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  • 1. Ambort, D.
    et al.
    Johansson, M.E.V.
    Gustafsson, J. K.
    Nilsson, Harriet E.
    Ermund, A.
    Johansson, B.R.
    Koeck, Philip J. B.
    KTH, School of Technology and Health (STH), Structural Biotechnology (Closed 20130701).
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology (Closed 20130701).
    Hansson, G.C.
    Calcium and pH-dependent packing and release of the gel-forming MUC2 mucin2012In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, no 15, p. 5645-5650Article in journal (Refereed)
    Abstract [en]

    MUC2, the major colonic mucin, forms large polymers by N-terminal trimerization and C-terminal dimerization. Although the assembly process for MUC2 is established, it is not known how MUC2 is packed in the regulated secretory granulae of the goblet cell. When the N-terminal VWD1-D2-D'D3 domains (MUC2-N) were expressed in a goblet-like cell line, the protein was stored together with full-length MUC2. By mimicking the pH and calcium conditions of the secretory pathway we analyzed purified MUC2-N by gel filtration, density gradient centrifugation, and transmission electron microscopy. At pH 7.4 the MUC2-N trimer eluted as a single peak by gel filtration. At pH 6.2 with Ca2+ it formed large aggregates that did not enter the gel filtration column but were made visible after density gradient centrifugation. Electron microscopy studies revealed that the aggregates were composed of rings also observed in secretory granulae of colon tissue sections. TheMUC2-N aggregates were dissolved by removing Ca2+ and raising pH. After release from goblet cells, the unfolded full-length MUC2 formed stratified layers. These findings suggest a model for mucin packing in the granulae and the mechanism for mucin release, unfolding, and expansion.

  • 2. Busenlehner, Laura S.
    et al.
    Alander, Johan
    Jegerscohld, Caroline
    Holm, Peter J.
    Bhakat, Priyaranjan
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Morgenstern, Ralf
    Armstrong, Richard N.
    Location of substrate binding sites within the integral membrane protein microsomal glutathione transferase-12007In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 10, p. 2812-2822Article in journal (Refereed)
    Abstract [en]

    Microsomal glutathione transferase-1 (MGST1) is a trimeric, membrane-bound enzyme with both glutathione (GSH) transferase and hydroperoxidase activities. As a member of the MAPEG superfamily, MGST1 aids in the detoxication of numerous xenobiotic substrates and in cellular protection from oxidative stress through the GSH-dependent reduction of phospholipid hydroperoxides. However, little is known about the location of the different substrate binding sites, including whether the transferase and peroxidase activities overlap structurally. Although molecular density attributed to GSH has been observed in the 3.2 A resolution electron crystallographic structure of MGST1, the electrophilic and phospholipid hydroperoxide substrate binding sites remain elusive. Amide H-D exchange kinetics and H-D ligand footprinting experiments indicate that GSH and hydrophobic substrates bind within similar, but distinct, regions of MGST1. Site-directed mutagenesis, guided by the H-D exchange results, demonstrates that specific residues within the GSH footprint effect transferase activity toward 1-chloro-2,4-dinitrobenzene. In addition, cytosolic residues surrounding the chemical stress sensor C49 but not modeled in the crystal structure appear to play an important role in the formation of the binding site for hydrophobic substrates. Although the fatty acid/phospholipid binding site structurally overlaps that for GSH, it does not appear to be localized to the same region as other hydrophobic substrates. Finally, H-D exchange mass spectrometry reveals a specific conformational transition that may mediate substrate binding and/or product release. Such structural changes in MGST1 are essential for activation of the enzyme and are important for its biological function.

  • 3.
    Cheng, Kimberley
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Single-particle cryo-electron microscopy of macromolecular assemblies2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In this thesis, single-particle cryo-electron microscopy (cryo-EM) was used to study the structure of three macromolecular assemblies: the two hemocyanin isoforms from Rapana thomasiana, the Pyrococcus furiosus chaperonin, and the ribosome from Escherichia coli.

    Hemocyanins are large respiratory proteins in arthropods and molluscs. Most molluscan hemocyanins exist as two distinct isoforms composed of related polypeptides. In most species the two isoforms differ in terms of their oligomeric stability, and thus we set out to investigate the two Rapana thomasiana hemocyanins (RtH) in order to explain this behaviour. Our findings showed that the two RtHio forms are identical at the experimental resolution. Furthermore, three previously unreported connections that most likely contribute to the oligomeric stability were identified.

    Chaperonins are double-ring protein complexes that assist the folding process of nascent, non-native polypeptide chains. The chaperonin from the hyperthermophilic archaea Pyrococcus furiosus belongs to Group II chaperonins, and unlike most othergroup II chaperonins it appears to be homo-oligomeric. The 3D reconstruction of the Pyrococcus furiousus chaperonin revealed a di-octameric structure in a partially closed/open state, something in between the closed folding-active state and the open substrate-accepting state.

    The ribosome is the molecular machine where protein synthesis takes place. In bacteria there is a unique RNA molecule called transfer-messenger RNA (tmRNA) that together with its helper protein SmpB rescues ribosomes trapped on defective messenger RNAs (mRNAs) through a process called trans-translation. tmRNA is about 4 times the size of a normal tRNA, and it is composed of a tRNA-like domain (TLD) that is connected to the mRNA-like domain (MLD) by several pseudoknots (PKs) and RNA helices. During trans-translation, tmRNA utilize its TLD to receive the incomplete polypeptide from the peptidyl-tRNA in the ribosomal P site of the stalledribosome. Subsequently, its MLD is used to tag the incomplete polypeptide with adegradation signal. When tmRNA enters a stalled ribosome the MLD and pseudoknots form a highly structured arc that encircles the beak of the small ribosomal subunit. Byutilizing maximum-likelihood based methods for heterogeneity analysis we could observe the Escherichia coli ribosome in a number of different tmRNA·SmpB-boundstates. The cryo-EM map of the post-accommodated state revealed that the TLD·SmpBpart of the tmRNA·SmpB complex mimics native tRNAs in the A site of stalled ribosomes. The density map also showed that the tmRNA arc remains well structuredand that it is still attached to the beak of the small ribosomal subunit. Thereconstructions of the double-translocation tmRNA-bound ribosome complex showed that the pseudoknots of tmRNA still form an arc, and that they are located at positions similar to the ones assigned for the pseudoknots in the post-accommodated state. In addition, the tmRNA arc exists in two states; one stable and highly structured and another more flexible and disorganized.

  • 4.
    Cheng, Kimberley
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Ivanova, Natalia
    Biomedicinskt centrum, Uppsala.
    Scheres, S.H.W
    Pavlov, Michael
    Biomedicinskt centrum.
    Carazo, J.M.
    Herbert, Heinz
    Ehrenberg, Måns
    Lindahl, Martin
    KTH, School of Technology and Health (STH).
    Structural analysis of double translocated tmRNA on the 70S ribosome indicates flexibility of the tmRNA structure.Manuscript (preprint) (Other academic)
  • 5.
    Cheng, Kimberley
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Ivanova, Natalia
    Biomedicinskt centrum, Uppsala.
    Scheres, Sjores
    CSIC, Natl Biotechnol Ctr, Biocomp Unit, E-28049 Madrid, Spain .
    Pavlov, Michael Y
    Biomedicinskt centrum, Uppsala.
    Maria Carazo, Jose
    Lund Univ, Mol Biophys KILU.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Ehrenberg, Måns
    Biomedicinskt centrum, Uppsala.
    Lindahl, Martin
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    tmRNA-SmpB complex mimics native aminoacyl-tRNAs in the A site of stalled ribosomes2010In: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 169, no 3, p. 342-348Article in journal (Refereed)
    Abstract [en]

    Bacterial ribosomes stalled on faulty, often truncated, mRNAs lacking stop codons are rescued by trans-translation. It relies on an RNA molecule (tmRNA) capable of replacing the faulty mRNA with its own open reading frame (ORF). Translation of tmRNA ORF results in the tagging of faulty protein for degradation and its release from the ribosome. We used single-particle cryo-electron microscopy to visualize tmRNA together with its helper protein SmpB on the 70S Escherichia coli ribosome in states subsequent to GTP hydrolysis on elongation factor Tu (EF-Tu). Three-dimensional reconstruction and heterogeneity analysis resulted in a 15 A resolution structure of the tmRNA-SmpB complex accommodated in the A site of the ribosome, which shows that SmpB mimics the anticodon- and D-stem of native tRNAs missing in the tRNA-like domain of tmRNA. We conclude that the tmRNA-SmpB complex accommodates in the ribosomal A site very much like an aminoacyl-tRNA during protein elongation. (C) 2009 Elsevier Inc. All rights reserved.

  • 6.
    Cheng, Kimberley
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology (Closed 20130701).
    Karlström, M
    Purhonen, P
    Ladenstein, R.
    Herbert, Hans
    Koeck, Philip J.B.
    Low resolution structure and apparent melting temperature of the chaperonin from Pyrococcus furiosusManuscript (preprint) (Other academic)
  • 7.
    Cheng, Kimberley
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Koeck, Philip J. B.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Elmlund, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Idakieva, Krassimira
    Parvanova, Katja
    Schwarz, Heinz
    Ternström, Tomas
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Comparison of the two Rapana thomasiana Hemocyanin isoforms: RtH1 and RtH22006In: Proc 16. International Microscopy Conference, 2006Conference paper (Refereed)
  • 8.
    Cheng, Kimberley
    et al.
    Department of Biosciences at NOVUM, Karolinska Institutet and School of Technology and Health, Royal Institute of Technology, S-141 57 Huddinge, Sweden.
    Koeck, Philip J. B.
    Department of Biosciences at NOVUM, Karolinska Institutet and School of Technology and Health, Royal Institute of Technology, S-141 57 Huddinge, Sweden.
    Elmund, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Ternström, Tomas
    Schwarz, Heinz
    Idakieva, Krassimira
    Parvanova, Katja
    Rapana thomasiana hemocyanin (RtH): Comparison of the two isoforms, RtH1 and RtH2, at 19 Å and 16 Å resolution2006In: Micron, ISSN 0968-4328, E-ISSN 1878-4291, Vol. 37, no 6, p. 566-576Article in journal (Refereed)
    Abstract [en]

    Three-dimensional (3D) reconstructions of the two 8.4 MDa Rapana thomasiana hemocyanin isoforms, RtH1 and RtH2, have been obtained by cryoelectron microscopy of molecules embedded in vitreous ice and single particle image processing. The final 3D structures of the RtH1 and RtH2 didecamers at 19 angstrom and 16 angstrom resolution, respectively, are very similar to earlier reconstructions of gastropodan hemocyanins, revealing structural features such as the obliquely oriented subunits, the five- and two-fold symmetrical axes. Three new interactions are defined; two of them connecting the arch and the wall while the third is formed between the collar and the wall. The collar-wall connection and one of the arch-wall connections are positioned between two individual subunit dimers, while the second arch-wall connection is located between two subunits within the subunit dimer. All three interactions establish connections to the first tier of the wall. Furthermore, for each interaction we have allocated two first tier functional units most likely involved in forming the connections.

  • 9.
    Elmlund, Dominika
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Towards unbiased 3D reconstruction: in single-particle cryo-electron microscopy2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Cryo-electron microscopy of freestanding molecules (single-particles) plays a pivotal role in the difficult and pressing challenge of determining the structures of large macromolecular complexes. Molecular volumes are generated by aligning large sets of randomly oriented two-dimensional (2D) projection images in three dimensions (3D) before reconstruction is performed using tomographic techniques. The increasing popularity of the single-particle method is highly correlated with technical advances in instrumentation and computation. This thesis introduces new computational methods for 3D structure determination from electron microscopic projection images of single molecules. The algorithms have been developed to fill a gap in the single particle methodology – the lack of methods for ab initio 3D reconstruction of asymmetrical or low-symmetry molecules co-existing in different functional states. The proposed approach does not rely on a priori information about the structure or the character of the sample heterogeneity, which minimizes template dependence and makes the methods applicable to a wide range of single molecules. The presented algorithms constitute the basis of a new open source software package - SIMPLE (Single-particle IMage Processing Linux Engine). SIMPLE is an efficient and easy-to-use image processing system for semi-automated ab initio 3D reconstruction from challenging single-particle data sets (asymmetrical particles, significant degree of heterogeneity).

  • 10.
    Elmlund, Dominika
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Davis, Ralph
    Elmlund, Hans
    Ab initio structure determination from electron microscopic images of single molecules coexisting in different functional states2010In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 18, no 7, p. 777-786Article in journal (Refereed)
    Abstract [en]

    We have developed methods for ab initio three-dimensional (3D) structure determination from projection images of randomly oriented single molecules coexisting in multiple functional states, to aid the study of complex samples of macromolecules and nanoparticles by electron microscopy (EM). New algorithms for the determination of relative 3D orientations and conformational state assignment of single-molecule projection images are combined with well-established techniques for alignment and statistical image analysis. We describe how the methodology arrives at homogeneous groups of images aligned in 3D and discuss application to experimental EM data sets of the Escherichia coli ribosome and yeast RNA polymerase II.

  • 11.
    Elmlund, Dominika
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Elmlund, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    High-resolution single-particle orientation refinement based on spectrally self-adapting common lines2009In: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 167, no 1, p. 83-94Article in journal (Refereed)
    Abstract [en]

    Three-dimensional (3D) structure determination from electron microscopic images of single molecules can be difficult for particles with low or no internal symmetry, and for images with low signal-to-noise ratio (SNR), due to the existence of false maxima in the scoring function used for orientation search. In attempt to improve robustness of orientation parameter refinement towards noise and poor starting reconstruction quality, we have developed a method for common lines-based orientation search in Fourier space. The Fourier-space formulation enables inclusion of resolution (spatial frequency of the low-pass limit) as a variable that is adjusted in a particle-dependent, self-adaptive manner. The method allows for the underlying 3D structure to be estimated to high resolution, and requires only a crude, low-resolution reconstruction as starting-point for refinement. Benchmarking of the method is performed on experimental and synthetic data.

  • 12.
    Elmlund, Dominika
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Elmlund, Hans
    SIMPLE: an image processing system for ab initio 3D reconstruction in single-particleelectron microscopyManuscript (preprint) (Other academic)
  • 13.
    Elmlund, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Protein structure dynamics and interplay: by single-particle electron microscopy2008Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    Single-particle cryo-electron microscopy (cryo-EM) is a method capable of obtaining information about the structural organization and dynamics of large macromolecular assemblies. In the late nineties, the method was suggested to have the potential of generating “atomic resolution” reconstructions of particles above a certain mass. However, visualization of secondary structure elements in cryo-EM reconstructions has so far been achieved mainly for highly symmetrical macromolecular assemblies or by using previously existing X-ray structures to solve the initial alignment problem. A factor that severely limits the resolution for low-symmetry (point group symmetry Cn) particles is the problem of ab initio three-dimensional alignment of cryo-EM projection images of proteins in vitreous ice.

    A more general problem in the field of molecular biology is the study of heterogeneous structural properties of particles in preparations of purified macromolecular complexes. If not resolved, structural heterogeneity limits the achievable resolution of a cryo-EM reconstruction and makes correct biological interpretation difficult. If resolved, the heterogeneity instead offers a tremendous biological insight into the dynamic behaviour of a structure, and statistical information about partitioning over subpopulations with distinct structural features within the ensemble of particles may be gained.

    This thesis adds to the existing body of methods in the field of single-particle cryo-EM by addressing the problem of ab initio rotational alignment and the problem of resolving structural heterogeneity without using a priori information about the structural variability within large populations of cryo-EM projections of unstained proteins. The thesis aims at making the single-particle cryo-EM method a generally applicable tool for generating subnanometer resolution reconstructions and perform heterogeneity analysis of biological macromolecules.

  • 14.
    Elmlund, Hans
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Baraznenok, Vera
    Division of Metabolic Diseases, Karolinska Institutet.
    Lindahl, Martin
    Department of Molecular Biophysics, Lund University.
    Samuelsen, Camilla O.
    Department of Genetics, Institute of Molecular Biology, Copenhagen.
    Koeck, Philip J. B.
    KTH, School of Technology and Health (STH).
    Holmberg, Steen
    Department of Genetics, Institute of Molecular Biology, Copenhagen.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Gustafsson, Claes M.
    Division of Metabolic Diseases, Karolinska Institutet.
    The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II2006In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 103, no 43, p. 15788-15793Article in journal (Refereed)
    Abstract [en]

    CDK8 (cyclin-dependent kinase 8), along with CycC, Med12, and Med13, form a repressive module (the Cdk8 module) that prevents RNA polymerase II (pol II) interactions with Mediator. Here, we report that the ability of the Cdk8 module to prevent pol II interactions is independent of the Cdk8-dependent kinase activity. We use electron microscopy and single-particle reconstruction to demonstrate that the Cdk8 module forms a distinct structural entity that binds to the head and middle region of Mediator, thereby sterically blocking interactions with pol II.

  • 15.
    Elmlund, Hans
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Baraznenok, Vera
    Division of Metabolic Diseases, Karolinska Institutet.
    Linder, Tomas
    Division of Metabolic Diseases, Karolinska Institutet.
    Rofougaran, Reza
    Dept. of Medical Biochemistry and Biophysics, Umeå University.
    Hofer, Anders
    Dept. of Medical Biochemistry and Biophysics, Umeå University.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Lindahl, Martin
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Gustafsson, Claes M.
    Dept. of Medical Biochemistry and Cell Biology, Göteborg University.
    Visualization of a massive TBP-binding coupled histone-fold domain rearrangement within the general transcription factor IIDArticle in journal (Other academic)
  • 16. Elmlund, Hans
    et al.
    Baraznenok, Vera
    Linder, Tomas
    Szilagyi, Zsolt
    Rofougaran, Reza
    Hofer, Anders
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Lindahl, Martin Joakim
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Gustafsson, Claes M.
    Cryo-EM Reveals Promoter DNA Binding and Conformational Flexibility of the General Transcription Factor TFIID2009In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 17, no 11, p. 1442-1452Article in journal (Refereed)
    Abstract [en]

    The general transcription factor IID (TFIID) is required for initiation of RNA polymerase II-dependent transcription at many eukaryotic promoters. TFIID comprises the TATA-binding protein (TBP) and several conserved TBP-associated factors (TAFs). Recognition of the core promoter by TFIID assists assembly of the preinitiation complex. Using cryo-electron microscopy in combination with methods for ab initio single-particle reconstruction and heterogeneity analysis, we have produced density maps of two conformational states of Schizosaccharomyces pombe TFIID, containing and lacking TBP. We report that TBP-binding is coupled to a massive histone-fold domain rearrangement. Moreover, docking of the TBP-TAF1(N-terminus) atomic structure to the THID map and reconstruction of a TAF-promoter DNA complex helps to account for TAF-dependent regulation of promoter-TBP and promoter-TAF interactions.

  • 17.
    Elmlund, Hans
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Lundqvist, Joakirn
    Lund Univ, Dept Mol Biophys.
    Al-Karadaghi, Salam
    Lund Univ, Dept Mol Biophys.
    Hansson, Mats
    Lund Univ, Dept Biochem.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Lindahl, Martin
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    A new cryo-EM single-particle ab initio reconstruction method visualizes secondary structure elements in an ATP-fueled AAA+ motor2008In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 375, no 4, p. 934-947Article in journal (Refereed)
    Abstract [en]

    The generation of ab initio three-dimensional (3D) models is a bottleneck in the studies of large macromolecular assemblies by single-particle cryo-electron microscopy. We describe here a novel method, in which established methods for two-dimensional image processing are combined with newly developed programs for joint rotational 3D alignment of a large number of class averages (RAD) and calculation of 3D volumes from aligned projections (VolRec). We demonstrate the power of the method by reconstructing an similar to 660-kDa ATP-fueled AAA+ motor to 7.5 angstrom resolution, with secondary structure elements identified throughout the structure. We propose the method as a generally applicable automated strategy to obtain 3D reconstructions from unstained single particles imaged in vitreous ice.

  • 18. 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.

  • 19.
    Hebert, Hans
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Jegerschold, Caroline
    The structure of membrane associated proteins in eicosanoid and glutathione metabolism as determined by electron crystallography2007In: Current opinion in structural biology, ISSN 0959-440X, E-ISSN 1879-033X, Vol. 17, no 4, p. 396-404Article, review/survey (Refereed)
    Abstract [en]

    Membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG) are involved in biosynthesis of arachidonic-derived mediators of pain, fever, and inflammation as well as in biotransformation and detoxification of electrophilic substances. Structure determination of microsomal glutathione transferase 1 using electron crystallography has provided the first atomic model of an MAPEG member. The homotrimer consists of three repeats of a four-helix transmembrane bundle with the largest extramembranous domain connecting the first and second helix and with a short proline rich loop on the same side between helices three and four. Residues of importance for intramolecular or intermolecular contacts as well as for stabilizing the active site have been identified and the results can be applied for interpreting structure-function relationship for similar MAPEG members.

  • 20. Holm, Peter J.
    et al.
    Bhakat, Priyaranjan
    Jegerschold, Caroline
    Gyobu, Nobuhiko
    Mitsuoka, Kaoru
    Fujiyoshi, Yoshinori
    Morgenstern, Ralf
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Structural basis for detoxification and oxidative stress protection in membranes2006In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 360, no 5, p. 934-945Article in journal (Refereed)
    Abstract [en]

    Synthesis of mediators of fever, pain and inflammation as well as protection against reactive molecules and oxidative stress is a hallmark of the MAPEG superfamily (membrane associated proteins in eicosanoid and glutathione metabolism). The structure of a MAPEG member, rat mictosomal glutathione transferase 1, at 3.2 angstrom resolution, solved here in complex with glutathione by electron crystallography, defines the active site location and a cytosolic domain involved in enzyme activation. The glutathione binding site is found to be different from that of the canonical soluble glutathione transferases. The architecture of the homotrimer supports a catalytic mechanism involving subunit interactions and reveals both cytosolic and membraneous substrate entry sites, providing a rationale for the membrane location of the enzyme.

  • 21.
    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.

  • 22.
    Koeck, Philip J. B.
    et al.
    Karolinska Institutet.
    Purhonen, P.
    Alvang, R.
    Grundberg, B.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    3D-correlation-averaging for membrane-protein-crystals2008In: EMC 2008 14th European Microscopy Congress, 2008, p. 55-56Conference paper (Refereed)
    Abstract [en]

    Few 2-dimensional protein crystals can be used to determine high-resolution structures, whereas most electron crystallography projects remain at a resolution around 10 Ångström. This might be partly due to lack of flatness of many two-dimensional crystals [1]. We have investigated this problem and suggest single particle projection matching (3D-correlation averaging) of locally averaged unit cells to improve the quality of three-dimensional maps. Theoretical considerations and tests on simulated data demonstrate the feasibility of this refinement method [2].

  • 23. Koeck, Philip J. B.
    et al.
    Purhonen, P
    Alvang, R
    Grundberg, B
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Single-particle refinement in electron crystallography of membrane-proteins2007Conference paper (Refereed)
  • 24. Kumar, Ramakrishnan B.
    et al.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Jegerschold, Caroline
    Deciphering the Interaction of FLAP and 5LO2013In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 104, no 2, p. 540A-540AArticle in journal (Other academic)
  • 25.
    Köck, Philip J. B.
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology (Closed 20130701).
    Purhonen, Pasi
    Alvang, Ronny
    KTH, School of Technology and Health (STH).
    Grundberg, Björn
    KTH, School of Technology and Health (STH), Structural Biotechnology (Closed 20130701).
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology (Closed 20130701).
    Single particle refinement in electron crystallography: A pilot study2007In: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 160, no 3, p. 344-352Article in journal (Refereed)
    Abstract [en]

    Electron crystallography can be used to determine the structures of membrane proteins at near-atomic resolution in some cases. However, most electron crystallography projects remain at a resolution around 10 angstrom. This might be partly due to lack of flatness of many two-dimensional crystals. We have investigated this problem and suggest single particle processing of locally averaged unit cells to improve the quality and possibly the resolution of three-dimensional maps. Applying this method to the secondary transporter melibiose permease we have calculated a three-dimensional map that is clearer and easier to interpret than the map derived using purely electron-crystallographic methods.

  • 26.
    Lindahl, Martin
    KTH, School of Technology and Health (STH), Structural Biotechnology (Closed 20130701).
    tmRNA to the rescue: Structural motives for the salvage of stalled ribosomes2010In: RNA Biology, ISSN 1547-6286, Vol. 7, no 5, p. 577-581Article in journal (Other academic)
    Abstract [en]

    During translation, mRNA molecules are incidentally damaged, leaving the ribosome unable to reach or recognize the stop codon and thus stalled with mRNA and a potentially harmful polypeptide product attached to tRNA in the ribosomal P-site. In bacteria, a process called trans-translation has evolved, where a protein-RNA complex (smpB-tmRNA) mimicks the role of aminoacyl charged tRNA in the ribosomal A-site. The ribosome then resumes protein synthesis guided by an mRNA-like portion of the tmRNA which ends with a stop codon and codes for a peptide sequence susceptible to proteolysis, thus allowing the bacteria to salvage stalled ribosomes and degrade ill-defined and potentially harmful protein products. In this article, we will recollect how structural studies have yielded a model for how the pre-translocation stages of trans-translation employing structural mimicry. We will also discuss possible models for

  • 27.
    Lundbäck, Anna-Karin
    et al.
    KTH, School of Technology and Health (STH).
    Mueller, Shirley A.
    Engel, Andreas
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology (Closed 20130701).
    Assembly of Kch, a putative potassium channel from Escherichia coli2009In: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 168, no 2, p. 288-293Article in journal (Refereed)
    Abstract [en]

    Attempts to explore the structure and function of Kch, a putative potassium channel of Escherichia coli have yielded varying results; potassium-associated functions have been found in vivo but not in vitro. Here the kch gene is shown to produce two proteins, full-length Kch and the large C-terminal cytosolic domain (the RCK domain). Further, these two proteins are associated at the initial stages of purification. Previous structural studies of full-length Kch claim that the isolated protein forms large aggregates that are not suitable for analysis. The results presented here show that the purified protein sample, although heterogeneous, has one major population with a mass of about 400 kDa, implying the presence of two Kch tetramers in a complex form. A three dimensional reconstruction at 25 angstrom based on electron microscopy data from negatively stained particles, revealed a 210 angstrom long and 95 angstrom wide complex in which the two tetrameric Kch units are linked by their RCK domains, giving rise to a large central ring of density. The formation of this dimer of tetramers on expression or during purification, may explain why attempts to reconstitute Kch into liposomes for activity measurements have failed.

  • 28. Lundbäck, Anna-Karin
    et al.
    van den Berg, Susanne
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Berglund, Helena
    Eshaghi, Said
    Exploring the activity of tobacco etch virus protease in detergent solutions2008In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 382, no 1, p. 69-71Article in journal (Refereed)
    Abstract [en]

    Tobacco etch virus (TEV) protease is generally used to remove affinity tags from target proteins. It has been reported that some detergents inhibit the activity of this protease, and therefore should be avoided when removing affinity tags from membrane proteins. The aim of this study was to explore and evaluate this further. Hence, affinity tag removal with TEV protease was tested from three membrane proteins (a Pgp synthase and two CorA homologs) in the presence of 16 different detergents commonly used in membrane protein purification and crystallization. We observed that in the presence of the same detergent (Triton X-100), TEV protease could remove the affinity tag completely from one protein (CorA) but not from another protein (Pgp synthase). There was also a large variation in yield of cleaved membrane protein in different detergents, which probably depends on features of the protein-detergent complex. These observations show that, contrary to an earlier report, detergents do not inhibit the enzymatic activity of the TEV protease.

  • 29.
    Lundqvist, Joakim
    et al.
    Dept. of Molecular Biophysics, Lund University.
    Elmlund, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Kasperska, Dominika
    Division of Metabolic Diseases, Karolinska Institutet.
    Axelsson, Eva
    Dept. of Biochemistry, Lund University.
    Sirijovski, Nick
    Dept. of Biochemistry, Lund University.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Willows, Robert
    Department of Chemistry and Biomolecular Sciences, Macquarie University.
    Hansson, Mats
    Dept. of Biochemistry, Lund University.
    Lindahl, Martin
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Al-Karadaghi, Salam
    Dept. of Molecular Biophysics, Lund University.
    Cryo-electron microscopy reveals an ATP-fueled and Integrin-I mediated conformational transition of the AAA+ activation complex in R. capsulatus Mg-chelataseManuscript (Other academic)
  • 30. Lundqvist, Joakim
    et al.
    Elmlund, Hans
    KTH, School of Technology and Health (STH).
    Wulff, Ragna Peterson
    Berglund, Lisa
    Elmlund, Dominika
    KTH, School of Technology and Health (STH).
    Emanuelsson, Cecilia
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Willows, Robert D.
    Hansson, Mats
    Lindahl, Martin
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Al-Karadaghi, Salam
    ATP-Induced Conformational Dynamics in the AAA plus Motor Unit of Magnesium Chelatase2010In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 18, no 3, p. 354-365Article in journal (Refereed)
    Abstract [en]

    Mg-chelatase catalyzes the first committed step of the chlorophyll biosynthetic pathway, the ATP-dependent insertion of Mg2+ into protoporphyrin IX (PPIX). Here we report the reconstruction using single-particle cryo-electron microscopy of the complex between subunits BchD and BchI of Rhodobacter capsulatus Mg-chelatase in the presence of ADP, the nonhydrolyzable ATP analog AMPPNP, and ATP at 7.5 angstrom, 14 angstrom, and 13 angstrom resolution, respectively. We show that the two AAA+ modules of the subunits form a unique complex of 3 dimers related by a three-fold axis. The reconstructions demonstrate substantial differences between the conformations of the complex in the presence of ATIP and ADP, and suggest that the C-terminal integrin-I domains of the BchD subunits play a central role in transmitting conformational changes of BchI to BchD. Based on these data a model for the function of magnesium chelatase is proposed.

  • 31. 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.

  • 32. Petrlova, Jitka
    et al.
    Zhu, Lin
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Morgelin, Matthias
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Jegerschold, Caroline
    Voss, John C.
    Lagerstedt, Jens O.
    Structural properties of functional HDL and variants of apoA-I2012In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 26Article in journal (Other academic)
  • 33. 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)
  • 34. Purhonen, P.
    et al.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Thomsen, K.
    Maunsbach, A. B.
    Structure of Na, K-ATPase as analyzed by cryo-electron microscopy2005In: J. Gen. Physiol., 2005Conference paper (Refereed)
  • 35. Purhonen, P
    et al.
    Koeck, Philip J. B.
    Thomsen, K
    Maunsbach, AB
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Cryo-EM studies of renal Na,K-ATPase in native membranes2008In: Proc of the 12th International ATPase Conference. Na,K-ATPase and related transport ATPases of P-type, 2008Conference paper (Refereed)
  • 36. Purhonen, P
    et al.
    Lundbäck, AK
    Lemonier, R
    Leblanc, G
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Three-dimensional Structure of the Sugar Symporter Melibiose Permease from Cryo-electron Microscopy2005In: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 152, p. 76-83Article in journal (Refereed)
    Abstract [en]

    Melibiose permease (MelB) of Escherichia coli is a secondary transporter that couples the uptake of melibiose and various other galactosides to symport of cations that can be Na+, Li+ or H+. MelB belongs to the glycoside-pentoside-hexuronide: cation symporter family of porters and is suggested to have 12 transmembrane helices. We have determined the three-dimensional structure of MelB at 10 angstrom resolution in the membrane plane with cryo-electron microscopy from two-dimensional crystals. The three-dimensional map shows a heart-shaped molecule composed of two domains with a large central cavity between them. The structure is constricted at one side of the membrane while it is open to the other. The overall molecular shape resembles those of lactose permease and glycerol-3-phosphate transporter. However, organization of helices in MOB seems less symmetrical than in these two members of the major facilitator superfamily.

  • 37. Purhonen, P
    et al.
    Thomsen, K
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Maunsbach, AB
    The structure of Na,K-ATPase in native pig kidney membranes2006In: Proceedings of the 16th International Microscopy Congress, 2006Conference paper (Refereed)
  • 38. Purhonen, P
    et al.
    Thomsen, K
    Koeck, Philip J. B.
    Maunsbach, AB
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Three-dimensional structure of renal Na,K-ATPase as determined by cryo-electron microscopy2007Conference paper (Refereed)
  • 39. Purhonen, P.
    et al.
    Thomsen, K.
    Maunsbach, A. B.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Association of renal Na,K-ATPase alpha-subunit with the beta- and gamma-subunits based on cryoelectron microscopy2006In: Journal of Membrane Biology, ISSN 0022-2631, E-ISSN 1432-1424, Vol. 214, no 3, p. 139-146Article in journal (Refereed)
    Abstract [en]

    Na,K-ATPase transports Na+ and K+ across cell membranes and consists of alpha- and beta-subunits. Na,K-ATPase also associates with small FXYD proteins that regulate the activity of the pump. We have used cryoelectron microscopy of two-dimensional crystals including data to 8 A resolution to determine the three-dimensional (3-D) structure of renal Na,K-ATPase containing FXYD2, the gamma-subunit. A homology model for the a- subunit was calculated from a Ca2+-ATPase structure and used to locate the additional beta- and gamma-subunits present in the 3-D map of Na,K-ATPase. Based on the 3-D map, the beta- subunit is located close to transmembrane helices M8 and M10 and the gamma-subunit is adjacent to helices M2 and M9 of the alpha-subunit.

  • 40. Schagerlof, Ulrika
    et al.
    Wilson, Greer
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Al-Karadaghi, Salam
    Hagerhall, Cecilia
    Transmembrane topology of FRO2, a ferric chelate reductase from Arabidopsis thaliana2006In: Plant Molecular Biology, ISSN 0167-4412, E-ISSN 1573-5028, Vol. 62, no 02-jan, p. 215-221Article in journal (Refereed)
    Abstract [en]

    Iron uptake in Arabidopsis thaliana is mediated by ferric chelate reductase FRO2, a transmembrane protein belonging to the flavocytochrome b family. There is no high resolution structural information available for any member of this family. We have determined the transmembrane topology of FRO2 experimentally using the alkaline phosphatase fusion method. The resulting topology is different from that obtained by theoretical predictions and contains 8 transmembrane helices, 4 of which build up the highly conserved core of the protein. This core is present in the entire flavocytochrome b family. The large water soluble domain of FRO2, which contains NADPH, FAD and oxidoreductase sequence motifs, was located on the inside of the membrane.

  • 41. Schagerlöf, Ulrika
    et al.
    Elmlund, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Gakh, Oleksandr
    Nordlund, Gustav
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Lindahl, Martin
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Isaya, Grazia
    Al-Karadaghi, Salam
    Structural basis of the iron storage function of frataxin from single-particle reconstruction of the iron-loaded oligomer2008In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 17, p. 4948-4954Article in journal (Refereed)
    Abstract [en]

    The mitochondrial protein frataxin plays a central role in mitochondrial iron homeostasis, and frataxin deficiency is responsible for Friedreich ataxia, a neurodegenerative and cardiac disease that affects 1 in 40000 children. Here we present a single-particle reconstruction from cryoelectron microscopic images of iron-loaded 24-subunit oligomeric frataxin particles at 13 and 17 angstrom resolution. Computer-aided classification of particle images showed heterogeneity in particle size, which was hypothesized to result from gradual accumulation of iron within the core structure. Thus, two reconstructions were created from two classes of particles with iron cores of different sizes. The reconstructions show the iron core of frataxin for the first time. Compared to the previous reconstruction of iron-free particles from negatively stained images, the higher resolution of the present reconstruction allowed a more reliable analysis of the overall three-dimensional structure of the 24-meric assembly. This was done after docking the X-ray structure of the frataxin trimer into the EM reconstruction. The structure revealed a close proximity of the suggested ferroxidation sites of different monomers to the site proposed to serve in iron nucleation and mineralization. The model also assigns a new role to the N-terminal helix of frataxin in controlling the channel at the 4-fold axis of the 24-subunit oligomer. The reconstructions show that, together with some common features, frataxin has several unique features which distinguish it from ferritin. These include the overall organization of the oligomers, the way they are stabilized, and the mechanisms of iron core nucleation.

  • 42. Sirijovski, Nickolche
    et al.
    Lundqvist, Joakim
    Rosenback, Matilda
    Elmlund, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Al-Karadaghi, Salam
    Willows, Robert D.
    Hansson, Mats
    Substrate-binding model of the chlorophyll biosynthetic magnesium chelatase BchH subunit2008In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 17, p. 11652-11660Article in journal (Refereed)
    Abstract [en]

    Photosynthetic organisms require chlorophyll and bacteriochlorophyll to harness light energy and to transform water and carbon dioxide into carbohydrates and oxygen. The biosynthesis of these pigments is initiated by magnesium chelatase, an enzyme composed of BchI, BchD, and BchH proteins, which catalyzes the insertion of Mg2+ into protoporphyrin IX ( Proto) to produce Mg-protoporphyrin IX. BchI and BchD form an ATP-dependent AAA(+) complex that transiently interacts with the Proto-binding BchH subunit, at which point Mg2+ is chelated. In this study, controlled proteolysis, electron microscopy of negatively stained specimens, and single-particle three-dimensional reconstruction have been used to probe the structure and substrate-binding mechanism of the BchH subunit to a resolution of 25 angstrom. The apo structure contains three major lobeshaped domains connected at a single point with additional densities at the tip of two lobes termed the "thumb" and "finger." With the independent reconstruction of a substratebound BchH complex (BchH.Proto), we observed a distinct conformational change in the thumb and finger subdomains. Prolonged proteolysis of native apo-BchH produced a stable C-terminal fragment of 45 kDa, and Proto was shown to protect the full-length polypeptide from degradation. Fitting of a truncated BchH polypeptide reconstruction identified the Nand C-terminal domains. Our results show that the N- and C-terminal domains play crucial roles in the substrate-binding mechanism.

  • 43. Wulff, Ragna
    et al.
    Lundqvist, Joakim
    Rutsdottir, Gudrun
    Hansson, Andreas
    Stenbaek, Anders
    Elmlund, Dominika
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Elmlund, Hans
    Jensen, Paul Erik
    Hansson, Mats
    NADPH-dependent thioredoxin reductase C and its possible involvement in the cyclase reaction of the chlorophyll biosynthetic pathwayManuscript (preprint) (Other academic)
  • 44. Wulff, Ragna Peterson
    et al.
    Lundqvist, Joakim
    Rutsdottir, Gudrun
    Hansson, Andreas
    Stenbaek, Anne
    Elmlund, Dominika
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Elmlund, Hans
    Jensen, Poul Erik
    Hansson, Mats
    The Activity of Barley NADPH-Dependent Thioredoxin Reductase C Is Independent of the Oligomeric State of the Protein: Tetrameric Structure Determined by Cryo-Electron Microscopy2011In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 50, no 18, p. 3713-3723Article in journal (Refereed)
    Abstract [en]

    Thioredoxin and thioredoxin reductase can regulate cell metabolism through redox regulation of disulfide bridges or through removal of H2O2. These two enzymatic functions are combined in NADPH-dependent thioredoxin reductase C (NTRC), which contains an N-terminal thioredoxin reductase domain fused with a C-terminal thioredoxin domain. Rice NTRC exists in different oligomeric states, depending on the absence or presence of its NADPH cofactor. It has been suggested that the different oligomeric states may have diverse activity. Thus, the redox status of the chloroplast could influence the oligomeric state of NTRC and thereby its activity. We have characterized the oligomeric states of NTRC from barley (Hard rum vulgare L.). This also includes a structural model of the tetrameric NTRC derived from cryo-electron microscopy and single-particle reconstruction. We conclude that the tetrameric NTRC is a dimeric arrangement of two NTRC homodimers. Unlike that of rice NTRC, the quaternary structure of barley NTRC complexes is unaffected by addition of NADPH. The activity of NTRC was tested with two different enzyme assays. The N-terminal part of NTRC was tested in a thioredoxin reductase assay. A peroxide sensitive Mg-protoporphyrin IX monomethyl ester (MPE) cyclase enzyme system of the chlorophyll biosynthetic pathway was used to test the catalytic ability of both the N- and C-terminal parts of NTRC. The different oligomeric assembly states do not exhibit significantly different activities. Thus, it appears that the activities are independent of the oligomeric state of barley NTRC.

1 - 44 of 44
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