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  • 1.
    Amrein, Beat Anton
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Extending the Reach of Computational Approaches to Model Enzyme Catalysis2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Recent years have seen tremendous developments in methods for computational modeling of (bio-) molecular systems. Ever larger reactive systems are being studied with high accuracy approaches, and high-level QM/MM calculations are being routinely performed. However, applying high-accuracy methods to large biological systems is computationally expensive and becomes problematic when conformational sampling is needed. To address this challenge, classical force field based approaches such as free energy perturbation (FEP) and empirical valence bond calculations (EVB) have been employed in this work. Specifically:

    1. Force-field independent metal parameters have been developed for a range of alkaline earth and transition metal ions, which successfully reproduce experimental solvation free energies, metal-oxygen distances, and coordination numbers. These are valuable for the computational study of biological systems.

    2. Experimental studies have shown that the epoxide hydrolase from Solanum tuberosum (StEH1) is not only an enantioselective enzyme, but for smaller substrates, displays enantioconvergent behavior. For StEH1, two detailed studies, involving combined experimental and computational efforts have been performed: We first used trans-stilbene oxide to establish the basic reaction mechanism of this enzyme. Importantly, a highly conserved and earlier ignored histidine was identified to be important for catalysis. Following from this, EVB and experiment have been used to investigate the enantioconvergence of the StEH1-catalyzed hydrolysis of styrene oxide. This combined approach involved wildtype StEH1 and an engineered enzyme variant, and established a molecular understanding of enantioconvergent behavior of StEH1.

    3. A novel framework was developed for the Computer-Aided Directed Evolution of Enzymes (CADEE), in order to be able to quickly prepare, simulate, and analyze hundreds of enzyme variants. CADEE’s easy applicability is demonstrated in the form of an educational example.

    In conclusion, classical approaches are a computationally economical means to achieve extensive conformational sampling. Using the EVB approach has enabled me to obtain a molecular understanding of complex enzymatic systems. I have also increased the reach of the EVB approach, through the implementation of CADEE, which enables efficient and highly parallel in silico testing of hundreds-to-thousands of individual enzyme variants.

  • 2.
    Amrein, Beat Anton
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Steffen-Munsberg, Fabian
    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.
    Szeler, Ireneusz
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Purg, Miha
    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.
    Kulkarni, Yashraj
    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.
    Kamerlin, Shina Caroline Lynn
    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.
    CADEE: Computer-Aided Directed Evolution of Enzymes2017In: IUCrJ, ISSN 0972-6918, E-ISSN 2052-2525, Vol. 4, no 1, 50-64 p.Article in journal (Refereed)
    Abstract [en]

    The tremendous interest in enzymes as biocatalysts has led to extensive work in enzyme engineering, as well as associated methodology development. Here, a new framework for computer-aided directed evolution of enzymes (CADEE) is presented which allows a drastic reduction in the time necessary to prepare and analyze in silico semi-automated directed evolution of enzymes. A pedagogical example of the application of CADEE to a real biological system is also presented in order to illustrate the CADEE workflow.

  • 3.
    Anandapadmanaban, Madhanagopal
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, Faculty of Science & Engineering.
    Pilstål, Robert
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, Faculty of Science & Engineering.
    Andrésen, Cecilia
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Trewhella, Jill
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. University of Sydney, Australia.
    Moche, Martin
    Karolinska Institute, Sweden.
    Wallner, Björn
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, Faculty of Science & Engineering.
    Sunnerhagen, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Mutation-Induced Population Shift in the MexR Conformational Ensemble Disengages DNA Binding: A Novel Mechanism for MarR Family Derepression2016In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 24, no 8, 1311-1321 p.Article in journal (Refereed)
    Abstract [en]

    MexR is a repressor of the MexAB-OprM multidrug efflux pump operon of Pseudomonas aeruginosa, where DNA-binding impairing mutations lead to multidrug resistance (MDR). Surprisingly, the crystal structure of an MDR-conferring MexR mutant R21W (2.19 angstrom) presented here is closely similar to wildtype MexR. However, our extended analysis, by molecular dynamics and small-angle X-ray scattering, reveals that the mutation stabilizes a ground state that is deficient of DNA binding and is shared by both mutant and wild-type MexR, whereas the DNA-binding state is only transiently reached by the more flexible wild-type MexR. This population shift in the conformational ensemble is effected by mutation-induced allosteric coupling of contact networks that are independent in the wild-type protein. We propose that the MexR-R21W mutant mimics derepression by small-molecule binding to MarR proteins, and that the described allosteric model based on population shifts may also apply to other MarR family members.

  • 4. Andersen, Birgit
    et al.
    Lundgren, Stina
    Dobritzsch, Doreen
    Karolinska Institutet.
    Piskur, Jure
    A recruited protease is involved in catabolism of pyrimidines2008In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 379, no 2, 243-250 p.Article in journal (Refereed)
    Abstract [en]

    In nature, the same biochemical reaction can be catalyzed by enzymes having fundamentally different folds, reaction mechanisms and origins. For example, the third step of the reductive catabolism of pyrimidines, the conversion of N-carbamyl-beta-alanine to beta-alanine, is catalyzed by two beta-alanine synthase (beta ASase, EC 3.5.1.6) subfamilies. We show that the "prototype" eukaryote beta ASases, such as those from Drosophila melanogaster and Arabidopsis thaliana, are relatively efficient in the conversion of N-carbamyl-beta A compared with a representative of fungal beta ASases, the yeast Saccharomyces kluyveri beta ASase, which has a high K(m) value (71 mM). S. kluyveri beta ASase is specifically inhibited by dipeptides and tripeptides, and the apparent K(i) value of glycyl-glycine is in the same range as the substrate K(m). We show that this inhibitor binds to the enzyme active center in a similar way as the substrate. The observed structural similarities and inhibition behavior, as well as the phylogenetic relationship, suggest that the ancestor of the fungal beta ASase was a protease that had modified its profession and become involved in the metabolism of nucleic acid precursors.

  • 5.
    Andersson, Charlotta S.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Berthold, Catrine
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    A Dynamic C-terminal Segment in the Mycobacterium tuberculosis Mn/Fe R2lox Protein can Assume a Helical Structure with Possible Functional ConsequencesManuscript (preprint) (Other academic)
  • 6.
    Andersson, Charlotta S.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Öhrström, Maria
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Popović-Bijelić, Ana
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The manganese ion of the heterodinuclear Mn/Fe cofactor in Chlamydia trachomatis ribonucleotide reductase R2c is located at metal position 1.2012In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 134, no 1, 123-125 p.Article in journal (Refereed)
    Abstract [en]

    The essential catalytic radical of Class-I ribonucleotide reductase is generated and delivered by protein R2, carrying a dinuclear metal cofactor. A new R2 subclass, R2c, prototyped by the Chlamydia trachomatis protein was recently discovered. This protein carries an oxygen-activating heterodinuclear Mn(II)/Fe(II) metal cofactor and generates a radical-equivalent Mn(IV)/Fe(III) oxidation state of the metal site, as opposed to the tyrosyl radical generated by other R2 subclasses. The metal arrangement of the heterodinuclear cofactor remains unknown. Is the metal positioning specific, and if so, where is which ion located? Here we use X-ray crystallography with anomalous scattering to show that the metal arrangement of this cofactor is specific with the manganese ion occupying metal position 1. This is the position proximal to the tyrosyl radical site in other R2 proteins and consistent with the assumption that the high-valent Mn(IV) species functions as a direct substitute for the tyrosyl radical.

  • 7.
    Andersson, Charlotta Selina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structural studies of R2 and R2–like proteins with a heterodinuclear Mn/Fe cofactor and enzymes involved in Mycobacterium tuberculosis lipid metabolism2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Tuberculosis is a notorious disease responsible for the deaths of 1.4 million people worldwide. A third of the world's population is infected with Mycobacterium tuberculosis, the bacterium causing the disease. The increase of multi drug-resistant strains worsens the situation, and the World Health Organization has declared tuberculosis to be a global emergency. The bacterium envelopes itself with a unique set of very long-chain lipids that play an important role in virulence and drug resistance. Therefore enzymes involved in lipid metabolism are putative drug targets. 

    To allow entry into different metabolic pathways and transmembrane transport, fatty acids have to be activated. This is done primarily by fatty acyl-CoA synthetases (ACSs). We identified an ACS possibly involved in the bacterium’s virulence and solved its structure. Structural interpretation combined with previously reported data gives us insights into the details of its function. This enzyme is known to harbor lipid substrates longer than the enzyme itself, and we now propose how this peripheral membrane protein accommodates its substrates. 

    Some of the most chemically challenging oxidations are performed by dinuclear metalloproteins belonging to the ferritin-like superfamily. We show that the ferritin-like protein, R2lox, from M. tuberculosis contains a new type of heterodinuclear Mn/Fe cofactor. This protein cofactor is capable of performing potent 2-electron oxidations as demonstrated by a novel tyrosine-valine crosslink observed in the protein. 

    Recently a new subclass of ribonucleotide reductase (RNR) R2 proteins, was identified in the intracellular pathogen Chlamydia trachomatis containing the same type of Mn/Fe cofactor mentioned above. The RNR R2 proteins use their metal site to generate a stable radical, essential for the reduction of ribonucleotides to their deoxy forms, the building blocks of DNA. With this work, we were able to characterize the architecture of this metal cofactor.

  • 8.
    Andér, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Computational Analysis of Molecular Recognition Involving the Ribosome and a Voltage Gated K+ Channel2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Over the last few decades, computer simulation techniques have been established as an essential tool for understanding biochemical processes. This thesis deals mainly with the application of free energy calculations to ribosomal complexes and a cardiac ion channel.

    The linear interaction energy (LIE) method is used to explore the energetic properties of the essential process of codon–anticodon recognition on the ribosome. The calculations show the structural and energetic consequences and effects of first, second, and third position mismatches in the ribosomal decoding center.

    Recognition of stop codons by ribosomal termination complexes is fundamentally different from sense codon recognition. Free energy perturbation simulations are used to study the detailed energetics of stop codon recognition by the bacterial ribosomal release factors RF1 and RF2. The calculations explain the vastly different responses to third codon position A to G substitutions by RF1 and RF2. Also, previously unknown highly specific water interactions are identified.

    The GGQ loop of ribosomal RFs is essential for its hydrolytic activity and contains a universally methylated glutamine residue. The structural effect of this methylation is investigated. The results strongly suggest that the methylation has no effect on the intrinsic conformation of the GGQ loop, and, thus, that its sole purpose is to enhance interactions in the ribosomal termination complex.

    A first microscopic, atomic level, analysis of blocker binding to the pharmaceutically interesting potassium ion channel Kv1.5 is presented. A previously unknown uniform binding mode is identified, and experimental binding data is accurately reproduced. Furthermore, problems associated with pharmacophore models based on minimized gas phase ligand conformations are highlighted.

    Generalized Born and Poisson–Boltzmann continuum models are incorporated into the LIE method to enable implicit treatment of solvent, in an effort to improve speed and convergence. The methods are evaluated and validated using a set of plasmepsin II inhibitors.

  • 9. Antuch, W
    et al.
    Berndt, Kurt D
    Eidgenössische Technische Hochschule–Hönggerberg, Zürich, Switzerland.
    Chávez, M A
    Delfín, J
    Wüthrich, K
    The NMR solution structure of a Kunitz-type proteinase inhibitor from the sea anemone Stichodactyla helianthus.1993In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 212, 675-684 p.Article in journal (Refereed)
    Abstract [en]

    The solution structure of a 55-amino-acid Kunitz-type proteinase inhibitor, ShPI, purified from the Caribbean sea anemone Stichodactyla helianthus, was determined by NMR spectroscopy. Nearly complete sequence-specific 1H-NMR assignments were obtained at pH 4.6 and 36 degrees C, and stereo-specific assignments were determined for 23 pairs of diastereotopic substituents. A data set of 666 upper distance limit constraints and 122 dihedral angle constraints collected on this basis was used as input for a structure calculation with the program DIANA. Following energy minimization with the program OPAL, the average root-mean-square diviation (RMSD) of the 20 DIANA conformers used to represent the solution structure relative to the mean structure is 61 pm for all backbone atoms N, C alpha and C', and 106 pm for all heavy atoms of residues 2-53. This high-quality solution structure of ShPI has a nearly identical molecular architecture as the bovine pancreatic trypsin inhibitor (BPTI), despite a mere 35% of sequence similarity between the two proteins. Exchange rates measured for 48 out of the 51 backbone amide protons showed that the positions of 20 slowly exchanging amide protons correlate well with hydrogen bonds involving these protons in the energy-minimized solution structure. The solution structure of ShPI is compared to the four homologous proteins for which the three-dimensional structure is also available.

  • 10. Aquila, A.
    et al.
    Barty, A.
    Bostedt, C.
    Boutet, S.
    Carini, G.
    dePonte, D.
    Drell, P.
    Doniach, S.
    Downing, K. H.
    Earnest, T.
    Elmlund, H.
    Elser, V.
    Gühr, M.
    Hajdu, Janos
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Hastings, J.
    Hau-Riege, S. P.
    Huang, Z.
    Lattman, E. E.
    Maia, F. R. N. C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Marchesini, S.
    Ourmazd, A.
    Pellegrini, C.
    Santra, R.
    Schlichting, I.
    Schroer, C.
    Spence, J. C. H.
    Vartanyants, I. A.
    Wakatsuki, S.
    Weis, W. I.
    Williams, G. J.
    The linac coherent light source single particle imaging road map2015In: Structural Dynamics, Vol. 2, no 4, 041701Article in journal (Refereed)
    Abstract [en]

    Intense femtosecond x-ray pulses from free-electron laser sources allow the imag-ing of individual particles in a single shot. Early experiments at the Linac CoherentLight Source (LCLS) have led to rapid progress in the field and, so far, coherentdiffractive images have been recorded from biological specimens, aerosols, andquantum systems with a few-tens-of-nanometers resolution. In March 2014, LCLSheld a workshop to discuss the scientific and technical challenges for reaching theultimate goal of atomic resolution with single-shot coherent diffractive imaging. This paper summarizes the workshop findings and presents the roadmap towardreaching atomic resolution, 3D imaging at free-electron laser sources.

  • 11. Aquila, Andrew
    et al.
    Hunter, Mark S.
    Doak, R. Bruce
    Kirian, Richard A.
    Fromme, Petra
    White, Thomas A.
    Andreasson, Jakob
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Arnlund, David
    Bajt, Saša
    Barends, Thomas R. M.
    Barthelmess, Miriam
    Bogan, Michael J.
    Bostedt, Christoph
    Bottin, Hervé
    Bozek, John D.
    Caleman, Carl
    Coppola, Nicola
    Davidsson, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Physical Chemistry.
    DePonte, Daniel P.
    Elser, Veit
    Epp, Sascha W.
    Erk, Benjamin
    Fleckenstein, Holger
    Foucar, Lutz
    Frank, Matthias
    Fromme, Raimund
    Graafsma, Heinz
    Grotjohann, Ingo
    Gumprecht, Lars
    Hajdu, Janos
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Hampton, Christina Y.
    Hartmann, Andreas
    Hartmann, Robert
    Hau-Riege, Stefan
    Hauser, Günter
    Hirsemann, Helmut
    Holl, Peter
    Holton, James M.
    Hömke, André
    Johansson, Linda
    Kimmel, Nils
    Kassemeyer, Stephan
    Krasniqi, Faton
    Kühnel, Kai-Uwe
    Liang, Mengning
    Lomb, Lukas
    Malmerberg, Erik
    Marchesini, Stefano
    Martin, Andrew V.
    Maia, Filipe R.N.C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Messerschmidt, Marc
    Nass, Karol
    Reich, Christian
    Neutze, Richard
    Rolles, Daniel
    Rudek, Benedikt
    Rudenko, Artem
    Schlichting, Ilme
    Schmidt, Carlo
    Schmidt, Kevin E.
    Schulz, Joachim
    Seibert, M. Marvin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Soltau, Heike
    Shoeman, Robert L.
    Sierra, Raymond
    Starodub, Dmitri
    Stellato, Francesco
    Stern, Stephan
    Strüder, Lothar
    Timneanu, Nicusor
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Ullrich, Joachim
    Wang, Xiaoyu
    Williams, Garth J.
    Weidenspointner, Georg
    Weierstall, Uwe
    Wunderer, Cornelia
    Barty, Anton
    Spence, John C. H.
    Chapman, Henry N.
    Time-resolved protein nanocrystallography using an X-ray free-electron laser2012In: Optics Express, ISSN 1094-4087, Vol. 20, no 3, 2706-2716 p.Article in journal (Refereed)
    Abstract [en]

    We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 µs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.

  • 12.
    Arndt, Anton
    Swedish School of Sport and Health Sciences, GIH, Department of Sport and Health Sciences, Laboratory for Biomechanics and Motor Control.
    Achilles tendon biomechanics and functional anatomy2012Conference paper (Other academic)
  • 13.
    Arndt, Anton
    et al.
    Swedish School of Sport and Health Sciences, GIH, Department of Sport and Health Sciences, Laboratory for Biomechanics and Motor Control.
    Bengtsson, Ann-Sophie
    Peolsson, Michael
    Thorstensson, Alf
    Swedish School of Sport and Health Sciences, GIH, Department of Sport and Health Sciences, Laboratory for Biomechanics and Motor Control.
    Movin, Tomas
    Non-uniform displacement within the Achilles tendon durig passive ankle joint motion.2012In: Knee Surgery, Sports Traumatology, Arthroscopy, ISSN 0942-2056, E-ISSN 1433-7347, Vol. 20, no 9, 1868-74 p.Article in journal (Refereed)
    Abstract [en]

    PURPOSE:

    An initial step in the understanding of Achilles tendon dynamics is to investigate the effects of passive motion, thereby minimising muscle activation and reducing internal joint forces. Internal tendon dynamics during passive ankle joint motion have direct implications for clinical rehabilitation protocols after Achilles tendon surgery. The aim of this study was to test the hypothesis that tendon tissue displacement is different in different layers of the Achilles tendon during controlled passive ankle joint movements.

    METHODS:

    Ultrasound imaging was conducted on the right Achilles tendon of nine healthy recreationally active males. Standardised isokinetic passive dorsi-plantar-flexion movements were performed with a total range of motion of 35°. The tendon was divided into superficial, central and deep layers in the resulting B-mode ultrasound images viewed in the sagittal plane. A block-matching speckle tracking algorithm was applied post-process, with kernels for the measurement of displacement placed in each of the layers.

    RESULTS:

    The mean (SD) displacement of the Achilles tendon during passive dorsiflexion was 8.4 (1.9) mm in the superficial layer, 9.4 (1.9) mm in the central portion and 10.4 (2.1) mm in the deep layer, respectively. In all cases, the movement of the deep layer of the tendon was greater than that of the superficial one (P < 0.01).

    CONCLUSIONS:

    These results, achieved in vivo with ultrasonographic speckle tracking, indicated complex dynamic differences in different layers of the Achilles tendon, which could have implications for the understanding of healing processes of tendon pathologies and also of normal tendon function.

  • 14. Arnlund, David
    et al.
    Johansson, Linda C
    Wickstrand, Cecilia
    Barty, Anton
    Williams, Garth J
    Malmerberg, Erik
    Davidsson, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Physical Chemistry.
    Milathianaki, Despina
    DePonte, Daniel P
    Shoeman, Robert L
    Wang, Dingjie
    James, Daniel
    Katona, Gergely
    Westenhoff, Sebastian
    White, Thomas A
    Aquila, Andrew
    Bari, Sadia
    Berntsen, Peter
    Bogan, Mike
    van Driel, Tim Brandt
    Doak, R Bruce
    Kjær, Kasper Skov
    Frank, Matthias
    Fromme, Raimund
    Grotjohann, Ingo
    Henning, Robert
    Hunter, Mark S
    Kirian, Richard A
    Kosheleva, Irina
    Kupitz, Christopher
    Liang, Mengning
    Martin, Andrew V
    Nielsen, Martin Meedom
    Messerschmidt, Marc
    Seibert, M Marvin
    Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, California, USA..
    Sjöhamn, Jennie
    Stellato, Francesco
    Weierstall, Uwe
    Zatsepin, Nadia A
    Spence, John C H
    Fromme, Petra
    Schlichting, Ilme
    Boutet, Sébastien
    Groenhof, Gerrit
    Chapman, Henry N
    Neutze, Richard
    Visualizing a protein quake with time-resolved X-ray scattering at a free-electron laser2014In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 11, no 9, 923-926 p.Article in journal (Refereed)
    Abstract [en]

    We describe a method to measure ultrafast protein structural changes using time-resolved wide-angle X-ray scattering at an X-ray free-electron laser. We demonstrated this approach using multiphoton excitation of the Blastochloris viridis photosynthetic reaction center, observing an ultrafast global conformational change that arises within picoseconds and precedes the propagation of heat through the protein. This provides direct structural evidence for a 'protein quake': the hypothesis that proteins rapidly dissipate energy through quake-like structural motions.

  • 15.
    Aurelius, Oskar
    et al.
    Lund University.
    Johansson, Renzo
    Lund University.
    Bågenholm, Viktoria
    Lund University.
    Lundin, Daniel
    Stockholm University.
    Tholander, Fredrik
    Karolinska Institutet.
    Balhuizen, Alexander
    Lund University.
    Beck, Tobias
    University of Göttingen, Germany.
    Sahlin, Margareta
    Stockholm University.
    Sjöberg, Britt-Marie
    Stockholm University.
    Mulliez, Etienne
    Institut de Recherches en Technologies et Sciences pour le Vivant (iRTSV), France.
    Logan, Derek T.
    Lund University.
    The Crystal Structure of Thermotoga maritima Class III Ribonucleotide Reductase Lacks a Radical Cysteine Pre-Positioned in the Active Site2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 7, e0128199Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, the building blocks for DNA synthesis, and are found in all but a few organisms. RNRs use radical chemistry to catalyze the reduction reaction. Despite RNR having evolved several mechanisms for generation of different kinds of essential radicals across a large evolutionary time frame, this initial radical is normally always channelled to a strictly conserved cysteine residue directly adjacent to the substrate for initiation of substrate reduction, and this cysteine has been found in the structures of all RNRs solved to date. We present the crystal structure of an anaerobic RNR from the extreme thermophile Thermotoga maritima (tmNrdD), alone and in several complexes, including with the allosteric effector dATP and its cognate substrate CTP. In the crystal structure of the enzyme as purified, tmNrdD lacks a cysteine for radical transfer to the substrate pre-positioned in the active site. Nevertheless activity assays using anaerobic cell extracts from T. maritima demonstrate that the class III RNR is enzymatically active. Other genetic and microbiological evidence is summarized indicating that the enzyme is important for T. maritima. Mutation of either of two cysteine residues in a disordered loop far from the active site results in inactive enzyme. We discuss the possible mechanisms for radical initiation of substrate reduction given the collected evidence from the crystal structure, our activity assays and other published work. Taken together, the results suggest either that initiation of substrate reduction may involve unprecedented conformational changes in the enzyme to bring one of these cysteine residues to the expected position, or that alternative routes for initiation of the RNR reduction reaction may exist. Finally, we present a phylogenetic analysis showing that the structure of tmNrdD is representative of a new RNR subclass IIIh, present in all Thermotoga species plus a wider group of bacteria from the distantly related phyla Firmicutes, Bacteroidetes and Proteobacteria.

  • 16.
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology.
    The RAPID crystallization strategy for structure-based inhibitor design2009In: From Molecules to Medicines: Structure of Biological Macromolecules and Its Relevance in Combating New Diseases and Bioterrorism / [ed] J. Sussman and P. Spadon, Dordrecht, The Netherlands: Springer , 2009, 11-19 p.Chapter in book (Other academic)
  • 17.
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Succeeding with seeding: some practical advice2007In: Evolving Methods for Macromolecular Crystallography / [ed] Read, Randy J.; Sussman, Joel L., 2007, 1-10 p.Conference paper (Refereed)
    Abstract [en]

    Seeding is a powerful and versatile method for optimizing crystal growth conditions. This article discusses, from a practical point of view, what seeding is, the selection and transfer of seeds, and into what conditions they should be transferred. The most common causes of failures in seeding experiments are also analyzed.

  • 18.
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Automated liquid-handling systems for submicroliter crystallization2007In: Protein Crystallization Strategies for Structural Genomics / [ed] Naomi E. Chayen, La Jolla, California: International University Line , 2007, 57-73 p.Chapter in book (Other academic)
  • 19.
    Berndt, Kurt D
    et al.
    Eidgenössische Technische Hochschule-Hönggerberg, Zürich, Switzerland.
    Beunink, J
    Schröder, W
    Wüthrich, K
    Designed replacement of an internal hydration water molecule in BPTI: structural and functional implications of a glycine-to-serine mutation.1993In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 32, 4564-4570 p.Article in journal (Refereed)
    Abstract [en]

    The three-dimensional structure of the basic pancreatic trypsin inhibitor (BPTI) contains four internal water molecules, which form a total of nine intermolecular hydrogen bonds with the BPTI polypeptide chain. To investigate the effect of such internal hydration on protein structure and stability, we displaced one of the internal water molecules in a recombinant BPTI analogue, BPTI(G36S), in which Gly 36 is replaced by serine. The replacement of a water molecule by the seryl side chain was established by the absence of the protein-water nuclear Overhauser effects (NOE) that had been attributed to the water molecule near Gly 36 in wild-type BPTI and by the presence of new, intramolecular NOEs to the hydroxyl proton of Ser 36. BPTI(G36S) has slightly reduced thermal stability compared to BPTI, corresponding to a destabilization by delta (delta G) approximately 0.7 kcal/M in 6 M guanidinium hydrochloride solution. Additionally, the stabilities of the complexes formed between BPTI(G36S) and trypsin, plasmin, or kallikrein are significantly reduced when compared to the corresponding complexes with wild-type BPTI.

  • 20.
    Berndt, Kurt D
    et al.
    Eidgenossische TH-Honggerberg, Zürich, Switzerland.
    Güntert, P
    Wüthrich, K
    Nuclear-Magnetic-Resonance Solution Structure of Dendrotoxin-K from the Venom of Dendroaspis-Polylepis-Polylepis1993In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 234, no 3, 735-750 p.Article in journal (Refereed)
  • 21.
    Berndt, Kurt D
    et al.
    Eidgenösische Technische Hochschule-Hönggerberg, Zürich, Switzerland.
    Güntert, Peter
    Orbons, Leonard P.M.
    Wüthrich, Kurt
    Determination of a high-quality nuclear magnetic resonance solution structure of the bovine pancreatic trypsin inhibitor and comparison with three crystal structures1992In: Journal of Molecular Biology, Vol. 227, 757-775 p.Article in journal (Refereed)
    Abstract [en]

    A high-quality three-dimensional structure of the bovine pancreatic trypsin inhibitor (BPTI) in aqueous solution was determined by 1H nuclear magnetic resonance (n.m.r.) spectroscopy and compared to the three available high-resolution X-ray crystal structures. A newly collected input of 642 distance constraints derived from nuclear Overhauser effects and 115 dihedral angle constraints was used for the structure calculations with the program DIANA, followed by restrained energy minimization with the program AMBER. The BPTI solution structure is represented by a group of 20 conformers with an average root-mean-square deviation (RMSD) relative to the mean solution structure of 0.43 A for backbone atoms and 0.92 A for all heavy atoms of residues 2 to 56. The pairwise RMSD values of the three crystal structures relative to the mean solution structure are 0.76 to 0.85 A for the backbone atoms and 1.24 to 1.33 A for all heavy atoms of residues 2 to 56. Small local differences in backbone atom positions between the solution structure and the X-ray structures near residues 9, 25 to 27, 46 to 48 and 52 to 58, and conformational differences for individual amino acid side-chains were analyzed for possible correlations with intermolecular protein-protein contacts in the crystal lattices, using the pairwise RMSD values among the three crystal structures as a reference.

  • 22.
    Berndt, Kurt D
    et al.
    Eidgenössische TH-Hönggerberg, Zürich, Switzerland.
    Güntert, Peter
    Wüthrich, Kurt
    Conformational sampling by NMR solution structures calculated with the program DIANA evaluated by comparison with long-time molecular dynamics calculations in explicit water1996In: Proteins: Structure, Function, and Genetics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 24, 304-313 p.Article in journal (Refereed)
    Abstract [en]

    The NMR solution structure of bovine pancreatic trypsin inhibitor (BPTI) obtained by distance geometry calculations with the program DIANA is compared with groups of conformers generated by molecular dynamics (MD) simulations in explicit water at ambient temperature and pressure. The MD simulations started from a single conformer and were free or restrained either by the experimental NOE distance restraints or by time-averaged restraints; the groups of conformers were collected either in 10 ps intervals during 200 ps periods of simulation, or in 50 ps intervals during a 1 ns period of simulation. Overall, these comparisons show that the standard protein structure determination protocol with the program DIANA provides a picture of the protein structure that is in agreement with MD simulations using "realistic" potential functions over a nanosecond timescale. For well-constrained molecular regions there is a trend in the free MD simulation of duration 1 ns that the sampling of the conformation space is slightly increased relative to the DIANA calculations. In contrast, for surface-exposed side-chains that are less extensively constrained by the NMR data, the DIANA conformers tend to sample larger regions of conformational space than conformers selected from any of the MD trajectories. Additional insights into the behavior of surface side-chains come from comparison of the MD runs of 200 ps or 1 ns duration. In this time range the sampling of conformation space by the protein surface depends strongly on the length of the simulation, which indicates that significant side-chain transitions occur on the nanosecond timescale and that much longer simulations will be needed to obtain statistically significant data on side-chain dynamics.

  • 23.
    Björkelid, Christofer
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Enzymes in the Mycobacterium tuberculosis MEP and CoA Pathways Targeted for Structure-Based Drug Design2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Tuberculosis, caused by the pathogenic bacteria Mycobacterium tuberculosis, is one of the most widespread and deadly infectious diseases today. Treatment of tuberculosis relies on antibiotics that were developed more than 50 years ago. These are now becoming ineffective due to the emergence of antibiotic resistant strains of the bacteria.

    The aim of the research in this thesis was to develop new antibiotics for tuberculosis treatment. To this end, we targeted enzymes from two essential biosynthetic pathways in M. tuberculosis for drug development. The methylerythritol phosphate (MEP) pathway synthesizes a group of compounds called isoprenoids. These compounds have essential roles in all living organisms. The fact that humans utilize a different pathway for isoprenoid synthesis makes the MEP pathway enzymes attractive targets for drug development. We have determined the structures of two essential enzymes from this pathway by X-ray crystallography: 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) and 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD). These are the first structures of these enzymes from M. tuberculosis. Additionally, structures of the IspD enzyme from the related bacteria Mycobacterium smegmatis were determined. We have characterized these enzymes and evaluated the efficiency of a number of inhibitors of the DXR enzyme by biochemical methods. Crystal structures of DXR in complex with some of these inhibitors were also determined.

    The second pathway of interest for drug development is the universal pathway for Coenzyme A biosynthesis. Enzymes in this pathway have essential roles in all living organisms. However, the bacterial enzymes have little similarity to the human homologues. We have determined a number of structures of the M. tuberculosis pantothenate kinase (PanK), the regulatory enzyme of this pathway, in complex with two new classes of inhibitory compounds, and evaluated these by biochemical methods.

    The structures and biochemical characterization of these enzymes provide us with detailed information about their functions and broadens our knowledge of these bacteria. Biochemical and structural information about new inhibitors of these enzymes serve as a starting point for future development of antibiotics against tuberculosis.

  • 24.
    Björkelid, Christofer
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Raichurkar, Anand Kumar V.
    AstraZeneca India Private Limited.
    Mukherjee, Kakoli
    AstraZeneca India Private Limited.
    Malolanarasimhan, Krishnan
    AstraZeneca India Private Limited.
    Bandodkar, Balachandra
    AstraZeneca India Private Limited.
    Jones, T. Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Structural and biochemical characterization of compounds inhibiting Mycobacterium tuberculosis Pantothenate Kinase2013In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 25, 18260-18270 p.Article in journal (Refereed)
    Abstract [en]

    Mycobacterium tuberculosis, the bacterial causative agent oftuberculosis, currently affects millions of people. The emergence of drug-resistant strains makes development of new antibiotics targeting the bacterium a global health priority. Pantothenate kinase, a key enzyme in the universal biosynthesis of the essential cofactor CoA, was targeted in this study to find new tuberculosis drugs. The biochemicalcharacterizations of two new classes of compounds that inhibitpantothenate kinase from M. tuberculosis are described, along with crystal structures of their enzyme-inhibitor complexes. These represent the first crystal structures of this enzyme with engineered inhibitors. Both classes of compounds bind in the active site of the enzyme, overlapping with the binding sites of the natural substrate and product, pantothenateand phosphopantothenate, respectively. One class of compounds also interferes with binding of the cofactor ATP. The complexes were crystallized in two crystal forms, one of which is in a new space group for this enzyme and diffracts to the highest resolution reported for anypantothenate kinase structure. These two crystal forms allowed, for the first time, modeling of the cofactor-binding loop in both open and closed conformations. The structures also show a binding mode of ATP different from that previously reported for the M. tuberculosis enzyme but similar to that in the pantothenate kinases of other organisms.

  • 25.
    Boukharta, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Computational Modelling of Ligand Complexes with G-Protein Coupled Receptors, Ion Channels and Enzymes2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Accurate predictions of binding free energies from computer simulations are an invaluable resource for understanding biochemical processes and drug action. The primary aim of the work described in the thesis was to predict and understand ligand binding to several proteins of major pharmaceutical importance using computational methods.

    We report a computational strategy to quantitatively predict the effects of alanine scanning and ligand modifications based on molecular dynamics free energy simulations. A smooth stepwise scheme for free energy perturbation calculations is derived and applied to a series of thirteen alanine mutations of the human neuropeptide Y1 G-protein coupled receptor and a series of eight analogous antagonists. The robustness and accuracy of the method enables univocal interpretation of existing mutagenesis and binding data. We show how these calculations can be used to validate structural models and demonstrate their ability to discriminate against suboptimal ones. Site-directed mutagenesis, homology modelling and docking were further used to characterize agonist binding to the human neuropeptide Y2 receptor, which is important in feeding behavior and an obesity drug target.  In a separate project, homology modelling was also used for rationalization of mutagenesis data for an integron integrase involved in antibiotic resistance.

    Blockade of the hERG potassium channel by various drug-like compounds, potentially causing serious cardiac side effects, is a major problem in drug development. We have used a homology model of hERG to conduct molecular docking experiments with a series of channel blockers, followed by molecular dynamics simulations of the complexes and evaluation of binding free energies with the linear interaction energy method. The calculations are in good agreement with experimental binding affinities and allow for a rationalization of three-dimensional structure-activity relationships with implications for design of new compounds. Docking, scoring, molecular dynamics, and the linear interaction energy method were also used to predict binding modes and affinities for a large set of inhibitors to HIV-1 reverse transcriptase. Good agreement with experiment was found and the work provides a validation of the methodology as a powerful tool in structure-based drug design. It is also easily scalable for higher throughput of compounds.

  • 26. Boutet, Sébastien
    et al.
    Lomb, Lukas
    Williams, Garth J
    Barends, Thomas R M
    Aquila, Andrew
    Doak, R Bruce
    Weierstall, Uwe
    DePonte, Daniel P
    Steinbrener, Jan
    Shoeman, Robert L
    Messerschmidt, Marc
    Barty, Anton
    White, Thomas A
    Kassemeyer, Stephan
    Kirian, Richard A
    Seibert, M Marvin
    Montanez, Paul A
    Kenney, Chris
    Herbst, Ryan
    Hart, Philip
    Pines, Jack
    Haller, Gunther
    Gruner, Sol M
    Philipp, Hugh T
    Tate, Mark W
    Hromalik, Marianne
    Koerner, Lucas J
    van Bakel, Niels
    Morse, John
    Ghonsalves, Wilfred
    Arnlund, David
    Bogan, Michael J
    Caleman, Carl
    Fromme, Raimund
    Hampton, Christina Y
    Hunter, Mark S
    Johansson, Linda C
    Katona, Gergely
    Kupitz, Christopher
    Liang, Mengning
    Martin, Andrew V
    Nass, Karol
    Redecke, Lars
    Stellato, Francesco
    Timneanu, Nicusor
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Wang, Dingjie
    Zatsepin, Nadia A
    Schafer, Donald
    Defever, James
    Neutze, Richard
    Fromme, Petra
    Spence, John C H
    Chapman, Henry N
    Schlichting, Ilme
    High-resolution protein structure determination by serial femtosecond crystallography2012In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 337, no 6092, 362-364 p.Article in journal (Refereed)
    Abstract [en]

    Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules.

  • 27. Brunne, R M
    et al.
    Berndt, Kurt D
    ETH Hönggerberg, Zürich, Switzerland.
    Güntert, P
    Wüthrich, K
    van Gunsteren, W F
    Güntert, P
    Wüthrich, K
    Van Gunsteren, W F
    Structure and internal dynamics of the bovine pancreatic trypsin inhibitor in aqueous solution from long-time molecular dynamics simulations1995In: Proteins: Structure, Function, and Genetics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 23, no 1, 49-62 p.Article in journal (Refereed)
    Abstract [en]

    Structural and dynamic properties of bovine pancreatic trypsin inhibitor (BPTI) in aqueous solution are investigated using two molecular dynamics (MD) simulations: one of 1.4 ns length and one of 0.8 ns length in which atom-atom distance bounds derived from NMR spectroscopy are included in the potential energy function to make the trajectory satisfy these experimental data more closely. The simulated properties of BPTI are compared with crystal and solution structures of BPTI, and found to be in agreement with the available experimental data. The best agreement with experiment was obtained when atom-atom distance restraints were applied in a time-averaged manner in the simulation. The polypeptide segments found to be most flexible in the MD simulations coincide closely with those showing differences between the crystal and solution structures of BPTI.

  • 28.
    Buijs, Jos
    et al.
    Uppsala University, Sweden.
    Ramström, Margareta
    Uppsala University, Sweden.
    Danfelter, Mikael
    Uppsala University, Sweden.
    Larsericsdotter, Helén
    Mälardalen University, Department of Biology and Chemical Engineering.
    Håkansson, Per
    Mälardalen University, Department of Biology and Chemical Engineering.
    Oscarsson, Sven
    Mälardalen University, Department of Biology and Chemical Engineering.
    Localized changes in the structural stability of myoglobin upon adsorption onto silica particles, as studied with hydrogen/deuterium exchange mass spectrometry2003In: Journal of Colloid and Interface Science, ISSN 0021-9797, Vol. 263, no 2, 441-448 p.Article in journal (Refereed)
    Abstract [en]

    A new method is presented for monitoring the conformational stability of various parts of a protein that is physically adsorbed onto nanometer-sized silica particles. The method employs hydrogen/deuterium (H/D) exchange of amide hydrogens, a process that is extremely sensitive to structural features of proteins. The resulting mass increase is analyzed with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. Higher structural specificity is obtained by enzymatically cleaving the adsorbed proteins prior to mass spectrometric analysis. The mass increases of four peptic fragments of myoglobin are followed as a function of the H/D exchange time. The four peptic fragments cover 90% of the myoglobin structure. Two of the peptic fragments, located in the middle of the myoglobin sequence and close to the heme group, do not show any adsorption-induced changes in their structural stability, whereas the more stable C- and N-terminal fragments are destabilized. Interestingly, for the N-terminal fragment, comprising residues 1–29, two distinct and equally large conformational populations are observed. One of these populations has a stability similar to that in solution (−23 kJ/mol), whereas the other population is highly destabilized upon adsorption (−11 kJ/mol).

  • 29.
    Castell, Alina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Fighting Tuberculosis –: Structural Studies of Three Mycobacterial Proteins2008Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis presents the cloning, purification, crystallization, and structural studies of two unknown proteins from Mycobacterium tuberculosis, and of an aminotransferase from Mycobacterium smegmatis. Structural knowledge of these proteins is of highest interest for structure-based drug design, which is one of the approaches that can be used in order to fight tuberculosis (TB).

    The structure of the conserved hypothetical protein Rv0216 was refined to a resolution of 1.9 Å. The structure exhibits a so-called double hotdog-fold, similar to known hydratases. However, only parts of the hydratase active site are conserved in Rv0216, and no function could be assigned to the protein. Several Rv0216-like protein sequences were found in a variety of actino- and proteobacteria, suggesting that these proteins form a new protein family. Furthermore, other hotdog-folded proteins in M. tuberculosis were identified, of which a few are likely to be hydratases or dehydratases involved in the fatty acid metabolism.

    The structure of Rv0130 exhibits a single hotdog-fold and contains a highly conserved R-hydratase motif. Rv0130 was shown to hydrate fatty acid coenzyme A derivatives with a length of six to eight carbons. The Rv0130 active site is situated in a long tunnel, formed by a kink in the central hotdog-helix, which indicate that it can utilize long fatty acid chains as well. A number of previously predicted hotdog-folded proteins also feature a similar tunnel.

    The structure of branched chain aminotransferase (BCAT) of M. smegmatis was determined in the apo-form and in complex with an aminooxy inhibitor. Mycobacterial BCAT is very similar to the human BCAT, apart for one important difference in the active site. Gly243 is a threonine in the human BCAT, a difference that offers specificity in inhibition and substrate recognition of these proteins. The aminooxy compound and MES were found to inhibit the mycobacterial BCAT activities. The aminooxy compound inhibits by blocking the substrate-pocket. A second inhibitor-binding site was identified through the binding of a MES molecule. Therefore, both the MES-binding site and the substrate-pocket of M. smegmatis BCAT are suggested to be potential sites for the development of new inhibitors against tuberculosis.

  • 30.
    Chen, Yang
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Structural and Biochemical Studies of Antibiotic Resistance and Ribosomal Frameshifting2013Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Protein synthesis, translation, performed by the ribosome, is a fundamental process of life and one of the main targets of antibacterial drugs. This thesis provides structural and biochemical understanding of three aspects of bacterial translation.

    Elongation factor G (EF-G) is the target for the antibiotic fusidic acid (FA). FA binds to EF-G only on the ribosome after GTP hydrolysis and prevents EF-G dissociation from the ribosome. Point mutations in EF-G can lead to FA resistance but are often accompanied by a fitness cost in terms of slower growth of the bacteria. Secondary mutations can compensate for this fitness cost while resistance is maintained. Here we present the crystal structure of the clinical FA drug target, Staphylococcus aureus EF-G, together with the mapping and analysis of all known FA-resistance mutations in EF-G. We also present crystal structures of the FA-resistant mutant F88L, the FA-hypersensitive mutant M16I and the FA-resistant but fitness-compensated double mutant F88L/M16I. Analysis of mutant structures together with biochemical data allowed us to propose that fitness loss and compensation are caused by effects on the conformational dynamics of EF-G on the ribosome.

    Aminoglycosides are another group of antibiotics that target the decoding region of the 30S ribosomal subunit. Resistance to aminoglycosides can be acquired by inactivation of the drugs via enzymatic modification. Here, we present the first crystal structure an aminoglycoside 3’’ adenyltransferase, AadA from Salmonella enterica. AadA displays two domains and unlike related structures most likely functions as a monomer.

    Frameshifts are deviations the standard three-base reading frame of translation. -1 frameshifting can be caused by normal tRNASer3 at GCA alanine codons and tRNAThr3 at CCA/CCG proline codons. This process has been proposed to involve doublet decoding using non-standard codon-anticodon interactions. In our study, we showed by equilibrium binding that these tRNAs bind with low micromolar Kd to the frameshift codons. Our results support the doublet-decoding model and show that non-standard anticodon loop structures need to be adopted for the frameshifts to happen.

    These findings provide new insights in antibiotic resistance and reading-frame maintenance and will contribute to a better understanding of the translation elongation process. 

  • 31.
    Chen, Yang
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Näsvall, Joakim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I.
    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.
    Crystal structure of AadA at 2.5 Å resolution - an aminoglycoside 3" adenyltransferaseManuscript (preprint) (Other academic)
  • 32.
    Chen, Yang
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Näsvall, Joakim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Wu, Shiying
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Andersson, Dan I.
    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.
    Structure of AadA from Salmonella enterica: a monomeric aminoglycoside (3'')(9) adenyltransferase2015In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 71, 2267-2277 p.Article in journal (Refereed)
    Abstract [en]

    Aminoglycoside resistance is commonly conferred by enzymatic modification of drugs by aminoglycoside-modifying enzymes such as aminoglycoside nucleo\-tidyltransferases (ANTs). Here, the first crystal structure of an ANT(3$^\prime$$^\prime$)(9) adenyltransferase, AadA from Salmonella enterica, is presented. AadA catalyses the magnesium-dependent transfer of adenosine monophosphate from ATP to the two chemically dissimilar drugs streptomycin and spectinomycin. The structure was solved using selenium SAD phasing and refined to 2.5Å resolution. AadA consists of a nucleotidyltransferase domain and an α-helical bundle domain. AadA crystallizes as a monomer and is a monomer in solution as confirmed by small-angle X-ray scattering, in contrast to structurally similar homodimeric adenylating enzymes such as kanamycin nucleotidyltransferase. Isothermal titration calorimetry experiments show that ATP binding has to occur before binding of the aminoglycoside substrate, and structure analysis suggests that ATP binding repositions the two domains for aminoglycoside binding in the interdomain cleft. Candidate residues for ligand binding and catalysis were subjected to site-directed mutagenesis. In vivo resistance and in vitro binding assays support the role of Glu87 as the catalytic base in adenylation, while Arg192 and Lys205 are shown to be critical for ATP binding.

  • 33. Chin, T M
    et al.
    Berndt, Kurt D
    Universtity of Chicago, USA.
    Yang, N C C
    Self-Assembling Hexameric Helical Bundle Forming Peptides1992In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 114, no 6, 2279-2280 p.Article in journal (Refereed)
  • 34.
    Chumnarnsilpa, Sakesit
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ma, Qing
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lin Lee, Wei
    Institute of Molecular and Cell Biology, A*STAR, Singapore.
    Burtnick, Leslie D.
    epartment of Chemistry and Centre for Blood Research, Life Sciences Institute, The University of British Columbia, Canada.
    Robinson, Robert C.
    Institute of Molecular and Cell Biology, A*STAR, Singapore.
    The crystal structure of the C-terminus of adseverin: Implications for actin bindingManuscript (Other (popular science, discussion, etc.))
    Abstract [en]

    Adseverin is a member of the calcium-regulated gelsolin superfamily of actin severing and capping proteins. Adseverin comprises six homologous domains (A1-A6) which share 60% homology with the six domains from gelsolin (G1-G6). Adseverin is truncated in comparison to gelsolin, lacking the C-terminal extension which masks the F-actin binding site in calcium-free gelsolin. Biochemical assays have indicated differences in the interaction of the C-terminus halves of adseverin and gelsolin with actin. Gelsolin contacts actin through a major site on G4 and a minor site on G6, while adseverin uses a site on A5. Here we present the X-ray structure of the activated C-terminal half of adseverin (A4-A6). This structure is highly similar to that of the activated form of the C-terminal half of gelsolin (G4-G6), both in arrangement of domains and in the three bound calcium ions. Comparative analysis of the actin-binding surfaces observed in the G4-G6/actin structure suggests that adseverin in this conformation will also be able to interact with actin through A4 and A6, while the A5 surface is obscured. A model of calcium-free adseverin constructed from the structure of gelsolin predicts that the interaction between A2 and A6 provides sterric inhibition to prevent interaction with F-actin in the absence of calcium. Actin-binding assays reveal that the minimal stoichiometry of adseverin to calcium needed to disassemble actin filaments is 1:1 as compared to the 1:2 that was previously observed for gelsolin. We propose that the absence of a gelsolin-like C-terminal extension in adseverin reduces the calcium requirement for activation.

  • 35. Claesson, Magnus
    et al.
    Siitonen, Vilja
    Dobritzsch, Doreen
    Karolinska Institutet.
    Metsä-Ketelä, Mikko
    Schneider, Gunter
    Crystal structure of the glycosyltransferase SnogD from the biosynthetic pathway of nogalamycin in Streptomyces nogalater2012In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 279, no 17, 3251-3263 p.Article in journal (Refereed)
    Abstract [en]

    The glycosyltransferase SnogD from Streptomyces nogalater transfers a nogalamine moiety to the metabolic intermediate 3',4'-demethoxynogalose-1-hydroxynogalamycinone during the final steps of biosynthesis of the aromatic polyketide nogalamycin. The crystal structure of recombinant SnogD, as an apo-enzyme and with a bound nucleotide, 2-deoxyuridine-5'-diphosphate, was determined to 2.6 Å resolution. Reductive methylation of SnogD was crucial for reproducible preparation of diffraction quality crystals due to creation of an additional intermolecular salt bridge between methylated lysine residue Lys384 and Glu374* from an adjacent molecule in the crystal lattice. SnogD is a dimer both in solution and in the crystal, and the enzyme subunit displays a fold characteristic of the GT-B family of glycosyltransferases. Binding of the nucleotide is associated with rearrangement of two active-site loops. Site-directed mutagenesis shows that two active-site histidine residues, His25 and His301, are critical for the glycosyltransferase activities of SnogD both in vivo and in vitro. The crystal structures and the functional data are consistent with a role for His301 in binding of the diphosphate group of the sugar donor substrate, and a function of His25 as a catalytic base in the glycosyl transfer reaction.

  • 36.
    Conti, Luca
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Renhorn, Jakob
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Gabrielsson, Anders
    KTH Royal Institute Technology, Sweden.
    Turesson, Fredrik
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Liin, Sara
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Lindahl, Erik
    KTH Royal Institute Technology, Sweden; Stockholm University, Sweden.
    Elinder, Fredrik
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Reciprocal voltage sensor-to-pore coupling leads to potassium channel C-type inactivation2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, 27562Article in journal (Refereed)
    Abstract [en]

    Voltage-gated potassium channels open at depolarized membrane voltages. A prolonged depolarization causes a rearrangement of the selectivity filter which terminates the conduction of ions - a process called slow or C-type inactivation. How structural rearrangements in the voltage-sensor domain (VSD) cause alteration in the selectivity filter, and vice versa, are not fully understood. We show that pulling the pore domain of the Shaker potassium channel towards the VSD by a Cd2+ bridge accelerates C-type inactivation. Molecular dynamics simulations show that such pulling widens the selectivity filter and disrupts the K+ coordination, a hallmark for C-type inactivation. An engineered Cd2+ bridge within the VSD also affect C-type inactivation. Conversely, a pore domain mutation affects VSD gating-charge movement. Finally, C-type inactivation is caused by the concerted action of distant amino acid residues in the pore domain. All together, these data suggest a reciprocal communication between the pore domain and the VSD in the extracellular portion of the channel.

  • 37. Dalhus, Bjørn
    et al.
    Saarinen, Markuu
    Sauer, Uwe
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Eklund, Pär
    Johansson, Kenth
    Karlsson, Andreas
    Ramaswamy, S
    Bjørk, Alexandra
    Synstad, Bjørnar
    Naterstad, Kristine
    Sirevåg, Reidun
    Eklund, Hans
    Structural basis for thermophilic protein stability: structures of thermophilic and mesophilic malate dehydrogenases.2002In: J Mol Biol, ISSN 0022-2836, Vol. 318, no 3, 707-21 p.Article in journal (Refereed)
    Abstract [en]

    The three-dimensional structure of four malate dehydrogenases (MDH) from thermophilic and mesophilic phototropic bacteria have been determined by X-ray crystallography and the corresponding structures compared. In contrast to the dimeric quaternary structure of most MDHs, these MDHs are tetramers and are structurally related to tetrameric malate dehydrogenases from Archaea and to lactate dehydrogenases. The tetramers are dimers of dimers, where the structures of each subunit and the dimers are similar to the dimeric malate dehydrogenases. The difference in optimal growth temperature of the corresponding organisms is relatively small, ranging from 32 to 55 degrees C. Nevertheless, on the basis of the four crystal structures, a number of factors that are likely to contribute to the relative thermostability in the present series have been identified. It appears from the results obtained, that the difference in thermostability between MDH from the mesophilic Chlorobium vibrioforme on one hand and from the moderate thermophile Chlorobium tepidum on the other hand is mainly due to the presence of polar residues that form additional hydrogen bonds within each subunit. Furthermore, for the even more thermostable Chloroflexus aurantiacus MDH, the use of charged residues to form additional ionic interactions across the dimer-dimer interface is favored. This enzyme has a favorable intercalation of His-Trp as well as additional aromatic contacts at the monomer-monomer interface in each dimer. A structural alignment of tetrameric and dimeric prokaryotic MDHs reveal that structural elements that differ among dimeric and tetrameric MDHs are located in a few loop regions. (c) 2002 Elsevier Science Ltd.

  • 38.
    Dobritzsch, Doreen
    et al.
    Karolinska Institutet.
    Andersen, Birgit
    Piskur, Jure
    Crystallization and X-ray diffraction analysis of dihydropyrimidinase from Saccharomyces kluyveri2005In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 61, no Pt 4, 359-362 p.Article in journal (Refereed)
    Abstract [en]

    Dihydropyrimidinase (EC 3.5.2.2) catalyzes the second step in the reductive pathway of pyrimidine degradation, the hydrolysis of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamylated beta-amino acids. Crystals of the recombinant enzyme from the yeast Saccharomyces kluyveri diffracting to 2.6 A at a synchrotron-radiation source have been obtained by the hanging-drop vapour-diffusion method. They belong to space group P2(1) (unit-cell parameters a = 91.0, b = 73.0, c = 161.4 A, beta = 91.4 degrees), with one homotetramer per asymmetric unit.

  • 39.
    Dobritzsch, Doreen
    et al.
    Karolinska Institutet.
    Gojković, Zoran
    Andersen, Birgit
    Piskur, Jure
    Crystallization and preliminary X-ray analysis of beta-alanine synthase from the yeast Saccharomyces kluyveri2003In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 59, no Pt 7, 1267-1269 p.Article in journal (Refereed)
    Abstract [en]

    In eukaryotes and some bacteria, the third step of reductive pyrimidine catabolism is catalyzed by beta-alanine synthase (EC 3.5.1.6). Crystals of the recombinant enzyme from the yeast Saccharomyces kluyveri were obtained using sodium citrate as a precipitant. The crystals belong to space group P2(1) (unit-cell parameters a = 117.2, b = 77.1, c = 225.5 A, beta = 95.0 degrees ) and contain four homodimers per asymmetric unit. Data were collected to 2.7 A resolution. Introduction of heavy atoms into the crystal lattice induced a different set of unit-cell parameters (a = 61.0, b = 77.9, c = 110.1 A, beta = 97.2 degrees ) in the same space group P2(1), with only one homodimer per asymmetric unit.

  • 40.
    Dobritzsch, Doreen
    et al.
    Martin-Luther-Universität Halle-Wittenberg.
    König, S
    Schneider, G
    Lu, G
    High resolution crystal structure of pyruvate decarboxylase from Zymomonas mobilis: Implications for substrate activation in pyruvate decarboxylases1998In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 273, no 32, 20196-20204 p.Article in journal (Refereed)
    Abstract [en]

    The crystal structure of tetrameric pyruvate decarboxylase from Zymomonas mobilis has been determined at 1.9 A resolution and refined to a crystallographic R-factor of 16.2% and Rfree of 19.7%. The subunit consists of three domains, all of the alpha/beta type. Two of the subunits form a tight dimer with an extensive interface area. The thiamin diphosphate binding site is located at the subunit-subunit interface, and the cofactor, bound in the V conformation, interacts with residues from the N-terminal domain of one subunit and the C-terminal domain of the second subunit. The 2-fold symmetry generates the second thiamin diphosphate binding site in the dimer. Two of the dimers form a tightly packed tetramer with pseudo 222 symmetry. The interface area between the dimers is much larger in pyruvate decarboxylase from Z. mobilis than in the yeast enzyme, and structural differences in these parts result in a completely different packing of the subunits in the two enzymes. In contrast to other pyruvate decarboxylases, the enzyme from Z. mobilis is not subject to allosteric activation by the substrate. The tight packing of the dimers in the tetramer prevents large rearrangements in the quaternary structure as seen in the yeast enzyme and locks the enzyme in an activated conformation. The architecture of the cofactor binding site and the active site is similar in the two enzymes. However, the x-ray analysis reveals subtle but significant structural differences in the active site that might be responsible for variations in the biochemical properties in these enzymes.

  • 41.
    Dobritzsch, Doreen
    et al.
    Karolinska Institutet.
    Lindh, Ingrid
    Uysal, Hüseyin
    Nandakumar, Kutty S
    Burkhardt, Harald
    Schneider, Gunter
    Holmdahl, Rikard
    Crystal structure of an arthritogenic anticollagen immune complex2011In: Arthritis and Rheumatism, ISSN 0004-3591, E-ISSN 1529-0131, Vol. 63, no 12, 3740-3748 p.Article in journal (Refereed)
    Abstract [en]

    OBJECTIVE: In rheumatoid arthritis, joint inflammation and cartilage destruction are mediated by autoantibodies directed to various self antigens. Type II collagen (CII)-specific antibodies are likely to play a role in this process and have been shown to induce experimental arthritis in susceptible animals. The purpose of this study was to reveal how arthritogenic autoantibodies recognize native CII in its triple-helical conformation.

    METHODS: Site-directed mutagenesis and crystallographic studies were performed to reveal crucial contact points between the CII antibody and the triple-helical CII peptide.

    RESULTS: The crystal structure of a pathogenic autoantibody bound to a major triple-helical epitope present on CII was determined, allowing a first and detailed description of the interactions within an arthritogenic complex that is frequently occurring in both mice and humans with autoimmune arthritis. The crystal structure emphasizes the role of arginine residues located in a commonly recognized motif on CII and reveals that germline-encoded elements are involved in the interaction with the epitope.

    CONCLUSION: The crystal structure of an arthritogenic antibody binding a triple-helical epitope on CII indicates a crucial role of germline-encoded and arginine residues as the target structures.

  • 42.
    Dobritzsch, Doreen
    et al.
    Karolinska Institutet.
    Persson, K
    Schneider, G
    Lindqvist, Y
    Crystallization and preliminary X-ray study of pig liver dihydropyrimidine dehydrogenase2001In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 57, no Pt 1, 153-155 p.Article in journal (Refereed)
    Abstract [en]

    Dihydropyrimidine dehydrogenase catalyzes the first and rate-limiting reaction in pyrimidine catabolism. The enzyme contains one FMN, one FAD and four Fe-S clusters per subunit of 1025 amino acids as prosthetic groups. It is also the major determinant of bioavailability and toxicity of 5-fluorouracil, a chemotherapeutic agent widely used in the treatment of solid tumors. Crystals of this enzyme diffracting to at least 2.5 A have been obtained by the hanging-drop vapour-diffusion method and belong to space group P2(1) (unit-cell parameters a = 82.0, b = 159.3, c = 163.6 A, beta = 96.1 degrees ), with two homodimers per asymmetric unit.

  • 43.
    Dobritzsch, Doreen
    et al.
    Karolinska Institutet.
    Ricagno, Stefano
    Schneider, Gunter
    Schnackerz, Klaus D
    Lindqvist, Ylva
    Crystal structure of the productive ternary complex of dihydropyrimidine dehydrogenase with NADPH and 5-iodouracil: Implications for mechanism of inhibition and electron transfer2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 15, 13155-13166 p.Article in journal (Refereed)
    Abstract [en]

    Dihydroprymidine dehydrogenase catalyzes the first and rate-limiting step in pyrimidine degradation by converting pyrimidines to the corresponding 5,6- dihydro compounds. The three-dimensional structures of a binary complex with the inhibitor 5-iodouracil and two ternary complexes with NADPH and the inhibitors 5-iodouracil and uracil-4-acetic acid were determined by x-ray crystallography. In the ternary complexes, NADPH is bound in a catalytically competent fashion, with the nicotinamide ring in a position suitable for hydride transfer to FAD. The structures provide a complete picture of the electron transfer chain from NADPH to the substrate, 5-iodouracil, spanning a distance of 56 A and involving FAD, four [Fe-S] clusters, and FMN as cofactors. The crystallographic analysis further reveals that pyrimidine binding triggers a conformational change of a flexible active-site loop in the alpha/beta-barrel domain, resulting in placement of a catalytically crucial cysteine close to the bound substrate. Loop closure requires physiological pH, which is also necessary for correct binding of NADPH. Binding of the voluminous competitive inhibitor uracil-4-acetic acid prevents loop closure due to steric hindrance. The three-dimensional structure of the ternary complex enzyme-NADPH-5-iodouracil supports the proposal that this compound acts as a mechanism-based inhibitor, covalently modifying the active-site residue Cys-671, resulting in S-(hexahydro-2,4-dioxo-5-pyrimidinyl)cysteine.

  • 44.
    Dobritzsch, Doreen
    et al.
    Karolinska Institutet.
    Schneider, G
    Schnackerz, K D
    Lindqvist, Y
    Crystal structure of dihydropyrimidine dehydrogenase, a major determinant of the pharmacokinetics of the anti-cancer drug 5-fluorouracil2001In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 20, no 4, 650-660 p.Article in journal (Refereed)
    Abstract [en]

    Dihydropyrimidine dehydrogenase catalyzes the first step in pyrimidine degradation: the NADPH-dependent reduction of uracil and thymine to the corresponding 5,6-dihydropyrimidines. Its controlled inhibition has become an adjunct target for cancer therapy, since the enzyme is also responsible for the rapid breakdown of the chemotherapeutic drug 5-fluorouracil. The crystal structure of the homodimeric pig liver enzyme (2x 111 kDa) determined at 1.9 A resolution reveals a highly modular subunit organization, consisting of five domains with different folds. Dihydropyrimidine dehydrogenase contains two FAD, two FMN and eight [4Fe-4S] clusters, arranged in two electron transfer chains that pass the dimer interface twice. Two of the Fe-S clusters show a hitherto unobserved coordination involving a glutamine residue. The ternary complex of an inactive mutant of the enzyme with bound NADPH and 5-fluorouracil reveals the architecture of the substrate-binding sites and residues responsible for recognition and binding of the drug.

  • 45.
    Duarte, Fernanda
    et al.
    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. Univ Oxford, Chem Res Lab, 12 Mansfield Rd, Oxford OX1 3TA, England.;Univ Oxford, Phys & Theoret Chem Lab, S Parks Rd, Oxford OX1 3QZ, England..
    Barrozo, Alexandre
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Williams, Nicholas H.
    Univ Sheffield, Dept Chem, Sheffield S3 7HF, S Yorkshire, England..
    Kamerlin, Shina C. Lynn
    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.
    The Competing Mechanisms of Phosphate Monoester Dianion Hydrolysis2016In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 138, no 33, 10664-10673 p.Article in journal (Refereed)
    Abstract [en]

    Despite the numerous experimental and theoretical studies on phosphate monoester hydrolysis, significant questions remain concerning the mechanistic details of these biologically critical reactions. In the present work we construct a linear free energy relationship for phosphate monoester hydrolysis to explore the effect of modulating leaving group plc on the competition between solvent- and substrate-assisted pathways for the hydrolysis of these compounds. Through detailed comparative electronic-structure studies of methyl phosphate and a series of substituted aryl phosphate monoesters, we demonstrate that the preferred mechanism is dependent on the nature of the leaving group. For good leaving groups, a strong preference is observed for a more dissociative solvent-assisted pathway. However, the energy difference between the two pathways gradually reduces as the leaving group pK(a) increases and creates mechanistic ambiguity for reactions involving relatively poor alkoxy leaving groups. Our calculations show that the transition-state structures vary smoothly across the range of pK(a)s studied and that the pathways remain discrete mechanistic alternatives. Therefore, while not impossible, a biological catalyst would have to surmount a significantly higher activation barrier to facilitate a substrate-assisted pathway than for the solvent-assisted pathway when phosphate is bonded to good leaving groups. For poor leaving groups, this intrinsic preference disappears.

  • 46. Dunny, Gary
    et al.
    Berntsson, Ronnie Per-Arne
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Enterococcal sex pheromones: evolutionary pathways to complex, two-signal systems2016In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 198, no 11, 1556-1562 p.Article, review/survey (Refereed)
    Abstract [en]

    Gram-positive bacteria carry out intercellular communication using secreted peptides. Important examples of this type of communication are the enterococcal sex pheromone systems, in which the transfer of conjugative plasmids is controlled by intercellular signaling among populations of donors and recipients. This review focuses on the pheromone response system of the conjugative plasmid pCF10. The peptide pheromones regulating pCF10 transfer act by modulating the ability of the PrgX transcription factor to repress the transcription of an operon encoding conjugation functions. Many Gram-positive bacteria regulate important processes, including the production of virulence factors, biofilm formation, sporulation, and genetic exchange using peptide-mediated signaling systems. The key master regulators of these systems comprise the RRNPP (RggRap/NprR/PlcR/PrgX) family of intracellular peptide receptors; these regulators show conserved structures. While many RRNPP systems include a core module of two linked genes encoding the regulatory protein and its cognate signaling peptide, the enterococcal sex pheromone plasmids have evolved to a complex system that also recognizes a second host-encoded signaling peptide. Additional regulatory genes not found in most RRNPP systems also modulate signal production and signal import in the enterococcal pheromone plasmids. This review summarizes several structural studies that cumulatively demonstrate that the ability of three pCF10 regulatory proteins to recognize the same 7-amino-acid pheromone peptide arose by convergent evolution of unrelated proteins from different families. We also focus on the selective pressures and structure/function constraints that have driven the evolution of pCF10 from a simple, single-peptide system resembling current RRNPPs in other bacteria to the current complex inducible plasmid transfer system.

  • 47.
    Egea-Jimenez, Antonio Luis
    et al.
    Aix Marseille Univ, Inst Paoli Calmettes, INSERM, CRCM,U1068,CNRS UMR7258, F-13009 Marseille, France; Katholieke Univ Leuven, Dept Human Genet, ON1 Herestr 49,Box 602, B-3000 Leuven, Belgium.
    Gallardo, Rodrigo
    Katholieke Univ Leuven, Dept Human Genet, ON1 Herestr 49,Box 602, B-3000 Leuven, Belgium;Katholieke Univ Leuven, Dept Mol Cellular & Mol Med, VIB, VIB Switch Lab, B-3000 Leuven, Belgium.
    Garcia-Pino, Abel
    Vrije Univ Brussel, Struct Biol Brussels, Dept Biotechnol DBIT, Pl Laan 2, B-1050 Brussels, Belgium; VIB, Mol Recognit Unit, Struct Biol Res Ctr, Pl Laan 2, B-1050 Brussels, Belgium; Univ Libre Bruxelles, Biol Struct & Biophys, CP300,Rue Prof Jeener & Brachet 12, B-6041 Gosselies, Belgium.
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Katholieke Univ Leuven, Dept Human Genet, ON1 Herestr 49,Box 602, B-3000 Leuven, Belgium.
    Wawrzyniak, Anna Maria
    Katholieke Univ Leuven, Dept Human Genet, ON1 Herestr 49,Box 602, B-3000 Leuven, Belgium.
    Kashyap, Rudra
    Aix Marseille Univ, Inst Paoli Calmettes, INSERM, CRCM,U1068,CNRS UMR7258, F-13009 Marseille, France; Katholieke Univ Leuven, Dept Human Genet, ON1 Herestr 49,Box 602, B-3000 Leuven, Belgium.
    Loris, Remy
    Vrije Univ Brussel, Struct Biol Brussels, Dept Biotechnol DBIT, Pl Laan 2, B-1050 Brussels, Belgium; VIB, Mol Recognit Unit, Struct Biol Res Ctr, Pl Laan 2, B-1050 Brussels, Belgium.
    Schymkowitz, Joost
    Katholieke Univ Leuven, Dept Mol Cellular & Mol Med, VIB, VIB Switch Lab, B-3000 Leuven, Belgium.
    Rousseau, Frederic
    Katholieke Univ Leuven, Dept Mol Cellular & Mol Med, VIB, VIB Switch Lab, B-3000 Leuven, Belgium.
    Zimmermann, Pascale
    Aix Marseille Univ, Inst Paoli Calmettes, INSERM, CRCM,U1068,CNRS UMR7258, F-13009 Marseille, France; Katholieke Univ Leuven, Dept Human Genet, ON1 Herestr 49,Box 602, B-3000 Leuven, Belgium.
    Frizzled 7 and PIP2 binding by syntenin PDZ2 domain supports Frizzled 7 trafficking and signalling2016In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 7, 12101Article in journal (Refereed)
    Abstract [en]

    PDZ domain-containing proteins work as intracellular scaffolds to control spatio-temporal aspects of cell signalling. This function is supported by the ability of their PDZ domains to bind other proteins such as receptors, but also phosphoinositide lipids important for membrane trafficking. Here we report a crystal structure of the syntenin PDZ tandem in complex with the carboxy-terminal fragment of Frizzled 7 and phosphatidylinositol 4,5-bisphosphate (PIP2). The crystal structure reveals a tripartite interaction formed via the second PDZ domain of syntenin. Biophysical and biochemical experiments establish co-operative binding of the tripartite complex and identify residues crucial for membrane PIP2-specific recognition. Experiments with cells support the importance of the syntenin-PIP2 interaction for plasma membrane targeting of Frizzled 7 and c-jun phosphorylation. This study contributes to our understanding of the biology of PDZ proteins as key players in membrane compartmentalization and dynamics.

  • 48.
    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.

  • 49.
    Ericsson, Daniel
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Tunnels and Grooves: Structure-Function Studies in Two Disparate Enzymes2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis describes structural and binding studies in enzymes from two different  organisms: ribonucleotide reductase from Mycobacterium tuberculosis (RNR) and lipase A from Candida antarctica (CalA).

    RNR is viable as a target for new drugs against the causative agent of tuberculosis. The biologically active form of RNR is a heterotetramer with an α2β2 substructure. Here we show that an N-acetylated heptapeptide based on the C-terminal sequence of the smaller RNR subunit can disrupt the formation of the holoenzyme sufficiently to inhibit its function. An N-terminal truncation, an alanine scan and a novel statistical molecular design approach based on the heptapeptide Ac-Glu-Asp-Asp-Asp-Trp-Asp-Phe-OH were applied. A full-length acetylated heptapeptide was necessary for inhibition, and Trp5 and Phe7 were also essential. Exchanging the acetyl for the N-terminal Fmoc protective-group increased the binding potency ten-fold. Based on this, several truncated and N-protected peptides were evaluated in a competitive fluorescence polarization assay. The single-amino acid Fmoc-Trp inhibits the RNR holoenzyme formation with a dissociation constant of 12µM, making it an attractive candidate for further development of non-peptidic inhibitors

    Lipases are enzymes with major biotechnological applications. We report the x-ray structure of CalA, the first member of a novel family of lipases. The fold includes a well-defined lid as well as a classical α/β hydrolase domain. The structure is that of the closed/inactive state of the enzyme, but loop movements near Phe431 will provide virtually unlimited access to solvent for the alcohol moiety of an ester substrate. The structure thus provides a basis for understanding the enzyme's preference for acyl moieties with long, straight tails, and for its highly promiscuous acceptance of widely different alcohol and amine moieties. An unconventional oxyanion hole is observed in the present structure, although the situation may change during interfacial activation.

  • 50.
    Fiz-Palacios, Omar
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology.
    Leander, Brian S.
    Heger, Thierry J.
    Old Lineages in a New Ecosystem: Diversification of Arcellinid Amoebae (Amoebozoa) and Peatland Mosses2014In: PLoS ONE, ISSN 1932-6203, Vol. 9, no 4, e95238- p.Article in journal (Refereed)
    Abstract [en]

    Arcellinid testate amoebae (Amoebozoa) form a group of free-living microbial eukaryotes with one of the oldest fossil records known, yet several aspects of their evolutionary history remain poorly understood. Arcellinids occur in a range of terrestrial, freshwater and even brackish habitats; however, many arcellinid morphospecies such as Hyalosphenia papilio are particularly abundant in Sphagnum-dominated peatlands, a relatively new ecosystem that appeared during the diversification of Sphagnum species in the Miocene (5-20 Myr ago). Here, we reconstruct divergence times in arcellinid testate amoebae after selecting several fossils for clock calibrations and then infer whether or not arcellinids followed a pattern of diversification that parallels the pattern described for Sphagnum. We found that the diversification of core arcellinids occurred during the Phanerozoic, which is congruent with most arcellinid fossils but not with the oldest known amoebozoan fossil (i.e. at ca. 662 or ca. 750 Myr). Overall, Sphagnum and the Hyalospheniidae exhibit different patterns of diversification. However, an extensive molecular phylogenetic analysis of distinct clades within H. papilio species complex demonstrated a correlation between the recent diversification of H. papilio, the recent diversification of Sphagnum mosses, and the establishment of peatlands.

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