-3490Article, review/survey (Refereed)
    Abstract [en]

    Nitrogen fixation is the vital biochemical process in which atmospheric molecular nitrogen is made available to the biosphere. The process is highly energetically costly and thus tightly regulated. The activity of the key enzyme, nitrogenase, is controlled by reversible mono-ADP-ribosylation of one of its components, the Fe protein. This protein provides the other component, the MoFe protein, with the electrons required for the reduction of molecular nitrogen. The Fe-protein is ADP-ribosylated and de-ADP-ribosylated by dinitrogenase reductase ADP-ribosyl transferase and dinitrogenase reductase activating glycohydrolase, respectively. Here we review the current biochemical and structural knowledge of this central regulatory reaction.

  • 62. Rapatskiy, Leonid
    et al.
    Ames, William M.
    Pérez-Navarro, Montserrat
    Savitsky, Anton
    Griese, Julia J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Weyhermüller, Thomas
    Shafaat, Hannah S.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Neese, Frank
    Pantazis, Dimitrios A.
    Cox, Nicholas
    Characterization of Oxygen Bridged Manganese Model Complexes Using Multifrequency (17)O-Hyperfine EPR Spectroscopies and Density Functional Theory2015In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 119, no 43, p. 13904-13921Article in journal (Refereed)
    Abstract [en]

    Multifrequency pulsed EPR data are reported for a series of oxygen bridged (μ-oxo/μ-hydroxo) bimetallic manganese complexes where the oxygen is labeled with the magnetically active isotope (17)O (I = 5/2). Two synthetic complexes and two biological metallocofactors are examined: a planar bis-μ-oxo bridged complex and a bent, bis-μ-oxo-μ-carboxylato bridge complex; the dimanganese catalase, which catalyzes the dismutation of H2O2 to H2O and O2, and the recently identified manganese/iron cofactor of the R2lox protein, a homologue of the small subunit of the ribonuclotide reductase enzyme (class 1c). High field (W-band) hyperfine EPR spectroscopies are demonstrated to be ideal methods to characterize the (17)O magnetic interactions, allowing a magnetic fingerprint for the bridging oxygen ligand to be developed. It is shown that the μ-oxo bridge motif displays a small positive isotropic hyperfine coupling constant of about +5 to +7 MHz and an anisotropic/dipolar coupling of -9 MHz. In addition, protonation of the bridge is correlated with an increase of the hyperfine coupling constant. Broken symmetry density functional theory is evaluated as a predictive tool for estimating hyperfine coupling of bridging species. Experimental and theoretical results provide a framework for the characterization of the oxygen bridge in Mn metallocofactor systems, including the water oxidizing cofactor of photosystem II, allowing the substrate/solvent interface to be examined throughout its catalytic cycle.

  • 63. Rollauer, Sarah E.
    et al.
    Tarry, Michael J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Graham, James E.
    Jaaskelainen, Mari
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jaeger, Franziska
    Johnson, Steven
    Krehenbrink, Martin
    Liu, Sai-Man
    Lukey, Michael J.
    Marcoux, Julien
    McDowell, Melanie A.
    Rodriguez, Fernanda
    Roversi, Pietro
    Stansfeld, Phillip J.
    Robinson, Carol V.
    Sansom, Mark S. P.
    Palmer, Tracy
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Berks, Ben C.
    Lea, Susan M.
    Structure of the TatC core of the twin-arginine protein transport system2012In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 492, no 7428, p. 210-+Article in journal (Refereed)
    Abstract [en]

    The twin-arginine translocation (Tat) pathway is one of two general protein transport systems found in the prokaryotic cytoplasmic membrane and is conserved in the thylakoid membrane of plant chloroplasts. The defining, and highly unusual, property of the Tat pathway is that it transports folded proteins, a task that must be achieved without allowing appreciable ion leakage across the membrane. The integral membrane TatC protein is the central component of the Tat pathway. TatC captures substrate proteins by binding their signal peptides. TatC then recruits TatA family proteins to form the active translocation complex. Here we report the crystal structure of TatC from the hyperthermophilic bacterium Aquifex aeolicus. This structure provides a molecular description of the core of the Tat translocation system and a framework for understanding the unique Tat transport mechanism.

  • 64. Schenberger Santos, Adrian Richard
    et al.
    Marques Gerhardt, Edileusa Cristina
    Moure, Vivian Rotuno
    Pedrosa, Fábio Oliveira
    Souza, Emanuel Maltempi
    Diamanti, Riccardo
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Huergo, Luciano Fernandes
    Kinetics and structural features of dimeric glutamine-dependent bacterial NAD(+) synthetases suggest evolutionary adaptation to available metabolites2018In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 293, no 19, p. 7397-7407Article in journal (Refereed)
    Abstract [en]

    NADH (NAD(+)) and its reduced form NADH serve as cofactors for a variety of oxidoreductases that participate in many metabolic pathways. NAD(+) also is used as substrate by ADP-ribosyl transferases and by sirtuins. NAD(+) biosynthesis is one of the most fundamental biochemical pathways in nature, and the ubiquitous NAD(+) synthetase (NadE) catalyzes the final step in this biosynthetic route. Two different classes of NadE have been described to date: dimeric single-domain ammonium-dependent NadE(NH3) and octameric glutamine-dependent NadE(Gln), and the presence of multiple NadE isoforms is relatively common in prokaryotes. Here, we identified a novel dimeric group of NadE(Gln) in bacteria. Substrate preferences and structural analyses suggested that dimeric NadE(Gln) enzymes may constitute evolutionary intermediates between dimeric NadE(NH3) and octameric NadE(Gln). The characterization of additional NadE isoforms in the diazotrophic bacterium Azospirillum brasilense along with the determination of intracellular glutamine levels in response to an ammonium shock led us to propose a model in which these different NadE isoforms became active accordingly to the availability of nitrogen. These data may explain the selective pressures that support the coexistence of multiple isoforms of NadE in some prokaryotes.

  • 65. Schütz, Patrick
    et al.
    Karlberg, Tobias
    van den Berg, Susanne
    Collins, Ruairi
    Lehtiö, Lari
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Holmberg-Schiavone, Lovisa
    Tempel, Wolfram
    Park, Hee-Won
    Hammarström, Martin
    Moche, Martin
    Thorsell, Ann-Gerd
    Schüler, Herwig
    Comparative structural analysis of human DEAD-box RNA helicases2010In: PloS one, ISSN 1932-6203, Vol. 5, no 9Article in journal (Refereed)
    Abstract [en]

    DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members.

  • 66.
    Segerstolpe, Åsa
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Granneman, Sander
    Björk, Petra
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Alves, Flavia de Lima
    Rappsilber, Juri
    Andersson, Charlotta S.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tollervey, David
    Wieslander, Lars
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Multiple rna interactions position mrd1 at the site of the small subunit pseudoknot within the 90s pre ribosome2013In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 41, no 2, p. 1178-1190Article in journal (Refereed)
    Abstract [en]

    Ribosomal subunit biogenesis in eukaryotes is a complex multistep process. Mrd1 is an essential and conserved small (40S) ribosomal subunit synthesis factor that is required for early cleavages in the 35S pre-ribosomal RNA (rRNA). Yeast Mrd1 contains five RNA-binding domains (RBDs), all of which are necessary for optimal function of the protein. Proteomic data showed that Mrd1 is part of the early pre-ribosomal complexes, and deletion of individual RBDs perturbs the pre-ribosomal structure. In vivo ultraviolet cross-linking showed that Mrd1 binds to the pre-rRNA at two sites within the 18S region, in helix 27 (h27) and helix 28. The major binding site lies in h27, and mutational analyses shows that this interaction requires the RBD1-3 region of Mrd1. RBD2 plays the dominant role in h27 binding, but other RBDs also contribute directly. h27 and helix 28 are located close to the sequences that form the central pseudoknot, a key structural feature of the mature 40S subunit. We speculate that the modular structure of Mrd1 coordinates pseudoknot formation with pre-rRNA processing and subunit assembly.

  • 67. Shafaat, Hannah S.
    et al.
    Griese, Julia J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pantazis, Dimitrios A.
    Roos, Katarina
    Stockholm University, Faculty of Science, Department of Physics.
    Andersson, Charlotta S.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Popovic-Bijelic, Ana
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Siegbahn, Per E. M.
    Stockholm University, Faculty of Science, Department of Physics.
    Neese, Frank
    Lubitz, Wolfgang
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Cox, Nicholas
    Electronic Structural Flexibility of Heterobimetallic Mn/Fe Cofactors: R2lox and R2c Proteins2014In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 136, no 38, p. 13399-13409Article in journal (Refereed)
    Abstract [en]

    The electronic structure of the Mn/Fe cofactor identified in a new class of oxidases (R2lox) described by Andersson and Hogbom [Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 5633] is reported. The R2lox protein is homologous to the small subunit of class Ic ribonucleotide reductase (R2c) but has a completely different in vivo function. Using multifrequency EPR and related pulse techniques, it is shown that the cofactor of R2lox represents an antiferromagnetically coupled Mn-III/Fe-III dimer linked by a mu-hydroxo/bis-mu-carboxylato bridging network. The Mn-III ion is coordinated by a single water ligand. The R2lox cofactor is photoactive, converting into a second form (R2lox(photo)) upon visible illumination at cryogenic temperatures (77 K) that completely decays upon warming. This second, unstable form of the cofactor more closely resembles the Mn-III/Fe-III cofactor seen in R2c. It is shown that the two forms of the R2lox cofactor differ primarily in terms of the local site geometry and electronic state of the Mn-III ion, as best evidenced by a reorientation of its unique Mn-55 hyperfine axis. Analysis of the metal hyperfine tensors in combination with density functional theory (DFT) calculations suggests that this change is triggered by deprotonation of the mu-hydroxo bridge. These results have important consequences for the mixed-metal R2c cofactor and the divergent chemistry R2lox and R2c perform.

  • 68.
    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.

  • 69.
    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.

  • 70.
    Skaar, Karin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Korza, Henryk J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tarry, Michael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sekyrova, Petra
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Expression and Subcellular Distribution of GFP-Tagged Human Tetraspanin Proteins in Saccharomyces cerevisiae2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 7, article id e0134041Article in journal (Refereed)
    Abstract [en]

    Tetraspanins are integral membrane proteins that function as organizers of multimolecular complexes and modulate function of associated proteins. Mammalian genomes encode approximately 30 different members of this family and remotely related eukaryotic species also contain conserved tetraspanin homologs. Tetraspanins are involved in a number of fundamental processes such as regulation of cell migration, fusion, immunity and signaling. Moreover, they are implied in numerous pathological states including mental disorders, infectious diseases or cancer. Despite the great interest in tetraspanins, the structural and biochemical basis of their activity is still largely unknown. A major bottleneck lies in the difficulty of obtaining stable and homogeneous protein samples in large quantities. Here we report expression screening of 15 members of the human tetraspanin superfamily and successful protocols for the production in Scerevisiae of a subset of tetraspanins involved in human cancer development. We have demonstrated the subcellular localization of overexpressed tetraspanin-green fluorescent protein fusion proteins in Scerevisiae and found that despite being mislocalized, the fusion proteins are not degraded. The recombinantly produced tetraspanins are dispersed within the endoplasmic reticulum membranes or localized in granule-like structures in yeast cells. The recombinantly produced tetraspanins can be extracted from the membrane fraction and purified with detergents or the poly (styrene-co-maleic acid) polymer technique for use in further biochemical or biophysical studies.

  • 71.
    Skoog, Karl
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Stenberg Bruzell, Filippa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ducroux, Aurélie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hellberg, Mårten
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Johansson, Henrik
    Lehtiö, Janne
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Daley, Daniel O.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Penicillin-binding protein 5 can form a homo-oligomeric complex in the inner membrane of Escherichia coli2011In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 20, no 9, p. 1520-1529Article in journal (Refereed)
    Abstract [en]

    Penicillin-binding protein 5 (PBP5) is a DD-carboxypeptidase, which cleaves the terminal D-alanine from the muramyl pentapeptide in the peptidoglycan layer of Escherichia coli and other bacteria. In doing so, it varies the substrates for transpeptidation and plays a key role in maintaining cell shape. In this study, we have analyzed the oligomeric state of PBP5 in detergent and in its native environment, the inner membrane. Both approaches indicate that PBP5 exists as a homo-oligomeric complex, most likely as a homo-dimer. As the crystal structure of the soluble domain of PBP5 (i.e., lacking the membrane anchor) shows a monomer, we used our experimental data to generate a model of the homo-dimer. This model extends our understanding of PBP5 function as it suggests how PBP5 can interact with the peptidoglycan layer. It suggests that the stem domains interact and the catalytic domains have freedom to move from the position observed in the crystal structure. This would allow the catalytic domain to have access to pentapeptides at different distances from the membrane.

  • 72.
    Smirnova, Irina A.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Moscow State University, Russian Federation.
    Sjöstrand, Dan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Li, Fei
    Björck, Markus
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Schäfer, Jacob
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Östbye, Henrik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stanford University, United States.
    von Ballmoos, Christoph
    Lander, Gabriel C.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Isolation of yeast complex IV in native lipid nanodiscs2016In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1858, no 12, p. 2984-2992Article in journal (Refereed)
    Abstract [en]

    We used the amphipathic styrene maleic acid (SMA) co-polymer to extract cytochrome c oxidase (CytcO) in its native lipid environment from S. cerevisiae mitochondria. Native nanodiscs containing one CytcO per disc were purified using affinity chromatography. The longest cross-sections of the native nanodiscs were 11 nm x 14 nm. Based on this size we estimated that each CytcO was surrounded by similar to 100 phospholipids. The native nanodiscs contained the same major phospholipids as those found in the mitochondrial inner membrane. Even though CytcO forms a supercomplex with cytochrome bc(1) in the mitochondria! membrane, cyt.bc(1) was not found in the native nanodiscs. Yet, the loosely-bound Respiratory SuperComplex factors were found to associate with the isolated CytcO. The native nanodiscs displayed an O-2-reduction activity of similar to 130 electrons CytcO(-1) s(-1) and the kinetics of the reaction of the fully reduced CytcO with 02 was essentially the same as that observed with CytcO in mitochondrial membranes. The kinetics of CO-ligand binding to the CytcO catalytic site was similar in the native nanodiscs and the mitochondrial membranes. We also found that excess SMA reversibly inhibited the catalytic activity of the mitochondrial CytcO, presumably by interfering with cyt. c binding. These data point to the importance of removing excess SMA after extraction of the membrane protein. Taken together, our data shows the high potential of using SMA-extracted CytcO for functional and structural studies.

  • 73.
    Srinivas, Vivek
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Banerjee, Rahul
    Lebrette, Hugo
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jones, Jason
    Aurelius, Oskar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    John, Juliane
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kim, In-Sik
    Pham, Cindy
    Gul, Sheraz
    Sutherlin, Kyle
    Bhowmick, Asmit
    Fransson, Thomas
    Aller, Pierre
    Butryn, Agata
    Tono, Kensuke
    Alonso-Mori, Roberto
    Fuller, Franklin
    Batyuk, Alexander
    Brewster, Aaron
    Sauter, Nicholas
    Orville, Allen
    Yachandra, Vittal
    Yano, Junko
    Lipscomb, John
    Kern, Jan
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    High Resolution XFEL Structure of the Methane Monooxygenase Hydroxylase Complex with its Regulatory Component at Ambient Temperature in Two Oxidation StatesManuscript (preprint) (Other academic)
  • 74.
    Srinivas, Vivek
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lebrette, Hugo
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lundin, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kutin, Yuri
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lerche, Michael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Eirich, Jürgen
    Branca, Rui M. M.
    Cox, Nicholas
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stanford University School of Medicine, USA.
    Metal-free ribonucleotide reduction powered by a DOPA radical in Mycoplasma pathogens2018In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 563, p. 416-420Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductase (RNR) catalyses the only known de novo pathway for the production of all four deoxyribonucleotides that are required for DNA synthesis1,2. It is essential for all organisms that use DNA as their genetic material and is a current drug target3,4. Since the discovery that iron is required for function in the aerobic, class I RNR found in all eukaryotes and many bacteria, a dinuclear metal site has been viewed as necessary to generate and stabilize the catalytic radical that is essential for RNR activity5,6,7. Here we describe a group of RNR proteins in Mollicutes—including Mycoplasma pathogens—that possess a metal-independent stable radical residing on a modified tyrosyl residue. Structural, biochemical and spectroscopic characterization reveal a stable 3,4-dihydroxyphenylalanine (DOPA) radical species that directly supports ribonucleotide reduction in vitro and in vivo. This observation overturns the presumed requirement for a dinuclear metal site in aerobic ribonucleotide reductase. The metal-independent radical requires new mechanisms for radical generation and stabilization, processes that are targeted by RNR inhibitors. It is possible that this RNR variant provides an advantage under metal starvation induced by the immune system. Organisms that encode this type of RNR—some of which are developing resistance to antibiotics—are involved in diseases of the respiratory, urinary and genital tracts. Further characterization of this RNR family and its mechanism of cofactor generation will provide insight into new enzymatic chemistry and be of value in devising strategies to combat the pathogens that utilize it. We propose that this RNR subclass is denoted class Ie.

  • 75. Stenmark, Pål
    et al.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Roshick, Christine
    McClarty, Grant
    Nordlund, Pär
    Crystals of the ribonucleotide reductase R2 protein from Chlamydia trachomatis obtained by heavy-atom co-crystallization.2004In: Acta Crystallogr D Biol Crystallogr, ISSN 0907-4449, Vol. 60, no Pt 2, p. 376-8Article in journal (Refereed)
  • 76. Suarez Covarrubias, Adrian
    et al.
    Larsson, Anna M
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lindberg, Jimmy
    Bergfors, Terese
    Björkelid, Christofer
    Mowbray, Sherry L
    Unge, Torsten
    Jones, T Alwyn
    Structure and function of carbonic anhydrases from Mycobacterium tuberculosis.2005In: J Biol Chem, ISSN 0021-9258, Vol. 280, no 19, p. 18782-9Article in journal (Refereed)
  • 77.
    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.
    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 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.

  • 78.
    Tarry, Michael
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jaaskelainen, Mari
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Paino, Annamari
    Tuominen, Heidi
    Ihalin, Riikka
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The Extra-Membranous Domains of the Competence Protein HofQ Show DNA Binding, Flexibility and a Shared Fold with Type I KH Domains2011In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 409, no 4, p. 642-653Article in journal (Refereed)
    Abstract [en]

    Secretins form large oligomeric assemblies in the membrane that control both macromolecular secretion and uptake. Several Pasteurellaceae are naturally competent for transformation, but the mechanism for DNA assimilation is largely unknown. In Haemophilus influenzae, the secretin ComE has been demonstrated to be essential for DNA uptake. In closely related Aggregatibacter actinomycetemcomitans, an opportunistic pathogen in periodontitis, the ComE homolog HofQ is believed to be the outer membrane DNA translocase. Here, we report the structure of the extramembranous domains of HofQ at 2.3 angstrom resolution by X-ray crystallography. We also show that the extra-membranous domains of HofQ are capable of DNA binding. The structure reveals two secretin-like folds, the first of which is formed by means of a domain swap. The second domain displays extensive structural similarity to K homology (KH) domains, including the presence of a GxxG motif, which is essential for the nucleotide-binding function of KH domains, suggesting a possible mechanism for DNA binding by HofQ. The data indicate a direct involvement in DNA acquisition and provide insight into the molecular basis for natural competence.

  • 79.
    Tarry, Michael
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Skaar, Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Draheim, Roger R.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Production of human tetraspanin proteins in Escherichia coli2012In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 82, no 2, p. 373-379Article in journal (Refereed)
    Abstract [en]

    Tetraspanins are found in multicellular eukaryotes and are generally thought to act as scaffolding proteins, localizing multiple proteins to a specific region of the cell membrane. Activities for tetraspanins have been identified in several fundamental processes such as motility, cell adhesion, proliferation and viral entry. Tetraspanins are also key players in cancer development and progression. However, structural and biochemical information on tetraspanins is decidely limited, due in no small part to the difficulties associated with expressing eukaryotic membrane proteins. In this study, we have used GFP fusions of a library of human tetraspanin proteins to identify growth conditions for expression in Escherichia coli. Three tetraspanin-GFP proteins could be produced at high enough levels to allow subsequent purification, paving the way for future structural and biochemical studies.

  • 80. Vécsey-Semjén, Beatrix
    et al.
    Kwak, Young-Keun
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Möllby, Roland
    Channel-Forming Abilities of Spontaneously Occurring alpha-Toxin Fragments from Staphylococcus aureus2010In: Journal of Membrane Biology, ISSN 0022-2631, E-ISSN 1432-1424, Vol. 234, no 3, p. 171-181Article in journal (Refereed)
    Abstract [en]

    Pore formation by four spontaneously occurring alpha-toxin fragments from Staphylococcus aureus were investigated on liposome and erythrocyte membranes. All the isolated fragments bound to the different types of membranes and formed transmembrane channels in egg-phosphatidyl glycerol vesicles. Fragments of amino acids (aa) 9-293 (32 kD) and aa 13-293 (31 kD) formed heptamers, similar to the intact toxin, while the aa 72-293 (26 kD) fragment formed heptamers, octamers, and nonamers, as judged by gel electrophoresis of the liposomes. All isolated fragments induced release of chloride ions from large unilamellar vesicles. Channel formation was promoted by acidic pH and negatively charged lipid head groups. Also, the fragments' hemolytic activity was strongly decreased under neutral conditions but could be partially restored by acidification of the medium. We paid special attention to the 26-kD fragment, which, despite the loss of about one-fourth of the N-terminal part of alpha-toxin, did form transmembrane channels in liposomes. In light of the available data on channel formation by alpha-toxin, our results suggest that proteolytic degradation might be better tolerated than previously reported. Channel opening could be inhibited and open channels could be closed by zinc in the medium. Channel closure could be reversed by addition of EDTA. In contrast, digestion at the C terminus led to premature oligomerization and resulted in species with strongly diminished activity and dependent on protonation.

  • 81.
    Wagner, Samuel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Klepsch, Mirjam M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Schlegel, Susan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Appel, Ansgar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Draheim, Roger
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tarry, Michael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    van Wijk, Klaas J.
    Slotboom, Dirk J.
    Persson, Jan O.
    Stockholm University, Faculty of Science, Department of Mathematics.
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tuning Escherichia coli for membrane protein overexpression2008In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 38, p. 14371-17376Article in journal (Refereed)
    Abstract [en]

    A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used “Walker strains” C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications.

  • 82.
    Wiseman, Benjamin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nitharwal, Ram Gopal
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Fedotovskaya, Olga
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Schäfer, Jacob
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Guo, Hui
    Kuang, Qie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Benlekbir, Samir
    Sjöstrand, Dan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rubinstein, John L.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structure of a functional obligate complex III2IV2 respiratory supercomplex from Mycobacterium smegmatis2018In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 25, no 12, p. 1128-1136Article in journal (Refereed)
    Abstract [en]

    In the mycobacterial electron-transport chain, respiratory complex III passes electrons from menaquinol to complex IV, which in turn reduces oxygen, the terminal acceptor. Electron transfer is coupled to transmembrane proton translocation, thus establishing the electrochemical proton gradient that drives ATP synthesis. We isolated, biochemically characterized, and determined the structure of the obligate III2IV2 supercomplex from Mycobacterium smegmatis, a model for Mycobacterium tuberculosis. The supercomplex has quinol:O-2 oxidoreductase activity without exogenous cytochrome c and includes a superoxide dismutase subunit that may detoxify reactive oxygen species produced during respiration. We found menaquinone bound in both the Q(o) and Q(i) sites of complex III. The complex III-intrinsic diheme cytochrome cc subunit, which functionally replaces both cytochrome c(1) and soluble cytochrome c in canonical electron-transport chains, displays two conformations: one in which it provides a direct electronic link to complex IV and another in which it serves as an electrical switch interrupting the connection.

  • 83.
    Xu, Hongyi
    et al.
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Lebrette, Hugo
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Clabbers, Max T. B.
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Zhao, Jingjing
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Griese, Julia J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Uppsala University, Sweden.
    Zou, Xiaodong
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Solving a new R2lox protein structure by microcrystal electron diffraction2019In: Science Advances, E-ISSN 2375-2548, Vol. 5, no 8, article id eaax4621Article in journal (Refereed)
    Abstract [en]

    Microcrystal electron diffraction (MicroED) has recently shown potential for structural biology. It enables the study of biomolecules from micrometer-sized 3D crystals that are too small to be studied by conventional x-ray crystallography. However, to date, MicroED has only been applied to redetermine protein structures that had already been solved previously by x-ray diffraction. Here, we present the first new protein structure-an R2lox enzyme-solved using MicroED. The structure was phased by molecular replacement using a search model of 35% sequence identity. The resulting electrostatic scattering potential map at 3.0-angstrom resolution was of sufficient quality to allow accurate model building and refinement. The dinuclear metal cofactor could be located in the map and was modeled as a heterodinuclear Mn/Fe center based on previous studies. Our results demonstrate that MicroED has the potential to become a widely applicable tool for revealing novel insights into protein structure and function.

  • 84.
    Xu, Hongyi
    et al.
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Lebrette, Hugo
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Yang, Taimin
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Srinivas, Vivek
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hovmöller, Sven
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Zou, Xiaodong
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    A Rare Lysozyme Crystal Form Solved Using Highly Redundant Multiple Electron Diffraction Datasets from Micron-Sized Crystals2018In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 26, no 4, p. 667-675Article in journal (Refereed)
    Abstract [en]

    Recent developments of novel electron diffraction techniques have shown to be powerful for determination of atomic resolution structures from micronand nano-sized crystals, too small to be studied by single-crystal X-ray diffraction. In this work, the structure of a rare lysozyme polymorph is solved and refined using continuous rotation MicroED data and standard X-ray crystallographic software. Data collection was performed on a standard 200 kV transmission electron microscope (TEM) using a highly sensitive detector with a short readout time. The data collection is fast (similar to 3 min per crystal), allowing multiple datasets to be rapidly collected from a large number of crystals. We show that merging data from 33 crystals significantly improves not only the data completeness, overall I/sigma and the data redundancy, but also the quality of the final atomic model. This is extremely useful for electron beam-sensitive crystals of low symmetry or with a preferred orientation on the TEM grid.

  • 85.
    Zhou, Shu
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pettersson, Pontus
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Huang, Jingjing
    Sjöholm, Johannes
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sjöstrand, Dan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pomes, Regis
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Solution NMR structure of yeast Rcf1, a protein involved in respiratory supercomplex formation2018In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 12, p. 3048-3053Article in journal (Refereed)
    Abstract [en]

    The Saccharomyces cerevisiae respiratory supercomplex factor 1 (Rcf1) protein is located in the mitochondrial inner membrane where it is involved in formation of supercomplexes composed of respiratory complexes III and IV. We report the solution structure of Rcf1, which forms a dimer in dodecylphosphocholine (DPC) micelles, where each monomer consists of a bundle of five transmembrane (TM) helices and a short flexible soluble helix (SH). Three TM helices are unusually charged and provide the dimerization interface consisting of 10 putative salt bridges, defining a charge zipper motif. The dimer structure is supported by molecular dynamics (MD) simulations in DPC, although the simulations show a more dynamic dimer interface than the NMR data. Furthermore, CD and NMR data indicate that Rcf1 undergoes a structural change when reconstituted in liposomes, which is supported by MD data, suggesting that the dimer structure is unstable in a planar membrane environment. Collectively, these data indicate a dynamic monomer-dimer equilibrium. Furthermore, the Rcf1 dimer interacts with cytochrome c, suggesting a role as an electron-transfer bridge between complexes III and IV. The Rcf1 structure will help in understanding its functional roles at a molecular level.

  • 86.
    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.))
  • 87.
    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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