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  • 51. Duarte, Fernanda
    et al.
    Barrozo, Alexandre
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Åqvist, Johan
    Williams, Nicholas
    Kamerlin, Shina
    Mechanistic Shifts Along the Linear Free Energy Relationship for ArylPhosphate Monoester Hydrolysis.Manuscript (preprint) (Other academic)
  • 52.
    Duarte, Fernanda
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Williams, Nicholas H
    Kamerlin, Shina C Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Resolving apparent conflicts between theoretical and experimental models of phosphate monoester hydrolysis.2015In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 137, no 3, p. 1081-93Article in journal (Refereed)
    Abstract [en]

    Understanding phosphoryl and sulfuryl transfer is central to many biochemical processes. However, despite decades of experimental and computational studies, a consensus concerning the precise mechanistic details of these reactions has yet to be reached. In this work we perform a detailed comparative theoretical study of the hydrolysis of p-nitrophenyl phosphate, methyl phosphate and p-nitrophenyl sulfate, all of which have served as key model systems for understanding phosphoryl and sulfuryl transfer reactions, respectively. We demonstrate the existence of energetically similar but mechanistically distinct possibilities for phosphate monoester hydrolysis. The calculated kinetic isotope effects for p-nitrophenyl phosphate provide a means to discriminate between substrate- and solvent-assisted pathways of phosphate monoester hydrolysis, and show that the solvent-assisted pathway dominates in solution. This preferred mechanism for p-nitrophenyl phosphate hydrolysis is difficult to find computationally due to the limitations of compressing multiple bonding changes onto a 2-dimensional energy surface. This problem is compounded by the need to include implicit solvation to at least microsolvate the system and stabilize the highly charged species. In contrast, methyl phosphate hydrolysis shows a preference for a substrate-assisted mechanism. For p-nitrophenyl sulfate hydrolysis there is only one viable reaction pathway, which is similar to the solvent-assisted pathway for phosphate hydrolysis, and the substrate-assisted pathway is not accessible. Overall, our results provide a unifying mechanistic framework that is consistent with the experimentally measured kinetic isotope effects and reconciles the discrepancies between theoretical and experimental models for these biochemically ubiquitous classes of reaction.

  • 53. Dubey, Mukesh K.
    et al.
    Broberg, Anders
    Sooriyaarachchi, Sanjeewani
    Ubhayasekera, Wimal
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Jensen, Dan Funck
    Karlsson, Magnus
    The glyoxylate cycle is involved in pleotropic phenotypes, antagonism and induction of plant defence responses in the fungal biocontrol agent Trichoderma atroviride2013In: Fungal Genetics and Biology, ISSN 1087-1845, E-ISSN 1096-0937, Vol. 58-59, p. 33-41Article in journal (Refereed)
    Abstract [en]

    Isocitrate lyase (ICL), a signature enzyme of the glyoxylate cycle, is required for metabolism of non-fermentable carbon compounds like acetate or ethanol, and virulence in bacteria and fungi. In the present study, we investigate the role of the glyoxylate cycle in the fungal biocontrol agent Trichoderma atroviride by generating id deletion and complementation mutants. Phenotypic analyses of the deletion mutant Delta icl suggest that ICL is required for normal growth, conidial pigmentation and germination, and abiotic stress tolerance. The Delta icl strain display reduced antagonism towards Botrytis cinerea in plate confrontation assays. Secretion and sandwich assays further show that secreted factors are partly responsible for the reduced antagonism. Furthermore, in vitro root colonization assays shows that the Delta icl strain retains the ability to internally colonize Arabidopsis thaliana roots. However, the Delta icl strain has a reduced ability to induce systemic defence in A. thaliana leaves that results in reduced protection against B. cinerea. These data shows that ICL and the glyoxylate cycle are important for biocontrol traits in T. atroviride, including direct antagonism and induction of defence responses in plants.

  • 54.
    Eckhard, Ulrich
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Univ British Columbia, Fac Dent, Dept Oral Biol & Med Sci, Inst Life Sci, 2350 Hlth Sci Mall, Vancouver, BC V6T 1Z3, Canada.
    Bandukwala, Hina
    Univ Waterloo, Dept Biol, 200 Univ Ave West, Waterloo, ON N2L 3G1, Canada..
    Mansfield, Michael J.
    Univ Waterloo, Dept Biol, 200 Univ Ave West, Waterloo, ON N2L 3G1, Canada..
    Marino, Giada
    Univ British Columbia, Fac Dent, Dept Oral Biol & Med Sci, Inst Life Sci, 2350 Hlth Sci Mall, Vancouver, BC V6T 1Z3, Canada..
    Cheng, Jiujun
    Univ Waterloo, Dept Biol, 200 Univ Ave West, Waterloo, ON N2L 3G1, Canada..
    Wallace, Iain
    Univ Waterloo, Dept Biol, 200 Univ Ave West, Waterloo, ON N2L 3G1, Canada..
    Holyoak, Todd
    Univ Waterloo, Dept Biol, 200 Univ Ave West, Waterloo, ON N2L 3G1, Canada..
    Charles, Trevor C.
    Univ Waterloo, Dept Biol, 200 Univ Ave West, Waterloo, ON N2L 3G1, Canada..
    Austin, John
    Hlth Canada, Hlth Prod & Food Branch, Bur Microbial Hazards, Ottawa, ON K1A 0K9, Canada..
    Overall, Christopher M.
    Univ British Columbia, Fac Dent, Dept Oral Biol & Med Sci, Inst Life Sci, 2350 Hlth Sci Mall, Vancouver, BC V6T 1Z3, Canada..
    Doxey, Andrew C.
    Univ Waterloo, Dept Biol, 200 Univ Ave West, Waterloo, ON N2L 3G1, Canada..
    Discovery of a proteolytic flagellin family in diverse bacterial phyla that assembles enzymatically active flagella2017In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 8, article id 521Article in journal (Refereed)
    Abstract [en]

    Bacterial flagella are cell locomotion and occasional adhesion organelles composed primarily of the polymeric protein flagellin, but to date have not been associated with any enzymatic function. Here, we report the bioinformatics-driven discovery of a class of enzymatic flagellins that assemble to form proteolytically active flagella. Originating by a metallopeptidase insertion into the central flagellin hypervariable region, this flagellin family has expanded to at least 74 bacterial species. In the pathogen, Clostridium haemolyticum, metallopeptidase-containing flagellin (which we termed flagellinolysin) is the second most abundant protein in the flagella and is localized to the extracellular flagellar surface. Purified flagellar filaments and recombinant flagellin exhibit proteolytic activity, cleaving nearly 1000 different peptides. With similar to 20,000 flagellin copies per similar to 10-mu m flagella this assembles the largest proteolytic complex known. Flagellum-mediated extracellular proteolysis expands our understanding of the functional plasticity of bacterial flagella, revealing this family as enzymatic biopolymers that mediate interactions with diverse peptide substrates.

  • 55.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Protein synthesis: Translocation in slow motion2010In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 466, no 7304, p. 325-326Article in journal (Refereed)
  • 56.
    Ehrenberg, Måns
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Bremer, Hans
    Dennis, Patrick P.
    Medium-dependent control of the bacterial growth rate2013In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 95, no 4, p. 643-658Article, review/survey (Refereed)
    Abstract [en]

    By combining results from previous studies of nutritional up-shifts we here re-investigate how bacteria adapt to different nutritional environments by adjusting their macromolecular composition for optimal growth. We demonstrate that, in contrast to a commonly held view the macromolecular composition of bacteria does not depend on the growth rate as an independent variable, but on three factors: (i) the genetic background (i.e. the strain used), (ii) the physiological history of the bacteria used for inoculation of a given growth medium, and (iii) the kind of nutrients in the growth medium. These factors determine the ribosome concentration and the average rate of protein synthesis per ribosome, and thus the growth rate. Immediately after a nutritional up-shift, the average number of ribosomes in the bacterial population increases exponentially with time at a rate which eventually is attained as the final post-shift growth rate of all cell components. After a nutritional up-shift from one minimal medium to another minimal medium of higher nutritional quality, ribosome and RNA polymerase syntheses are co-regulated and immediately increase by the same factor equal to the increase in the final growth rate. However, after an up-shift from a minimal medium to a medium containing all 20 amino acids, RNA polymerase and ribosome syntheses are no longer coregulated; a smaller rate of synthesis of RNA polymerase is compensated by a gradual increase in the fraction of free RNA polymerase, possibly due to a gradual saturation of mRNA promoters. We have also analyzed data from a recent publication, in which it was concluded that the macromolecular composition in terms of RNA/protein and RNA/DNA ratios is solely determined by the effector molecule ppGpp. Our analysis indicates that this is true only in special cases and that, in general, medium adaptation also depends on factors other than ppGpp.

  • 57.
    Ehrenberg, Måns
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Pavlov, Michael Yu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Optimal Strategy for Rapid Proteome Re-Arrangements in Bacterial Populations2013In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 104, no 2, p. 205A-206AArticle in journal (Other academic)
  • 58.
    Erdélyi, András
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    HisA mutants with minor structural differences display major functional deviations2016Independent thesis Advanced level (degree of Master (Two Years)), 40 credits / 60 HE creditsStudent thesis
    Abstract [en]

    Even though enzymes tend to specialize on one reaction during evolution, enzyme promiscuity is an abundant phenomenon. The subject of this thesis is the Salmonella enterica N’-[(5’-phosphosoribosyl)-formimino]-5-aminoimidazole-4 carboxamide-ribonucleotide (ProFAR) isomerase (SeHisA), a (βα)8-barrel enzyme from the histidine biosynthesis that catalyzes one reaction on one substrate in one organism. In Actinobacteria HisA has evolved to a bifunctional enzyme called phosphoribosyl isomerase A (PriA): it is capable of catalyzing the reaction normally done by the N’-(5’-phosphoribosyl) anthranilate (PRA) isomerase (TrpF) as well. The functional plasticity of PriA is possible due to the common reaction mechanism of HisA and TrpF, called Amadori rearrangement. The Amadori rearrangement is an acid-base catalyzed isomerization reaction where the aminoaldose (ProFAR or PRA) is converted into the corresponding ketose (PRFAR or CdRP). A SeHisA mutant with a glutamine to arginine mutation in position 18 (Q18R) shows a detectable TrpF activity, whereas another mutant with a duplication of residues from 13 to 15 (dup13-15) loses its HisA activity and gains TrpF activity.

    My first aim was to improve the TrpF activity of the Q18R mutant. A G79S mutation was introduced inspired by PriA. Proteins were purified and crystallized. In order to gain complex structures with either the TrpF reaction product analogue reduced CdRP (rCdRP) or ProFAR, co-crystallization and soaking were done. ProFAR is not commercially available and had to be synthetized and purified. In vitro TrpF activities of the Q18R and Q18R/G79S mutants were measured. My second aim was to compare the Q18R mutant with the dup13-15 mutant, since there is very little structural difference between them, but they show high difference in catalytic activity. Mutants, which would bridge the functional gap between them, were designed and by using lambda red recombineering were introduced into a Salmonella typhimurium genome. In vivo growth rate was measured and relative fitness was calculated for each mutant in respect to their HisA and TrpF activity.

    HisA mutants Q18R and Q18R/G79S showed very poor TrpF activity in in vitro assays. No dissociation constants could be measured for either of the mutants using microscale thermophoresis, and a very low kcat/KM value (~2 s-1M-1) with a high error rate could be determined for Q18R/G79S. Complex structures of Q18R and Q18R/G79S mutants with ProFAR were solved at a 2.47 Å and a 1.78 Å, respectively, from soaked crystals. No structure with rCdRP was obtained. Growth rate measurements in comparison with a strain with wild type HisA and TrpF, gave striking results pointing out the important role of the residue in the position 16 when three residues are inserted after the arginine in position 18. A leucine in position 16 yielded wild type HisA activity (94%) and poor TrpF activity (0-5%), with a valine, no HisA activity (0%) and a moderate TrpF activity (38-46%) were found compared to the wild type. These results pointed out how small the barrier between a specialist, a promiscuous and a bifunctional enzyme can be. SeHisA, being a specialized enzyme can easily be modified in order to gain TrpF function, and as I have showed in the present study, a single methyl group (the difference between a leucine and a valine) can turn the activity of an enzyme inside out.

  • 59.
    Fange, David
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Lovmar, Martin
    Pavlov, Michael Y.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Identification of enzyme inhibitory mechanisms from steady-state kinetics2011In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 93, no 9, p. 1623-1629Article in journal (Refereed)
    Abstract [en]

    Enzyme inhibitors are used in many areas of the life sciences, ranging from basic research to the combat of disease in the clinic. Inhibitors are traditionally characterized by how they affect the steady-state kinetics of enzymes, commonly analyzed on the assumption that enzyme-bound and free substrate molecules are in equilibrium. This assumption, implying that an enzyme-bound substrate molecule has near zero probability to form a product rather than dissociate, is valid only for very inefficient enzymes. When it is relaxed, more complex but also more information-rich steady-state kinetics emerges. Although solutions to the general steady-state kinetics problem exist, they are opaque and have been of limited help to experimentalists. Here we reformulate the steady-state kinetics of enzyme inhibition in terms of new parameters. These allow for assessment of ambiguities of interpretation due to kinetic scheme degeneracy and provide an intuitively simple way to analyze experimental data. We illustrate the method by concrete examples of how to assess scheme degeneracy and obtain experimental estimates of all available rate and equilibrium constants. We suggest simple, complementary experiments that can remove ambiguities and greatly enhance the accuracy of parameter estimation.

  • 60.
    Fange, David
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Mellenius, Harriet
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Dennis, Patrick P.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Thermodynamic Modeling of Variations in the Rate of RNA Chain Elongation of E-coli rrn Operons2014In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 106, no 1, p. 55-64Article in journal (Refereed)
    Abstract [en]

    Previous electron-microscopic imaging has shown high RNA polymerase occupation densities in the 16S and 23S encoding regions and low occupation densities in the noncoding leader, spacer, and trailer regions of the rRNA (rrn) operons in E. coli. This indicates slower transcript elongation within the coding regions and faster elongation within the noncoding regions of the operon. Inactivation of four of the seven rrn operons increases the transcript initiation frequency at the promoters of the three intact operons and reduces the time for RNA polymerase to traverse the operon. We have used the DNA sequence-dependent standard free energy variation of the transcription complex to model the experimentally observed changes in the elongation rate along the rrnB operon. We also model the stimulation of the average transcription rate over the whole operon by increasing rate of transcript initiation. Monte Carlo simulations, taking into account initiation of transcription, translocation, and backward and forward tracking of RNA polymerase, partially reproduce the observed transcript elongation rate variations along the rrn operon and fully account for the increased average rate in response to increased frequency of transcript initiation.

  • 61. Feng, Boya
    et al.
    Mandava, Chandra Sekhar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Guo, Qiang
    Wang, Jie
    Cao, Wei
    Li, Ningning
    Zhang, Yixiao
    Zhang, Yanqing
    Wang, Zhixin
    Wu, Jiawei
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Lei, Jianlin
    Gao, Ning
    Structural and Functional Insights into the Mode of Action of a Universally Conserved Obg GTPase2014In: PLoS biology, ISSN 1544-9173, E-ISSN 1545-7885, Vol. 12, no 5, p. e1001866-Article in journal (Refereed)
    Abstract [en]

    Obg proteins are a family of P-loop GTPases, conserved from bacteria to human. The Obg protein in Escherichia coli (ObgE) has been implicated in many diverse cellular functions, with proposed molecular roles in two global processes, ribosome assembly and stringent response. Here, using pre-steady state fast kinetics we demonstrate that ObgE is an anti-association factor, which prevents ribosomal subunit association and downstream steps in translation by binding to the 50S subunit. ObgE is a ribosome dependent GTPase; however, upon binding to guanosine tetraphosphate (ppGpp), the global regulator of stringent response, ObgE exhibits an enhanced interaction with the 50S subunit, resulting in increased equilibrium dissociation of the 70S ribosome into subunits. Furthermore, our cryo-electron microscopy (cryo-EM) structure of the 50S? ObgE? GMPPNP complex indicates that the evolutionarily conserved N-terminal domain (NTD) of ObgE is a tRNA structural mimic, with specific interactions with peptidyl-transferase center, displaying a marked resemblance to Class I release factors. These structural data might define ObgE as a specialized translation factor related to stress responses, and provide a framework towards future elucidation of functional interplay between ObgE and ribosome-associated (p) ppGpp regulators. Together with published data, our results suggest that ObgE might act as a checkpoint in final stages of the 50S subunit assembly under normal growth conditions. And more importantly, ObgE, as a (p) ppGpp effector, might also have a regulatory role in the production of the 50S subunit and its participation in translation under certain stressed conditions. Thus, our findings might have uncovered an under-recognized mechanism of translation control by environmental cues.

  • 62.
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Synthetic biology challenges long-held hypotheses in translation, codon bias and transcription2012In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 7, no 7, p. 835-845Article, review/survey (Refereed)
    Abstract [en]

    Synthetic biology is a powerful experimental approach, not only for developing new biotechnology applications, but also for testing hypotheses in basic biological science. Here, examples from our research using the best model system, Escherichia coli, are reviewed. New evidence drawn from synthetic biology has overturned several long-standing hypotheses regarding the mechanisms of transcription and translation: (i) all native aminoacyl-tRNAs are not equally efficient in translation at equivalent concentrations; (ii) accommodation is not always rate limiting in translation, and may not be for any aminoacyl-tRNA; (iii) proline is the only N-alkyl-amino acid in the genetic code not because of special suitability for protein structure, but because of its comparatively high nucleophilicity; (iv) the usages of most sense codons in E. coli do not correlate with cognate tRNA abundances and (v) class II transcriptional pausing and termination by T7 RNA polymerase cannot be assumed to occur in vivo based on in vitro data. Implications of these conclusions for the biotechnology field are discussed.

  • 63.
    Forster, Anthony C.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Lee, Sang Yup
    Editorial: NextGen SynBio has arrived...2012In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 7, no 7, p. 827-827Article in journal (Other academic)
  • 64.
    Fu, Ziao
    et al.
    Columbia Univ Coll Phys & Surg, Integrated Program Cellular Mol & Biomed Studies, 630 W 168th St, New York, NY 10032 USA..
    Kaledhonkar, Sandip
    Columbia Univ, Dept Biochem & Mol Biophys, 630 W 168th St, New York, NY 10027 USA..
    Borg, Anneli
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Sun, Ming
    Columbia Univ, Dept Biol Sci, New York, NY 10027 USA..
    Chen, Bo
    Columbia Univ, Dept Biol Sci, New York, NY 10027 USA..
    Grassucci, Robert A.
    Columbia Univ, Dept Biochem & Mol Biophys, 630 W 168th St, New York, NY 10027 USA.;Columbia Univ, Howard Hughes Med Inst, New York, NY 10032 USA..
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Frank, Joachim
    Columbia Univ, Dept Biochem & Mol Biophys, 630 W 168th St, New York, NY 10027 USA.;Columbia Univ, Dept Biol Sci, New York, NY 10027 USA.;Columbia Univ, Howard Hughes Med Inst, New York, NY 10032 USA..
    Key Intermediates in Ribosome Recycling Visualized by Time-Resolved Cryoelectron Microscopy2016In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 24, no 12, p. 2092-2101Article in journal (Refereed)
    Abstract [en]

    Upon encountering a stop codon on mRNA, polypeptide synthesis on the ribosome is terminated by release factors, and the ribosome complex, still bound with mRNA and P-site-bound tRNA (post-termination complex, PostTC), is split into ribosomal subunits, ready for a new round of translational initiation. Separation of post-termination ribosomes into subunits, or "ribosome recycling,'' is promoted by the joint action of ribosome-recycling factor (RRF) and elongation factor G (EF-G) in a guanosine triphosphate (GTP) hydrolysis-dependent manner. Here we used a mixing-spraying-based method of time-resolved cryo-electron microscopy (cryo-EM) to visualize the short-lived intermediates of the recycling process. The two complexes that contain (1) both RRF and EF-G bound to the PostTC or (2) deacylated tRNA bound to the 30S subunit are of particular interest. Our observations of the native form of these complexes demonstrate the strong potential of time-resolved cryo-EM for visualizing previously unobservable transient structures.

  • 65. Fullam, Elizabeth
    et al.
    Pojer, Florence
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Jones, T. Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Cole, Stewart T.
    Structure and function of the transketolase from Mycobacterium tuberculosis and comparison with the human enzyme2012In: Open Biology, ISSN 2046-2441, Vol. 2, p. 110026-Article in journal (Refereed)
    Abstract [en]

    The transketolase (TKT) enzyme in Mycobacterium tuberculosis represents a novel drug target for tuberculosis treatment and has low homology with the orthologous human enzyme. Here, we report on the structural and kinetic characterization of the transketolase from M. tuberculosis (TBTKT), a homodimer whose monomers each comprise 700 amino acids. We show that TBTKT catalyses the oxidation of donor sugars xylulose-5-phosphate and fructose-6-phosphate as well as the reduction of the acceptor sugar ribose-5-phosphate. An invariant residue of the TKT consensus sequence required for thiamine cofactor binding is mutated in TBTKT; yet its catalytic activities are unaffected, and the 2.5 angstrom resolution structure of full-length TBTKT provides an explanation for this. Key structural differences between the human and mycobacterial TKT enzymes that impact both substrate and cofactor recognition and binding were uncovered. These changes explain the kinetic differences between TBTKT and its human counterpart, and their differential inhibition by small molecules. The availability of a detailed structural model of TBTKT will enable differences between human and M. tuberculosis TKT structures to be exploited to design selective inhibitors with potential antitubercular activity.

  • 66.
    Gising, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Nilsson, Mikael T
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Odell, Luke R
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Yahiaoui, Samir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Lindh, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Iyer, Harini
    AstraZeneca India.
    Sinha, Achyut M
    AstraZeneca India.
    Srinivasa, Bachally R
    AstraZeneca India.
    Larhed, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Mowbray, Sherry L
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Karlén, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Trisubstituted Imidazoles as Mycobacterium tuberculosis Glutamine Synthetase Inhibitors2012In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 55, no 6, p. 2894-2898Article in journal (Refereed)
    Abstract [en]

    Mycobacterium tuberculosis glutamine synthetase (MtGS) is a promising target for antituberculosis drug discovery. In a recent high-throughput screening study we identified several classes of MtGS inhibitors targeting the ATP-binding site. We now explore one of these classes, the 2-tert-butyl-4,5-diarylimidazoles, and present the design, synthesis, and X-ray crystallographic studies leading to the identification of MtGS inhibitors with submicromolar IC(50) values and promising antituberculosis MIC values.

  • 67.
    Glas, Adrian
    et al.
    Max Planck Gesell, Chem Genom Ctr, Otto Hahn Str 15, D-44227 Dortmund, Germany..
    Wamhoff, Eike-Christian
    Max Planck Inst Colloids & Interfaces, Dept Biomol Syst, Muhlenberg 1, D-14424 Potsdam, Germany.;Free Univ Berlin, Dept Biol Chem & Pharm, Takustr 3, D-14195 Berlin, Germany..
    Krüger, Dennis M.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Max Planck Gesell, Chem Genom Ctr, Otto Hahn Str 15, D-44227 Dortmund, Germany..
    Rademacher, Christoph
    Max Planck Inst Colloids & Interfaces, Dept Biomol Syst, Muhlenberg 1, D-14424 Potsdam, Germany.;Free Univ Berlin, Dept Biol Chem & Pharm, Takustr 3, D-14195 Berlin, Germany..
    Grossmann, Tom N.
    Max Planck Gesell, Chem Genom Ctr, Otto Hahn Str 15, D-44227 Dortmund, Germany.;Vrije Univ Amsterdam, Dept Chem & Pharmaceut Sci, Boelelaan 1083, NL-1081 HV Amsterdam, Netherlands..
    Increased Conformational Flexibility of a Macrocycle-Receptor Complex Contributes to Reduced Dissociation Rates2017In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 23, no 64, p. 16157-16161Article in journal (Refereed)
    Abstract [en]

    Constraining a peptide in its bioactive conformation by macrocyclization represents a powerful strategy to design modulators of challenging biomolecular targets. This holds particularly true for the development of inhibitors of protein-protein interactions which often involve interfaces lacking defined binding pockets. Such flat surfaces are demanding targets for traditional small molecules rendering macrocyclic peptides promising scaffolds for novel therapeutics. However, the contribution of peptide dynamics to binding kinetics is barely understood which impedes the design process. Herein, we report unexpected trends in the binding kinetics of two closely related macrocyclic peptides that bind their receptor protein with high affinity. Isothermal titration calorimetry, F-19 NMR experiments and molecular dynamics simulations reveal that increased conformational flexibility of the macrocycle-receptor complex reduces dissociation rates and contributes to complex stability. This observation has impact on macrocycle design strategies that have so far mainly focused on the stabilization of bioactive ligand conformations.

  • 68.
    Guo, Xiaohu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Life will find a way: Structural and evolutionary insights into FusB and HisA2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    How do microbes adapt to challenges from the environment? In this thesis, two distinct cases were examined through structural and biochemical methods. In the first, we followed a real-time protein evolution of HisA to a novel function. The second case was fusidic acid (FA) resistance mediated by the protein FusB in Staphylococcus aureus.

    In the first study, the aim was to understand how mutants of HisA from the histidine biosynthetic pathway could evolve a novel TrpF activity and further evolve to generalist or specialist enzymes. We solved the crystal structure of wild type Salmonella enterica HisA in its apo-state and the structures of the mutants D7N and D7N/D176A in complex with the substrate ProFAR. These two distinct complex structures showed us the coupled conformational changes of HisA and ProFAR before catalysis. We also solved crystal structures of ten mutants, some in complex with substrate or product. The structures indicate that bi-functional mutants adopt distinct loop conformations linked to the two functions and that mutations in specialist enzymes favor one of the conformations. We also observed biphasic relationships in which small changes in the activities of low-performance enzymes had large effects on fitness, until a threshold, above which large changes in enzyme performance had little effect on fitness.

    Fusidic acid blocks protein translation by locking elongation factor G (EF-G) to the ribosome after GTP hydrolysis in elongation and recycling of bacterial protein synthesis. To understand the rescue mechanism, we solved the crystal structure of FusB at 1.6Å resolution. The structure showed that FusB is a two-domain protein and C-terminal domain contains a treble clef zinc finger. Using hybrid constructs between S. aureus EF-G that binds to FusB, and E. coli EF-G that does not, the binding determinants were located to domain IV of EF-G. This was further supported by small-angle X-ray scattering studies of the FusB·EF-G complex. Using single-molecule methods, we observed FusB frequently binding to the ribosome and rescue of FA-inhibited elongation by effects on the non-rotated state ribosome. Ribosome binding of FusB was confirmed by isothermal titration calorimetry.

    List of papers
    1. Two-step Ligand Binding in a (βα)8 Barrel Enzyme: Substrate-bound Structures Shed New Light on the Catalytic Cycle of HisA
    Open this publication in new window or tab >>Two-step Ligand Binding in a (βα)8 Barrel Enzyme: Substrate-bound Structures Shed New Light on the Catalytic Cycle of HisA
    Show others...
    2015 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 41, p. 24657-24668Article in journal (Refereed) Published
    Abstract [en]

    HisA is a (βα)8 barrel enzyme that catalyzes the Amadori rearrangement of ProFAR to PRFAR in the histidine biosynthesis pathway and it is a paradigm for the study of enzyme evolution. Still, its exact catalytic mechanism has remained unclear. Here, we present crystal structures of wild type Salmonella enterica HisA (SeHisA) in its apo state and of mutants D7N and D7N/D176A in complex with two different conformations of the labile substrate ProFAR, which was structurally visualized for the first time. Site-directed mutagenesis and kinetics demonstrated that Asp7 acts as the catalytic base and Asp176 as the catalytic acid. The SeHisA structures with ProFAR display two different states of the long loops on the catalytic face of the structure, and demonstrate that initial binding of ProFAR to the active site is independent of loop interactions. When the long loops enclose the substrate, ProFAR adopts an extended conformation where its non-reacting half is in a product-like conformation. This change is associated with shifts in a hydrogen-bond network including His47, Asp129, Thr171 and Ser202, all shown to be functionally important. The closed-conformation structure is highly similar to the bi-functional HisA homologue PriA in complex with PRFAR, thus proving that structure and mechanism are conserved between HisA and PriA. This study clarifies the mechanistic cycle of HisA and provides a striking example of how an enzyme and its substrate can undergo coordinated conformational changes before catalysis.

    National Category
    Structural Biology
    Identifiers
    urn:nbn:se:uu:diva-260701 (URN)10.1074/jbc.M115.678086 (DOI)000362598300003 ()26294764 (PubMedID)
    Funder
    Swedish Research CouncilSwedish Foundation for Strategic Research EU, FP7, Seventh Framework Programme, 283570
    Available from: 2015-08-23 Created: 2015-08-23 Last updated: 2018-11-22
    2. Structure and function of FusB: an elongation factor G-binding fusidic acid resistance protein active in ribosomal translocation and recycling
    Open this publication in new window or tab >>Structure and function of FusB: an elongation factor G-binding fusidic acid resistance protein active in ribosomal translocation and recycling
    Show others...
    2012 (English)In: Open Biology, ISSN 2046-2441, Vol. 2, p. 120016-Article in journal (Refereed) Published
    Abstract [en]

    Fusidic acid (FA) is a bacteriostatic antibiotic that locks elongation factor G (EF-G) to the ribosome after GTP hydrolysis during elongation and ribosome recycling. The plasmid pUB101-encoded protein FusB causes FA resistance in clinical isolates of Staphylococcus aureus through an interaction with EF-G. Here, we report 1.6 and 2.3 angstrom crystal structures of FusB. We show that FusB is a two-domain protein lacking homology to known structures, where the N-terminal domain is a four-helix bundle and the C-terminal domain has an alpha/beta fold containing a C4 treble clef zinc finger motif and two loop regions with conserved basic residues. Using hybrid constructs between S. aureus EF-G that binds to FusB and Escherichia coli EF-G that does not, we show that the sequence determinants for FusB recognition reside in domain IV and involve the C-terminal helix of S. aureus EF-G. Further, using kinetic assays in a reconstituted translation system, we demonstrate that FusB can rescue FA inhibition of tRNA translocation as well as ribosome recycling. We propose that FusB rescues S. aureus from FA inhibition by preventing formation or facilitating dissociation of the FA-locked EF-G-ribosome complex.

    Keywords
    FusB, elongation factor G, fusidic acid, antibiotic resistance
    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-181147 (URN)10.1098/rsob.120016 (DOI)000307111800002 ()
    Available from: 2012-09-18 Created: 2012-09-17 Last updated: 2016-01-13Bibliographically approved
    3. Characterization of interactions in FusB-mediated fusidic acid resistance
    Open this publication in new window or tab >>Characterization of interactions in FusB-mediated fusidic acid resistance
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    (English)Manuscript (preprint) (Other academic)
    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:uu:diva-265549 (URN)
    Available from: 2015-11-01 Created: 2015-11-01 Last updated: 2016-01-13
    4. Functional and structural innovations in the real-time evolution of new genes
    Open this publication in new window or tab >>Functional and structural innovations in the real-time evolution of new genes
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    (English)Manuscript (preprint) (Other academic)
    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:uu:diva-265716 (URN)
    Available from: 2015-11-02 Created: 2015-11-02 Last updated: 2016-01-13
  • 69.
    Guo, Xiaohu
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Peisker, Kristin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Bäckbro, Kristina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Chen, Yang
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Koripella, Ravi Kiran
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Mandava, Chandra Sekhar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Selmer, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Structure and function of FusB: an elongation factor G-binding fusidic acid resistance protein active in ribosomal translocation and recycling2012In: Open Biology, ISSN 2046-2441, Vol. 2, p. 120016-Article in journal (Refereed)
    Abstract [en]

    Fusidic acid (FA) is a bacteriostatic antibiotic that locks elongation factor G (EF-G) to the ribosome after GTP hydrolysis during elongation and ribosome recycling. The plasmid pUB101-encoded protein FusB causes FA resistance in clinical isolates of Staphylococcus aureus through an interaction with EF-G. Here, we report 1.6 and 2.3 angstrom crystal structures of FusB. We show that FusB is a two-domain protein lacking homology to known structures, where the N-terminal domain is a four-helix bundle and the C-terminal domain has an alpha/beta fold containing a C4 treble clef zinc finger motif and two loop regions with conserved basic residues. Using hybrid constructs between S. aureus EF-G that binds to FusB and Escherichia coli EF-G that does not, we show that the sequence determinants for FusB recognition reside in domain IV and involve the C-terminal helix of S. aureus EF-G. Further, using kinetic assays in a reconstituted translation system, we demonstrate that FusB can rescue FA inhibition of tRNA translocation as well as ribosome recycling. We propose that FusB rescues S. aureus from FA inhibition by preventing formation or facilitating dissociation of the FA-locked EF-G-ribosome complex.

  • 70.
    Hammarlöf, Disa L.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Liljas, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Temperature-sensitive mutants of RNase E in Salmonella enterica2011In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 193, no 23, p. 6639-6650Article in journal (Refereed)
    Abstract [en]

    RNase E has an important role in mRNA turnover and stable RNA processing although the reason for its essentiality is unknown. We isolated conditional mutants of RNase E to provide genetic tools to probe its essential function. In Salmonella enterica serovar Typhimurium an extreme slow-growth phenotype caused by mutant EF-Tu (Gln125Arg, tufA499) can be rescued by mutants of RNase E that have reduced activity. We exploited this phenotype to select mutations in RNase E and screened these for temperature sensitivity (ts) for growth. Four different ts mutations were identified, all in the N-terminal domain of RNase E: Gly66→Cys; Ile207→Ser; Ile207→Asn; Ala327→Pro. We also selected second-site mutations in RNase E that reversed temperature-sensitivity. The complete set of RNase E mutations (53 primary mutations including the ts mutations, and 23 double mutations) were analyzed for their possible effects on the structure and function of RNase E using the available 3-D structures. Most single mutations were predicted to destabilize the structure while second-site mutations that reversed the ts phenotype were predicted to restore stability to the structure. Three isogenic strain pairs carrying single or double mutations in RNase E (ts, and ts plus second-site mutation) were tested for their effects on the degradation, accumulation and processing of mRNA, rRNA and tRNA. The greatest defect was observed on rne mRNA autoregulation and this correlated with ability to rescue the tufA499-associated slow growth phenotype. This is consistent with the RNase E mutants being defective in initial binding or subsequent cleavage of an mRNA critical for fast growth.

  • 71.
    Haq, Syed Raza
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Jürgens, Maike C.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Chi N, Celestine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ETH.
    Koh, Cha San
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Elfström, Lisa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Selmer, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Gianni, Stefano
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    The plastic energy landscape of protein folding: a triangular folding mechanism with an equilibrium intermediate for a small protein domain2010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 23, p. 18051-18059Article in journal (Refereed)
    Abstract [en]

    Protein domains usually fold without or with only transiently populated intermediates, possibly to avoid misfolding, which could result in amyloidogenic disease. Whether observed intermediates are productive and obligatory species on the folding reaction pathway or dispensable by-products is a matter of debate. Here, we solved the crystal structure of a small protein domain, SAP97 PDZ2 I342W C378A, and determined its folding pathway. The presence of a folding intermediate was demonstrated both by single and double-mixing kinetic experiments using urea-induced (un) folding as well as ligand-induced folding. This protein domain was found to fold via a triangular scheme, where the folding intermediate could be either on-or off-pathway, depending on the experimental conditions. Furthermore, we found that the intermediate was present at equilibrium, which is rarely seen in folding reactions of small protein domains. The folding mechanism observed here illustrates the roughness and plasticity of the protein folding energy landscape, where several routes may be employed to reach the native state. The results also reconcile the folding mechanisms of topological variants within the PDZ domain family.

  • 72.
    Harish, Ajith
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Abroi, Aare
    Estonian Bioctr, Riia 23, EE-51010 Tartu, Estonia..
    Gough, Julian
    Univ Bristol, Computat Genom Grp, Dept Comp Sci, Merchant Venturers Bldg, Bristol BS8 1TH, Avon, England..
    Kurland, Charles
    Lund Univ, Microbial Ecol, Dept Biol, S-22100 Lund, Sweden..
    Did Viruses Evolve As a Distinct Supergroup from Common Ancestors of Cells?2016In: Genome Biology and Evolution, ISSN 1759-6653, E-ISSN 1759-6653, Vol. 8, no 8, p. 2474-2481Article in journal (Refereed)
    Abstract [en]

    The evolutionary origins of viruses according to marker gene phylogenies, as well as their relationships to the ancestors of host cells remains unclear. In a recent article Nasir and Caetano-Anolles reported that their genome-scale phylogenetic analyses based on genomic composition of protein structural-domains identify an ancient origin of the "viral supergroup" (Nasir et al. 2015. A phylogenomic data-driven exploration of viral origins and evolution. Sci Adv. 1(8):e1500527.). It suggests that viruses and host cells evolved independently from a universal common ancestor. Examination of their data and phylogenetic methods indicates that systematic errors likely affected the results. Reanalysis of the data with additional tests shows that small-genome attraction artifacts distort their phylogenomic analyses, particularly the location of the root of the phylogenetic tree of life that is central to their conclusions. These new results indicate that their suggestion of a distinct ancestry of the viral supergroup is not well supported by the evidence.

  • 73.
    Harish, Ajith
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kurland, Charles G.
    Lund Univ, Microbial Ecol Program, Dept Biol, Lund, Sweden..
    Akaryotes and Eukaryotes are independent descendants of a universal common ancestor2017In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 138, p. 168-183Article in journal (Refereed)
    Abstract [en]

    We reconstructed a global tree of life (ToL) with non-reversible and non-stationary models of genome evolution that root trees intrinsically. We implemented Bayesian model selection tests and compared the statistical support for four conflicting ToL hypotheses. We show that reconstructions obtained with a Bayesian implementation (Klopfstein et al., 2015) are consistent with reconstructions obtained with an empirical Sankoff parsimony (ESP) implementation (Harish et al., 2013). Both are based on the genome contents of coding sequences for protein domains (superfamilies) from hundreds of genomes. Thus, we conclude that the independent descent of Eukaryotes and Akaryotes (archaea and bacteria) from the universal common ancestor (UCA) is the most probable as well as the most parsimonious hypothesis for the evolutionary origins of extant genomes. Reconstructions of ancestral proteomes by both Bayesian and ESP methods suggest that at least 70% of unique domain-superfamilies known in extant species were present in the UCA. In addition, identification of a vast majority (96%) of the mitochondrial superfamilies in the UCA proteome precludes a symbiotic hypothesis for the origin of eukaryotes. Accordingly, neither the archaeal origin of eukaryotes nor the bacterial origin of mitochondria is supported by the data. The proteomic complexity of the UCA suggests that the evolution of cellular phenotypes in the two primordial lineages, Akaryotes and Eukaryotes, was driven largely by duplication of common superfamilies as well as by loss of unique superfamilies. Finally, innovation of novel superfamilies has played a surprisingly small role in the evolution of Akaryotes and only a marginal role in the evolution of Eukaryotes.

  • 74.
    Harish, Ajith
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kurland, Charles G.
    Lund Univ, Microbial Ecol Program, Dept Biol, Lund, Sweden..
    Empirical genome evolution models root the tree of life2017In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 138, p. 137-155Article in journal (Refereed)
    Abstract [en]

    A reliable phylogenetic reconstruction of the evolutionary history of contemporary species depends on a robust identification of the universal common ancestor (UCA) at the root of the Tree of Life (ToL). That root polarizes the tree so that the evolutionary succession of ancestors to descendants is discernable. In effect, the root determines the branching order and the direction of character evolution. Typically, conventional phylogenetic analyses implement time-reversible models of evolution for which character evolution is un-polarized. Such practices leave the root and the direction of character evolution undefined by the data used to construct such trees. In such cases, rooting relies on theoretic assumptions and/or the use of external data to interpret unrooted trees. The most common rooting method, the outgroup method is clearly inapplicable to the ToL, which has no outgroup. Both here and in the accompanying paper (Harish and Kurland, 2017) we have explored the theoretical and technical issues related to several rooting methods. We demonstrate (1) that Genome-level characters and evolution models are necessary for species phylogeny reconstructions. By the same token, standard practices exploiting sequence-based methods that implement gene-scale substitution models do not root species trees; (2) Modeling evolution of complex genomic characters and processes that are non-reversible and non-stationary is required to reconstruct the polarized evolution of the ToL; (3) Rooting experiments and Bayesian model selection tests overwhelmingly support the earlier finding that akaryotes and eukaryotes are sister clades that descend independently from UCA (Harish and Kurland, 2013); (4) Consistent ancestral state reconstructions from independent genome samplings confirm the previous finding that UCA features three fourths of the unique protein domain-superfamilies encoded by extant genomes.

  • 75.
    Holm, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    A tale of two antibiotics: Fusidic acid and Viomycin2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Antibiotics that target the bacterial ribosome make up about half of all clinically used antibiotics. We have studied two ribosome targeting drugs: Fusidic acid and Viomycin. Fusidic acid inhibits bacterial protein synthesis by binding to elongation factor G (EF-G) on the ribosome, thereby inhibiting translocation of the bacterial ribosome. Viomycin binds directly to the ribosome and inhibits both the fidelity of mRNA decoding and translocation. We found that the mechanisms of inhibition of these two antibiotics were unexpectedly complex. Fusidic acid can bind to EF-G on the ribosome during three separate stages of translocation. Binding of the drug to the first and most sensitive state does not lead to stalling of the ribosome. Rather the ribosome continues unhindered to a downstream state where it stalls for around 8 seconds. Dissociation of fusidic acid from this state allows the ribosome to continue translocating but it soon reaches yet another fusidic acid sensitive state where it can be stalled again, this time for 6 seconds. Viomycin inhibits translocation by binding to the pre-translocation ribosome in competition with EF-G. If viomycin binds before EF-G it stalls the ribosome for 44 seconds, much longer than a normal elongation cycle. Both viomycin and fusidic acid probably cause long queues of ribosomes to build up on the mRNA when they bind. Viomycin inhibits translational fidelity by binding to the ribosome during initial selection. We found that the concentration of viomycin required to bind to the ribosome with a given probability during decoding is proportional to the accuracy of the codon∙anticodon pair being decoded. This demonstrated that long standing models about ribosomal accuracy cannot be correct. Finally, we demonstrated that a common viomycin resistance mutation increases the drug binding rate and decreases its dissociation rate. Our results demonstrate that ribosome targeting drugs have unexpectedly complex mechanisms of action. Both fusidic acid and viomycin preferentially bind to conformations of the ribosome other than those that they stabilize. This suggests that determining the structures of stable drug-bound states may not give sufficient information for drug design.

    List of papers
    1. Fusidic Acid Targets Elongation Factor G in Several Stages of Translocation on the Bacterial Ribosome
    Open this publication in new window or tab >>Fusidic Acid Targets Elongation Factor G in Several Stages of Translocation on the Bacterial Ribosome
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    2015 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 6, p. 3440-3454Article in journal (Refereed) Published
    Abstract [en]

    The antibiotic fusidic acid (FA) targets elongation factor G (EF-G) and inhibits ribosomal peptide elongation and ribosome recycling, but deeper mechanistic aspects of FA action have remained unknown. Using quench flow and stopped flow experiments in a biochemical system for protein synthesis and taking advantage of separate time scales for inhibited (10 s) and uninhibited (100 ms) elongation cycles, a detailed kinetic model of FA action was obtained. FA targets EF-G at an early stage in the translocation process (I), which proceeds unhindered by the presence of the drug to a later stage (II), where the ribosome stalls. Stalling may also occur at a third stage of translocation(III), just before release of EF-G from the post-translocation ribosome. We show that FA is a strong elongation inhibitor (K-50% approximate to 1 mu M), discuss the identity of the FA targeted states, and place existing cryo-EM and crystal structures in their functional context.

    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-247496 (URN)10.1074/jbc.M114.611608 (DOI)000349456000020 ()25451927 (PubMedID)
    Available from: 2015-03-19 Created: 2015-03-19 Last updated: 2018-01-11Bibliographically approved
    2. Molecular mechanism of viomycin inhibition of peptide elongation in bacteria
    Open this publication in new window or tab >>Molecular mechanism of viomycin inhibition of peptide elongation in bacteria
    2016 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, no 4, p. 978-983Article in journal (Refereed) Published
    Abstract [en]

    Viomycin is a tuberactinomycin antibiotic essential for treating multi-drug-resistant tuberculosis. It inhibits bacterial protein synthesis by blocking elongation factor G (EF-G) catalyzed translocation of messenger RNA on the ribosome. Here we have clarified the molecular aspects of viomycin inhibition of the elongating ribosome using pre-steady-state kinetics. We found that the probability of ribosome inhibition by viomycin depends on competition between viomycin and EF-G for binding to the pretranslocation ribosome, and that stable viomycin binding requires an A-site bound tRNA. Once bound, viomycin stalls the ribosome in a pretranslocation state for a minimum of similar to 45 s. This stalling time increases linearly with viomycin concentration. Viomycin inhibition also promotes futile cycles of GTP hydrolysis by EF-G. Finally, we have constructed a kinetic model for viomycin inhibition of EF-G catalyzed translocation, allowing for testable predictions of tuberactinomycin action in vivo and facilitating in-depth understanding of resistance development against this important class of antibiotics.

    Keywords
    protein synthesis, viomycin, ribosome, antibiotics, tuberculosis
    National Category
    Microbiology in the medical area
    Identifiers
    urn:nbn:se:uu:diva-277786 (URN)10.1073/pnas.1517541113 (DOI)000368617900047 ()26755601 (PubMedID)
    Available from: 2016-02-23 Created: 2016-02-23 Last updated: 2018-01-10Bibliographically approved
    3. Insights into the fidelity mechanism of mRNA decoding from characterization of viomycin induced miscoding in translation
    Open this publication in new window or tab >>Insights into the fidelity mechanism of mRNA decoding from characterization of viomycin induced miscoding in translation
    (English)Article in journal (Refereed) Submitted
    Abstract [en]

    Using pre-steady state kinetics and an E. coli based in vitro translation system we have studied the effect of the antibiotic viomycin on mRNA decoding. We find that viomycin binds to the ribosome during initial selection of tRNA, after binding of ternary complex but before GTP hydrolysis by EF-Tu. Viomycin binding renders the ribosome completely incapable of rejecting incorrect A-site bound tRNAs in both initial selection and proofreading. Viomycin sensitivity correlates with the accuracy of initial selection for the four different codon·anticodon pairs tested here. Our results demonstrate that, in contrast to current ideas about ‘induced-fit’, accuracy in initial selection is achieved primarily by increased dissociation rates for near-cognate tRNAs rather than by decreased rates of GTP hydrolysis. Further, our results imply that the ‘monitoring’ bases A1492 and A1493 rapidly fluctuate between active and inactive conformations when a near-cognate tRNA is present in the A site.

    Place, publisher, year, edition, pages
    Uppsala:
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-300234 (URN)
    Available from: 2016-08-08 Created: 2016-08-08 Last updated: 2016-08-26
    4. Biochemical characterization of ΔTlyA mediated viomycin resistance
    Open this publication in new window or tab >>Biochemical characterization of ΔTlyA mediated viomycin resistance
    (English)Manuscript (preprint) (Other academic)
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-300235 (URN)
    Available from: 2016-08-08 Created: 2016-08-08 Last updated: 2016-08-26
  • 76.
    Holm, Mikael
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Borg, Anneli
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Molecular mechanism of viomycin inhibition of peptide elongation in bacteria2016In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, no 4, p. 978-983Article in journal (Refereed)
    Abstract [en]

    Viomycin is a tuberactinomycin antibiotic essential for treating multi-drug-resistant tuberculosis. It inhibits bacterial protein synthesis by blocking elongation factor G (EF-G) catalyzed translocation of messenger RNA on the ribosome. Here we have clarified the molecular aspects of viomycin inhibition of the elongating ribosome using pre-steady-state kinetics. We found that the probability of ribosome inhibition by viomycin depends on competition between viomycin and EF-G for binding to the pretranslocation ribosome, and that stable viomycin binding requires an A-site bound tRNA. Once bound, viomycin stalls the ribosome in a pretranslocation state for a minimum of similar to 45 s. This stalling time increases linearly with viomycin concentration. Viomycin inhibition also promotes futile cycles of GTP hydrolysis by EF-G. Finally, we have constructed a kinetic model for viomycin inhibition of EF-G catalyzed translocation, allowing for testable predictions of tuberactinomycin action in vivo and facilitating in-depth understanding of resistance development against this important class of antibiotics.

  • 77.
    Holm, Mikael
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ge, Xueliang
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Biochemical characterization of ΔTlyA mediated viomycin resistanceManuscript (preprint) (Other academic)
  • 78.
    Holm, Mikael
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Insights into the fidelity mechanism of mRNA decoding from characterization of viomycin induced miscoding in translationArticle in journal (Refereed)
    Abstract [en]

    Using pre-steady state kinetics and an E. coli based in vitro translation system we have studied the effect of the antibiotic viomycin on mRNA decoding. We find that viomycin binds to the ribosome during initial selection of tRNA, after binding of ternary complex but before GTP hydrolysis by EF-Tu. Viomycin binding renders the ribosome completely incapable of rejecting incorrect A-site bound tRNAs in both initial selection and proofreading. Viomycin sensitivity correlates with the accuracy of initial selection for the four different codon·anticodon pairs tested here. Our results demonstrate that, in contrast to current ideas about ‘induced-fit’, accuracy in initial selection is achieved primarily by increased dissociation rates for near-cognate tRNAs rather than by decreased rates of GTP hydrolysis. Further, our results imply that the ‘monitoring’ bases A1492 and A1493 rapidly fluctuate between active and inactive conformations when a near-cognate tRNA is present in the A site.

  • 79. Hsiao, An-Shan
    et al.
    Haslam, Richard P.
    Michaelson, Louise V.
    Liao, Pan
    Chen, Qin-Fang
    Sooriyaarachchi, Sanjeewani
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Napier, Johnathan A.
    Tanner, Julian A.
    Chye, Mee-Len
    Arabidopsis cytosolic acyl-CoA-binding proteins ACBP4, ACBP5 and ACBP6 have overlapping but distinct roles in seed development2014In: Bioscience Reports, ISSN 0144-8463, E-ISSN 1573-4935, Vol. 34, no 6, p. 865-877Article in journal (Refereed)
    Abstract [en]

    Eukaryotic cytosolic ACBPs (acyl-CoA-binding proteins) bind acyl-CoA esters and maintain a cytosolic acyl-CoA pool, but the thermodynamics of their protein-lipid interactions and physiological relevance in plants are not well understood. Arabidopsis has three cytosolic ACBPs which have been identified as AtACBP4, AtACBP5 and AtACBP6, and microarray data indicated that all of them are expressed in seeds; AtACBP4 is expressed in early embryogenesis, whereas AtACBP5 is expressed later. ITC (isothermal titration calorimetry) in combination with transgenic Arabidopsis lines were used to investigate the roles of these three ACBPs from Arabidopsis thaliana. The dissociation constants, stoichiometry and enthalpy change of AtACBP interactions with various acyl-CoA esters were determined using ITC. Strong binding of recombinant (r) AtACBP6 with long-chain acyl-CoA (C16-to C18-CoA) esters was observed with dissociation constants in the nanomolar range. However, the affinity of rAtACBP4 and rAtACBP5 to these acyl-CoA esters was much weaker (dissociation constants in the micromolar range), suggesting that they interact with acyl-CoA esters differently from rAtACBP6. When transgenic Arabidopsis expressing AtACBP6pro::GUS was generated, strong GUS (beta-glucuronidase) expression in cotyledonary-staged embryos and seedlings prompted us to measure the acyl-CoA contents of the acbp6 mutant. This mutant accumulated higher levels of C18:1-CoA and C18:1- and C18:2-CoAs in cotyledonary-staged embryos and seedlings, respectively, in comparison with the wild type. The acbp4acbp5acbp6 mutant showed the lightest seed weight and highest sensitivity to abscisic acid during germination, suggesting their physiological functions in seeds.

  • 80.
    Hultqvist, Greta
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Haq, Raza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Punekar, Avinash
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Chi, Celestine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ETH.
    Engström, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bach, Anders
    Strømgaard, Kristian
    University of Copenhagen.
    Selmer, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Gianni, Stefano
    Sapienza Università di Roma.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Energetic pathway sampling in a protein interaction domain2013In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 21, p. 1193-1202Article in journal (Other academic)
    Abstract [en]

    The affinity and specificity of protein-ligand interactions are influenced by energeticcrosstalk within the protein domain. However, the molecular details of such intradomain allostery are still unclear. Here, we have experimentally detected and computationally predicted interactionpathways in the postsynaptic density 95/discs large/zonula occludens 1 (PDZ)-peptide ligand model system using wild-type and circularly permuted PDZ proteins. The circular permutant introduced small perturbations in the tertiary structure and a concomitant rewiring of allosteric pathways, allowing us to describe how subtle changes may reshape energetic signaling. The results were analyzed in the context of other members of the PDZ family, which were found to contain distinct interaction pathways for different peptide ligands. The data reveal a fascinating scenario whereby several energetic pathways are sampled within one single domain and distinct pathways are activated by specific protein ligands. 

  • 81.
    Ieong, Ka-Weng
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Rate and Accuracy of Bacterial Protein Synthesis with Natural and Unnatural Amino Acids2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis addresses different questions regarding the rate, efficiency, and accuracy of peptide bond formation with natural as well as unnatural amino acids: Which step is rate-limiting during peptide bond formation? How does the accuracy vary with different transfer RNAs (tRNAs) and codons and how is it relevant to the living cells? Does proofreading selection of codon reading occur in a single- or multi-step manner as theoretically suggested? How does the E. coli translation system discriminate unnatural amino acids? Based on that, how to improve the incorporation efficiencies of unnatural amino acids?

    Based on the study on pH dependence of peptide bond formation, we show that the rate of the chemistry of peptidyl transfer to aminoacyl-tRNA (AA-tRNA) Gly-tRNAGly or Pro-tRNAPro limits the rate of peptide bond formation at physiological pH 7.5, and this could possibly be true for peptidyl transfer to all natural AA-tRNAs at physiological condition.

    By studying the efficiency-accuracy trade-off for codon reading by seven AA-tRNA containing ternary complexes, we observe a large variation on the accuracy of initial codon selection and identify several error hot-spots. The maximal accuracy varied 400-fold from 200 to 84000 depending on the tRNA identity, the type and position of the mismatches.

    We also propose a proofreading mechanism that contains two irreversible steps in sequence. This could be highly relevant to the living cells in relation to maintaining both high accuracy and high efficiency in protein synthesis.

    Finally, we show that peptide bond formation with small and large non-N-alkylated L- unnatural amino acids proceed at rates similar to those with natural amino acids Phe and Ala on the ribosome. Interestingly, the large side chain of the bulky unnatural amino acid only weakens its binding for elongation factor Tu (EF-Tu) but not slows down peptidyl transfer on the ribosome. Our results also suggest that the efficiency of unnatural amino acid incorporation could be improved in general by increasing EF-Tu concentration, lowering the reaction temperature and / or using tRNA bodies with optimal affinities for EF-Tu in the translation system.

    List of papers
    1. pH-sensitivity of the ribosomal peptidyl transfer reaction dependent on the identity of the A-site aminoacyl-tRNA
    Open this publication in new window or tab >>pH-sensitivity of the ribosomal peptidyl transfer reaction dependent on the identity of the A-site aminoacyl-tRNA
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    2011 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, no 1, p. 79-84Article in journal (Refereed) Published
    Abstract [en]

    We studied the pH-dependence of ribosome catalyzed peptidyl transfer from fMet-tRNA(fMet) to the aa-tRNAs Phe-tRNA(Phe), Ala-tRNA(Ala), Gly-tRNA(Gly), Pro-tRNA(Pro), Asn-tRNA(Asn), and Ile-tRNA(Ile), selected to cover a large range of intrinsic pK(a)-values for the α-amino group of their amino acids. The peptidyl transfer rates were different at pH 7.5 and displayed different pH-dependence, quantified as the pH-value, pK(a)(obs), at which the rate was half maximal. The pK(a)(obs)-values were downshifted relative to the intrinsic pK(a)-value of aa-tRNAs in bulk solution. Gly-tRNA(Gly) had the smallest downshift, while Ile-tRNA(Ile) and Ala-tRNA(Ala) had the largest downshifts. These downshifts correlate strongly with molecular dynamics (MD) estimates of the downshifts in pK(a)-values of these aa-tRNAs upon A-site binding. Our data show the chemistry of peptide bond formation to be rate limiting for peptidyl transfer at pH 7.5 in the Gly and Pro cases and indicate rate limiting chemistry for all six aa-tRNAs.

    Keywords
    Chromogranins, Granin-derived peptides, Granins, Immunohistochemistry, Neuroendocrine differentiation, Neuroendocrine tumours, Prohormone convertases, Secretogranins
    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-145815 (URN)10.1073/pnas.1012612107 (DOI)000285915000019 ()21169502 (PubMedID)
    Available from: 2011-02-11 Created: 2011-02-11 Last updated: 2017-12-11Bibliographically approved
    2. Large accuracy variation in initial codon selection by aminoacyl-tRNAs on the bacterial ribosome
    Open this publication in new window or tab >>Large accuracy variation in initial codon selection by aminoacyl-tRNAs on the bacterial ribosome