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  • 1.
    Divne, Christina
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    SINNING, I
    STAHLBERG, J
    PETTERSSON, G
    BAILEY, M
    SIIKAAHO, M
    MARGOLLESCLARK, E
    TEERI, T
    JONES, TA
    CRYSTALLIZATION AND PRELIMINARY-X-RAY STUDIES ON THE CORE PROTEINS OF CELLOBIOHYDROLASE-I AND ENDOGLUCANASE-I FROM TRICHODERMA-REESEI1993In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 234, no 3, 905-907 p.Article in journal (Refereed)
  • 2.
    Divne, Christina
    et al.
    KTH, School of Biotechnology (BIO).
    Stahlberg, J
    Teeri, T T
    Jones, T A
    High-resolution crystal structures reveal how a cellulose chain is bound in the 50 angstrom long tunnel of cellobiohydrolase I from Trichoderma reesei1998In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 275, no 2, 309-325 p.Article in journal (Refereed)
  • 3.
    Dogan, Jakob
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Lendel, Christofer
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Härd, Torleif
    Thermodynamics of folding and binding in an affibody:affibody complex2006In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 359, no 5, 1305-1315 p.Article in journal (Refereed)
    Abstract [en]

    Affibody binding proteins are selected from phage-displayed libraries of variants of the 58 residue Z domain. Z(Taq) is an affibody originally selected as a binder to Taq DNA polymerase. The anti-Z(Taq) affibody was selected as a binder to Z(Taq) and the Z(Taq):anti-Z(Taq) complex is formed with a dissociation constant K-d = 0.1 mu M. We have determined the structure of the Z(Taq):anti-Z(Taq) complex as well as the free state structures of Z(Taq) and anti-Z(Taq) using NMR. Here we complement the structural data with thermodynamic studies of Z(Taq) and anti-Z(Taq) folding and complex formation. Both affibody proteins show cooperative two-state thermal denaturation at melting temperatures T-M similar to 56 degrees C. Z(Taq):anti-Z(Taq) complex formation at 25 degrees C in 50 mM NaCl and 20 mM phosphate buffer (pH 6.4) is enthalpy driven with Delta H degrees(bind) = -9.0(+/- 0.1) kcal mol(-1). The heat capacity change Delta C-P degrees,(bind) = -0.43(+/- 0.01) kcal mol(-1) K-1 is in accordance with the predominantly non-polar character of the binding surface, as judged from calculations based on changes in accessible surface areas. A further dissection of the small binding entropy at 25 degrees C (-T Delta S degrees(bind) = -0.6(+/- 0.1) kcal mol(-1)) suggests that a favourable desolvation of non-polar surface is almost completely balanced by unfavourable conformational entropy changes and loss of rotational and translational entropy. Such effects can therefore be limiting for strong binding also when interacting protein components are stable and homogeneously folded. The combined structure and thermodynamics data suggest that protein properties are not likely to be a serious limitation for the development of engineered binding proteins based on the Z domain.

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

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

  • 5.
    Emanuelsson, Olof
    et al.
    Stockholms unviersitet.
    Elofsson, Arne
    von Heijne, Gunnar
    Cristóbal, Susana
    In silico prediction of the peroxisomal proteome in fungi, plants and animals.2003In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 330, no 2, 443-456 p.Article in journal (Refereed)
    Abstract [en]

    In an attempt to improve our abilities to predict peroxisomal proteins, we have combined machine-learning techniques for analyzing peroxisomal targeting signals (PTS1) with domain-based cross-species comparisons between eight eukaryotic genomes. Our results indicate that this combined approach has a significantly higher specificity than earlier attempts to predict peroxisomal localization, without a loss in sensitivity. This allowed us to predict 430 peroxisomal proteins that almost completely lack a localization annotation. These proteins can be grouped into 29 families covering most of the known steps in all known peroxisomal pathways. In general, plants have the highest number of predicted peroxisomal proteins, and fungi the smallest number.

  • 6.
    Emanuelsson, Olof
    et al.
    Stockholms unviersitet.
    Nielsen, H
    Center for Biological Sequence Analysis, Technical University of Denmark.
    Brunak, S
    Center for Biological Sequence Analysis, Technical University of Denmark.
    von Heijne, G
    Stockholms universitet.
    Predicting subcellular localization of proteins based on their N-terminal amino acid sequence.2000In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 300, no 4, 1005-1016 p.Article in journal (Refereed)
    Abstract [en]

    A neural network-based tool, TargetP, for large-scale subcellular location prediction of newly identified proteins has been developed. Using N-terminal sequence information only, it discriminates between proteins destined for the mitochondrion, the chloroplast, the secretory pathway, and "other" localizations with a success rate of 85% (plant) or 90% (non-plant) on redundancy-reduced test sets. From a TargetP analysis of the recently sequenced Arabidopsis thaliana chromosomes 2 and 4 and the Ensembl Homo sapiens protein set, we estimate that 10% of all plant proteins are mitochondrial and 14% chloroplastic, and that the abundance of secretory proteins, in both Arabidopsis and Homo, is around 10%. TargetP also predicts cleavage sites with levels of correctly predicted sites ranging from approximately 40% to 50% (chloroplastic and mitochondrial presequences) to above 70% (secretory signal peptides). TargetP is available as a web-server at http://www.cbs.dtu.dk/services/TargetP/.

  • 7.
    Friedman, Mikaela
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Orlova, Anna
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
    Johansson, Eva
    Affibody AB, Bromma.
    Eriksson, Tove L. J.
    Affibody AB, Bromma.
    Höidén-Guthenberg, Ingmarie
    Affibody AB, Bromma.
    Tolmachev, Vladimir
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
    Nilsson, Fredrik Y.
    Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding Affibody molecule2008In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 376, no 5, 1388-1402 p.Article in journal (Refereed)
    Abstract [en]

    The epidermal growth factor receptor 1 (EGFR) is overexpressed in various malignancies and is associated with a poor patient prognosis. A small, receptor-specific, high-affinity imaging agent would be a useful tool in diagnosing malignant tumors and in deciding upon treatment and assessing the response to treatment. We describe here the affinity maturation procedure for the generation of Affibody molecules binding with high affinity and specificity to EGFR. A library for affinity maturation was constructed by rerandomization of selected positions after the alignment of first-generation binding variants. New binders were selected with phage display technology, using a single oligonucleotide in a single-library effort, and the best second-generation binders had an approximately 30-fold improvement in affinity (K-d = 5-10 nM) for the soluble extracellular domain of EGFR in biospecific interaction analysis using Biacore. The dissociation equilibrium constant, Kd, was also determined for the Affibody with highest affinity using EGFR-expressing A431 cells in flow cytometric analysis (K-d = 2.8 nM). A retained high specificity for EGFR was verified by a dot blot assay showing staining only of EGFR proteins among a panel of serum proteins and other EGFR family member proteins (HER2, HER3, and HER4). The EGFR-binding Affibody molecules were radiolabeled with indium-111, showing specific binding to EGFR-expressing A431 cells and successful targeting of the A431 tumor xenografts with 4-6% injected activity per gram accumulated in the tumor 4 h postinjection.

  • 8. Gordley, Russll M.
    et al.
    Smith, Justin D.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Barbas, Carlos F., III
    Evolution of programmable zinc finger-recombinases with activity in human cells2007In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 367, no 3, 802-813 p.Article in journal (Refereed)
    Abstract [en]

    Site-specific recombinases are important tools for genomic engineering in many living systems. Applications of recombinases are, however, constrained by the DNA targeting endemic of the recombinase used. A tremendous range of recombinase applications can be envisioned if the targeting of recombinase specificity can be made readily programmable. To address this problem we sought to generate zinc finger-recombinase fusion proteins (ReCZFS) capable of site-specific function in a diversity of genetic contexts. Our first Rec(ZF), Tn3Ch15(X2), recombined substrates derived from the native Tn3 resolvase recombination site. Substrate Linked Protein Evolution (SLiPE) was used to optimize the catalytic domains of the enzymes Hin, Gin, and Tn3 for resolution between non-homologous sites, One of the evolved clones, GinL7C7, catalyzed efficient, site-specific recombination in a variety of sequence contexts. When introduced into human cells by retroviral transduction, GinL7C7 excised a 1.4 kb EGFP cassette out of the genome, diminishing fluorescence in similar to 17% of transduced cells. Following this template of rational design and directed evolution, Rec(ZF)S may eventually mediate gene therapies, facilitate the genetic manipulation of model organisms and cells, and mature into powerful new tools for molecular biology and medicine.

  • 9. Gregoretti, I. V.
    et al.
    Lee, Yun M.
    KTH, Superseded Departments, Biotechnology. Walther Cancer Center, Dept. of Chemistry and Biochemistry, University of Notre Dame, USA.
    Goodson, H. V.
    Molecular evolution of the histone deacetylase family: Functional implications of phylogenetic analysis2004In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 338, no 1, 17-31 p.Article in journal (Refereed)
    Abstract [en]

    Histone deacetylases (HDACs) modify core histones and participate in large regulatory complexes that both suppress and enhance transcription. Recent studies indicate that some HDACs can act on non-histone proteins as well. Interest in these enzymes is growing because HDAC inhibitors appear to be promising therapeutic agents against cancer and a variety of other diseases. Thus far, 11 members of the HDAC family have been identified in humans, but few have been characterized in detail. To better define the biological function of these proteins, make maximal use of studies performed in other systems, and assist in drug development efforts, we have performed a phylogenetic analysis of all HDAC-related proteins in all fully sequenced free-living organisms. Previous analyses have divided non-sirtuin HDACs into two groups, classes 1 and 2. We find that HDACs can be divided into three equally distinct groups: class 1, class 2, and a third class consisting of proteins related to the recently identified human HDAC11 gene. We term this novel group "class 4" to distinguish it from the unrelated "class 3" sirtuin deacetylases. Analysis of gene duplication events indicates that the common ancestor of metazoan organisms contained two class 1, two class 2, and a single class 4 HDAC. Examination of HDAC characteristics in light of these evolutionary relationships leads to functional predictions, among them that self-association is common among HDAC proteins. All three HDAC classes (including class 4) exist in eubacteria. Phylogenetic analysis of bacterial HDAC relatives suggests that all three HDAC classes precede the evolution of histone proteins and raises the possibility that the primary activity of some "histone deacetylase" enzymes is directed against non-histone substrates.

  • 10. Hallberg, B. M.
    et al.
    Henriksson, Gunnar
    KTH, Superseded Departments, Pulp and Paper Technology.
    Pettersson, G.
    Divne, Christina
    KTH, Superseded Departments, Biotechnology.
    Crystal structure of the flavoprotein domain of the extracellular flavocytochrome cellobiose dehydrogenase2002In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 315, no 3, 421-434 p.Article in journal (Refereed)
    Abstract [en]

    Cellobiose dehydrogenase (CDH) participates in the degradation of cellulose and lignin. The protein is an extracellular flavocytochrome with a b-type cytochrome domain (CYTcdh) connected to a flavodehydrogenase domain (DHcdh). DHcdh catalyses a two-electron oxidation at the anomeric C1 position of cellobiose to yield cellobiono-1,5-lactone, and the electrons are subsequently transferred from DHcdh to an acceptor, either directly or via CYTcdh. Here, we decribe the crystal structure of Phanerochaete chrysosporium DHcdh determined at 1.5 Angstrom resolution. DHcdh belongs to the GMC family of oxidoreductases, which includes glucose oxidase (GOX) and cholesterol oxidase (COX); however, the sequence identity with members of the family is low. The overall fold of DHcdh is p-hydroxybenzoate hydroxylase-like and is similar to, but also different from, that of GOX and COX. It is partitioned into an FAD-binding subdomain of alpha/beta type and a substrate-binding subdomain consisting of a seven-stranded beta sheet and six helices. Docking of CYTcdh, and DHcdh suggests that CYTcdh covers the active-site entrance in DHcdh, and that the resulting distance between the cofactors is within acceptable limits for inter-domain electron transfer. Based on docking of the substrate, cellobiose, in the active site of DHcdh, we propose that the enzyme discriminates against glucose by favouring interaction with the non-reducing end of cellobiose.

  • 11.
    Hallberg, Martin
    et al.
    KTH, Superseded Departments, Biotechnology.
    Leitner, C.
    Haltrich, D.
    Divne, Christina
    KTH, Superseded Departments, Biotechnology.
    Crystal structure of the 270 kDa homotetrameric lignin-degrading enzyme pyranose 2-oxidase2004In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 341, no 3, 781-796 p.Article in journal (Refereed)
    Abstract [en]

    Pyranose 2-oxidase (P2Ox) is a 270 kDa homotetramer localized preferentially in the hyphal periplasmic space of lignocellulolytic fungi and has a proposed role in lignocellulose degradation to produce the essential co-substrate, hydrogen peroxide, for lignin peroxidases. P2Ox oxidizes D-glucose and other aldopyranoses regioselectively at C2 to the corresponding 2-keto sugars; however, for some substrates, the enzyme also displays specificity for oxidation at C3. The crystal structure of P2Ox from Trametes multicolor has been determined using single anomalous dispersion with mercury as anomalous scatterer. The model was refined at 1.8 Angstrom resolution to R and R-free values of 0.134 and 0.171, respectively. The overall fold of the P2Ox subunit resembles that of members of the glucose-methanol-choline family of long-chain oxidoreductases, featuring a flavin-binding Rossmann domain of class alpha/beta and a substrate-binding subdomain with a six-stranded central beta sheet and three U helices. The homotetramer buries a large internal cavity of roughly 15,000 Angstrom(3), from which the four active sites are accessible. Four solvent channels lead from the surface into the cavity through which substrate must enter before accessing the active site. The present structure shows an acetate molecule bound in the active site with the carboxylate group positioned immediately below the flavin N5 atom, and with one carboxylate oxygen atom interacting with the catalytic residues His548 and Asn593. The entrance to the active site is blocked by a loop (residues 452 to 461) with excellent electron density but elevated temperature factors. We predict that this loop is dynamic and opens to allow substrate entry and exit. In silico docking of D-glucose in the P2Ox active site shows that with the active-site loop in the closed conformation, monosaccharides cannot be accommodated; however, after removing the loop from the model, a tentative set of protein-substrate interactions for beta-D-glucose have been outlined.

  • 12. Hard, T.
    et al.
    Rak, A.
    Allard, P.
    Kloo, Lars A.
    KTH, Superseded Departments, Chemistry.
    Garber, M.
    The solution structure of ribosomal protein L36 from Thermus thermophilus reveals a zinc-ribbon-like fold2000In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 296, no 1, 169-180 p.Article in journal (Refereed)
    Abstract [en]

    We have determined the solution NMR structure of the ribosomal protein L36 from Thermus thermophilus. L36 is the smallest protein in the large subunit of the prokaryotic ribosome. The sequence contains three completely conserved cysteine residues and one conserved histidine residue in a C-X-2-C-X-12-C-X-4-H motif. Extended X-ray absorption fine structure spectroscopy was used to confirm that a purified L36 sample contains an equimolar amount of zinc. The structure of L36 was determined using simulated annealing based on NOE distance restraints, dihedral angle restraints and hydrogen bond distance restraints derived from NMR spectra of N-15-labeled and non-labeled L36 samples at pH 7 and 12 degrees C, and by imposing tetrahedral zinc ion coordination geometry. The L36 fold is characterized by a triple-stranded antiparallel P-sheet with the zinc-binding site at one end. The structure of the zinc site is well-determined and shows that the three cysteine sulphur atoms are supported by hydrogen bonds to backbone amide protons. The conserved histidine residue is located in a short 3(10)-helix and coordinates zinc by the N-delta 1 atom. The electrostatic surface potential and location of conserved Arg, Lys and His side-chains suggest a large continuous L36-rRNA interaction interface. The folding topology as well as position and conformation of many conserved side-chains in L36 are very similar to those of zinc-ribbon domains found in the archaeal transcription factor TFIIB N terminus and the eukaryal transcription elongation factor hTFIIS C terminus. Given the relative antiquity of the ribosome it is possible that L36 reflects the parent of transcription-related zinc ribbons.

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

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

  • 14. Härd, Torleif
    et al.
    Lendel, Christofer
    Inhibition of amyloid formation.2012In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 421, no 4-5, 441-65 p.Article in journal (Refereed)
    Abstract [en]

    Amyloid is aggregated protein in the form of insoluble fibrils. Amyloid deposition in human tissue-amyloidosis-is associated with a number of diseases including all common dementias and type II diabetes. Considerable progress has been made to understand the mechanisms leading to amyloid formation. It is, however, not yet clear by which mechanisms amyloid and protein aggregates formed on the path to amyloid are cytotoxic. Strategies to prevent protein aggregation and amyloid formation are nevertheless, in many cases, promising and even successful. This review covers research on intervention of amyloidosis and highlights several examples of how inhibition of protein aggregation and amyloid formation has been achieved in practice. For instance, rational design can provide drugs that stabilize a native folded state of a protein, protein engineering can provide new binding proteins that sequester monomeric peptides from aggregation, small molecules and peptides can be designed to block aggregation or direct it into non-cytotoxic paths, and monoclonal antibodies have been developed for therapies towards neurodegenerative diseases based on inhibition of amyloid formation and clearance.

  • 15.
    Kaiser, Liselotte
    et al.
    Karolinska Institutet, Sweden.
    Velickovic, Tanja Cirkovic
    Karolinska Institutet, Sweden.
    Badia-Martinez, Daniel
    Karolinska Institutet, Sweden.
    Adedoyin, Justus
    Karolinska Institutet, Sweden.
    Thunberg, Sarah
    Karolinska Institutet, Sweden.
    Hallen, Dan
    Electrum, Sweden.
    Berndt, Kurt
    Karolinska Institutet, Sweden.
    Gronlund, Hans
    Karolinska Institutet, Sweden.
    Gafvelin, Guro
    Karolinska Institutet, Sweden.
    van Hage, Marianne
    Karolinska Institutet, Sweden.
    Achour, Adnane
    Karolinska Institutet, Sweden.
    Structural characterization of the tetrameric form of the major cat allergen Fel d 12007In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 370, no 4, 714-727 p.Article in journal (Refereed)
    Abstract [en]

    Felis domesticus allergen 1(Fel d 1) is a 35 kDa tetrameric glycoprotein formed by two heterodimers which elicits IgE responses in 95% of patients with allergy to cat. We have previously established in vitro conditions for the appropriate folding of recombinant Fel d 1 using a direct linkage of chain I to chain 2 (construct Fel d 1 (1 + 2)) and chain 2 to chain 1 (construct Fel d 1 (2 + 1)). Although the crystal structure of Fel d 1 (2 + 1) revealed a striking structural similarity to that of uteroglobin, a steroid-inducible cytokine-like molecule with anti-inflammatory and immunomodulatory properties, no functional tetrameric form of Fel d I could be identified. Here we present the crystal structure of the Fel d I (1 +2) tetramer at 1.6 angstrom resolution. Interestingly, the crystal structure of tetrameric Fel d I reveals two different calcium-binding sites. Symmetrically positioned on each side of the Fel d 1 tetramer, the external Ca2+ -binding sites correspond to a putative Ca2+- binding site previously suggested for uteroglobin. The second Ca2+-binding site lies within the dimerization interface, stabilizing the formation of the Fel d 1 tetramer, and inducing important local conformational changes that directly govern the shape of two water-filled cavities. The crystal structure suggests a potential portal for an unknown ligand. Alternatively, the two cavities could be used by the allergen as a conditional inner space allowing for the spatial rearrangement of centrally localized side-chains, such as Asp130, without altering the overall fold of the molecule. The striking structural similarity of the major cat allergen to uteroglobin, coupled to the identification in the present study of a common Ca2+ -binding site, let us speculate that Fel d I could provoke an allergic response through the modulation of phospholipase A2, by sequestering Ca ions in a similar manner as previously suggested for uteroglobin. (c) 2007 Elsevier Ltd. All rights reserved.

  • 16. Kleywegt, G J
    et al.
    Zou, J Y
    Divne, Christina
    KTH, School of Biotechnology (BIO).
    Davies, G J
    Sinning, I
    Stahlberg, J
    Reinikainen, T
    Srisodsuk, M
    Teeri, T T
    Jones, T A
    The crystal structure of the catalytic core domain of endoglucanase I from Trichoderma reesei at 3.6 angstrom resolution, and a comparison with related enzymes1997In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 272, no 3, 383-397 p.Article in journal (Refereed)
  • 17. Koski, Timo
    A fragment library based on gaussian mixtures predicting favorable molecular interactions2001In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 313, no 1, 197-214 p.Article in journal (Refereed)
    Abstract [en]

    Here, a protein atom-ligand fragment interaction library is described. The library is based on experimentally solved structures of protein-ligand and protein-protein complexes deposited in the Protein Data Bank (PDB) and it is able to characterize binding sites given a ligand structure suitable for a protein. A set of 30 ligand fragment types were defined to include three or more atoms in order to unambiguously define a frame of referencefor interactions of ligand atoms with their receptor proteins. Interactions between ligand fragments and 24 classes of protein target atoms plus a water oxygen atom were collected and segregated according to type. The spatial distributions of individual fragment - target atom pairs were visually inspected in order to obtain rough-grained constraints on the interaction volumes. Data fulfilling these constraints were given as input to an iterative expectation-maximization algorithm that produces as output maximum likelihood estimates of the parameters of the finite Gaussian mixture models. Concepts of statistical pattern recognition and the resulting mixture model densities are used (i) to predict the detailed interactions between Chlorella virus DNA ligase and the adenine ring of its ligand and (ii) to evaluate the "error" in prediction for both the training and validation sets of protein-ligand interaction found in the PDB. These analyses demonstrate that this approach can successfully narrow down the possibilities for both the interacting protein atom type and its location relative to a ligand fragment.

  • 18.
    Käll, Lukas
    et al.
    Ctr. for Genomics and Bioinformatics, Karolinska Institutet.
    Krogh, Anders
    Sonnhammer, Erik L. L.
    A combined transmembrane topology and signal peptide prediction method2004In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 338, no 5, 1027-1036 p.Article in journal (Refereed)
    Abstract [en]

    An inherent problem in transmembrane protein topology prediction and signal peptide prediction is the high similarity between the hydrophobic regions of a transmembrane helix and that of a signal peptide, leading to cross-reaction between the two types of predictions. To improve predictions further, it is therefore important to make a predictor that aims to discriminate between the two classes. In addition, topology information can be gained when successfully predicting a signal Peptide leading a trans' membrane protein since it dictates that the N terminus of the mature protein must be on the non-cytoplasmic side of the membrane. Here, we present Phobius, a combined transmembrane protein topology and signal peptide predictor. The predictor is based on a hidden Markov model (HMM) that models the different sequence regions of a signal peptide and the different regions of a transmembrane protein in a series of interconnected states. Training was done on a newly assembled and curated dataset. Compared to TMHMM and SignalP, errors coming from cross-prediction between transmembrane segments and signal peptides were reduced substantially by Phobius. False classifications of signal peptides were reduced from 26.1% to 3.9% and false classifications of transmembrane helices were reduced from 19.0%, to 7.7%. Phobius was applied to the proteomes of Honzo sapiens and Escherichia coli. Here we also noted a drastic reduction of false classifications compared to TMHMM/SignalP, suggesting that Phobius is well suited for whole-genome annotation of signal peptides and transmembrane regions. The method is available at http://phobius.cgb.ki.se/ as well as at http://phobius.binf.ku.dk/.

  • 19. Legendre, Daniel
    et al.
    Laraki, Nezha
    Gräslund, Torbjörn
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Björnvad, Mads E
    Bouchet, Michèle
    Nygren, Per-Åke
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Borchert, Torben V
    Fastrez, Jacques
    Display of active subtilisin 309 on phage: Analysis of parameters influencing the selection of subtilisin variants with changed substrate specificity from libraries using phosphonylating inhibitors2000In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 296, no 1, 87-102 p.Article in journal (Refereed)
    Abstract [en]

    Many attempts have been made to endow enzymes with new catalytic activities. One general strategy involves the creation of random combinatorial Libraries of mutants associated with an efficient screening or selection scheme. Phage display has been shown to greatly facilitate the selection of polypeptides with desired properties by establishing a close Link between the polypeptide and the gene that encodes it. Selection of phage displayed enzymes for new catalytic activities remains a challenge. The aim of this study was to display the serine protease subtilisin 309 (savinase) from Bacillus lentils on the surface of filamentous fd phage and to develop selection schemes that allow the extraction of subtilisin variants with a changed substrate specificity from libraries. Subtilisins are produced as secreted preproenzyme that mature in active enzyme autocatalytically. They have a broad substrate specificity but exhibit a significant preference for hydrophobic residues and very Limited reactivity toward charged residues at the P4 site in the substrate. Here, we show that savinase can be functionally displayed on phage in the presence of the proteic inhibitor CI2. The free enzyme is released from its complex with CI2 upon addition of the anionic detergent LAS. The phage-enzyme can be panned on streptavidin beads after labelling by reaction with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-L-Ala-L-Ala-L-Pro- Phe(P)-diphenyl ester. Reactions of libraries, in which residues 104 and 107 forming part of the S4 pocket have been randomised, with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-alpha-L-Lys-L-Ala- L-Pro-Phe(P)-diphenylester ter allowed us to select enzymes with increased specific activity for a substrate containing a lysine in P4. Parameters influencing the selection as for instance the efficiency of maturation of mutant enzymes in libraries have been investigated.

  • 20.
    Lindahl, E
    et al.
    KTH, Superseded Departments, Physics.
    Elofsson, A
    Stockholm University.
    Identification of related proteins on family, superfamily and fold level.2000In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 295, no 3, 613-25 p.Article in journal (Refereed)
    Abstract [en]

    Proteins might have considerable structural similarities even when no evolutionary relationship of their sequences can be detected. This property is often referred to as the proteins sharing only a "fold". Of course, there are also sequences of common origin in each fold, called a "superfamily", and in them groups of sequences with clear similarities, designated "family". Developing algorithms to reliably identify proteins related at any level is one of the most important challenges in the fast growing field of bioinformatics today. However, it is not at all certain that a method proficient at finding sequence similarities performs well at the other levels, or vice versa.Here, we have compared the performance of various search methods on these different levels of similarity. As expected, we show that it becomes much harder to detect proteins as their sequences diverge. For family related sequences the best method gets 75% of the top hits correct. When the sequences differ but the proteins belong to the same superfamily this drops to 29%, and in the case of proteins with only fold similarity it is as low as 15%. We have made a more complete analysis of the performance of different algorithms than earlier studies, also including threading methods in the comparison. Using this method a more detailed picture emerges, showing multiple sequence information to improve detection on the two closer levels of relationship. We have also compared the different methods of including this information in prediction algorithms. For lower specificities, the best scheme to use is a linking method connecting proteins through an intermediate hit. For higher specificities, better performance is obtained by PSI-BLAST and some procedures using hidden Markov models. We also show that a threading method, THREADER, performs significantly better than any other method at fold recognition.

  • 21.
    Lönneborg, Rosa
    et al.
    Stockholms universitet, Institutionen för biokemi och biofysik.
    Smirnova, Irina
    Dian, Cyril
    Leonard, Gordon A
    Brzezinski, Peter
    Stockholms universitet, Institutionen för biokemi och biofysik.
    In vivo and in vitro investigation of transcriptional regulation by DntR.2007In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 372, no 3, 571-82 p.Article in journal (Refereed)
    Abstract [en]

    DntR is a bacterial transcription factor that has been isolated from Burkholderia species that are able to degrade the nitro-aromatic compound 2,4-dinitrotoluene. We recently solved the X-ray crystal structure of DntR, which suggested a putative location of an inducer-binding cavity (IBC). In this study, we constructed mutants of DntR in which residues lining the proposed IBC were modified in order to identify the structural elements involved in inducer binding, to modulate the inducer binding specificity, and to investigate the mechanism of transcriptional regulation by DntR. The transcriptional activation of the reporter gene gfp induced by the wild-type and mutant DntRs was monitored by analysing whole-cell fluorescence using flow-cytometry after addition of a number of potential inducer compounds. Three of the mutant proteins (F111L; F111V/H169V and Y110S/F111V) were purified and the binding constants for several of the potential inducers to these mutants were estimated. Furthermore, crystal structures of the F111L and Y110S/F111V mutant proteins were solved and used to explain changes in the inducer binding specificity at an atomic level. A comparison of the inducing capability in the whole-cell system and binding constants for a number of potential inducers suggests a mechanism where binding of an inducer molecule is not the sole requirement for transcriptional activation. In addition, specific interactions between DntR and the inducer molecule resulting in a conformational change of the protein are needed.

  • 22. Nilsson, Harriet E.
    et al.
    Ambort, Daniel
    Bäckström, Malin
    Thomsson, Elisabeth
    Koeck, Philip J. B.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Department of Biosciences and Nutrition, Karolinska Institutet.
    Hansson, Gunnar C.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. epartment of Biosciences and Nutrition, Karolinska Institutet.
    Intestinal MUC2 Mucin Supramolecular Topology by Packing and Release Resting on D3 Domain Assembly2014In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 426, no 14, 2567-2579 p.Article in journal (Refereed)
    Abstract [en]

    MUC2 is the major gel-forming mucin of the colon forming a protective gel barrier organized into an inner stratified and an outer loose layer. The MUC2 N-terminus (D1-D2-D'D3 domains) has a dual function in building a net-like structure by disulfide-bonded trimerization and packing the MUC2 polymer into an N-terminal concatenated polygonal platform with the C-termini extending perpendicularly by pH- and calcium-dependent interactions. We studied the N-terminal D'D3 domain by producing three recombinant variants, with or without Myc tag and GFP (green fluorescent protein), and analyzed these by gel filtration, electron microscopy and single particle image processing. The three variants were all trimers when analyzed upon denaturing conditions but eluted as hexanners upon gel filtration under native conditions. Studies by electron microscopy and three-dimensional maps revealed cage-like structures with 2- and 3-fold symmetries. The structure of the MUC2 D3 domain confirms that the MUC2 mucin forms branched net-like structures. This suggests that the MUC2 mucin is stored with two N-terminal concatenated ring platforms turned by 180 against each other, implicating that every second unfolded MUC2 net in mature mucus is turned upside down.

  • 23. Perez, Jean-Baptiste
    et al.
    Segura, Jean-Manuel
    Abankwa, Daniel
    Piguet, Joachim
    Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland.
    Martinez, Karen L
    Vogel, Horst
    Monitoring the diffusion of single heterotrimeric G proteins in supported cell-membrane sheets reveals their partitioning into microdomains.2006In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 363, no 5Article in journal (Refereed)
    Abstract [en]

    Supported cell-membrane sheets are promising in vitro systems to investigate the properties of membranes with native protein/lipid composition, in particular their sub-compartmentalization and the differential localization of proteins associated to them. While such studies are usually performed using static microscopy techniques, we demonstrate here the potential offered by dynamic diffusion measurements. Whereas the overall fluidity of the lipid bilayer was preserved, the preparation of the membrane sheets led to the selective immobilization of extracellular and transmembrane (TM) glycosylated proteins and the anchored proteins/lipids associated with them. Taking advantage of this, we investigated the association of the G protein Gq with TM proteins, in particular G-protein coupled receptors (GPCRs), by monitoring the changes in diffusion occurring after preparation of the supported membranes. Two fluorescently tagged Galphaq proteins were constructed, which remained either mostly monomeric in the plasma membrane or associated with Gbetagamma in heterotrimers. While both constructs diffused similarly in living cells, the preparation of the supported membranes led to the selective immobilization of the heterotrimers with minimal changes of the diffusion of the monomeric Galphaq. The diverse mobility of monomeric and heterotrimeric Galphaq was a result of their different lipid anchors as demonstrated by monitoring the diffusion of the corresponding anchors alone. We propose that the immobilization of the heterotrimer was caused by its partitioning inside membrane microdomains surrounding GPCRs.

  • 24. Schlegel, Susan
    et al.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Lee, Chiara
    Hjelm, Anna
    Klepsch, Mirjam
    Strous, Marc
    Drew, David
    Slotboom, Dirk Jan
    de Gier, Jan-Willem
    Optimizing Membrane Protein Overexpression in the Escherichia coli strain Lemo21(DE3)2012In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 423, no 4, 648-659 p.Article in journal (Refereed)
    Abstract [en]

    Escherichia coli BL21(DE3) is widely used to overexpress proteins. In this overexpression host, the gene encoding the target protein is located on a plasmid and is under control of the T7 promoter, which is recognized exclusively by the T7 RNA polymerase (RNAP). The 17 RNAP gene is localized on the chromosome, and its expression is governed by the non-titratable, IPTG-inducible lacUV5 promoter. Recently, we constructed the Lemo21(DE3) strain, which allows improved control over the expression of genes from the 17 promoter. Lemo21(DE3) is a BL21(DE3) strain equipped with a plasmid harboring the gene encoding T7 lysozyme, an inhibitor of the T7 RNAP, under control of the exceptionally well-titratable rhamnose promoter. The overexpression yields of a large collection of membrane proteins in Lemo21(DE3) at different concentrations of rhamnose indicated that this strain may be very suitable for optimizing the production of membrane proteins. However, insight in the mechanism by which optimized expression yields are achieved in Lemo21(DE3) is lacking. Furthermore, whether the overexpressed proteins are suitable for functional and structural studies remains to be tested. Here, we show that in Lemo21(DE3), (i) the modulation of the activity of the 17 RNAP by the 17 lysozyme is key to optimizing the ratio of membrane proteins properly inserted in the cytoplasmic membrane to non-inserted proteins; (ii) maximizing the yields of membrane proteins is accompanied by reduction of the adverse effects of membrane protein overexpression, resulting in stable overexpression; and (iii) produced membrane proteins can be used for functional and structural studies.

  • 25. Seibert, M. Marvin
    et al.
    Patriksson, Alexandra
    Hess, Berk
    Max Planck Institute for Polymer Research.
    van der Spoel, David
    Reproducible polypeptide folding and structure prediction using molecular dynamics simulations2005In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 354, no 1, 173-183 p.Article in journal (Refereed)
    Abstract [en]

    The folding of a polypeptide from an extended state to a well-defined conformation is studied using microsecond classical molecular dynamics (MD) simulations and replica exchange molecular dynamics (REMD) simulations in explicit solvent and in vacuo. It is shown that the solvated peptide folds many times in the REMD simulations but only a few times in the conventional simulations. From the folding events in the classical simulations we estimate an approximate folding time of 1-2 micros. The REMD simulations allow enough sampling to deduce a detailed Gibbs free energy landscape in three dimensions. The global minimum of the energy landscape corresponds to the native state of the peptide as determined previously by nuclear magnetic resonance (NMR) experiments. Starting from an extended state it takes about 50 ns before the native structure appears in the REMD simulations, about an order of magnitude faster than conventional MD. The calculated melting curve is in good qualitative agreement with experiment. In vacuo, the peptide collapses rapidly to a conformation that is substantially different from the native state in solvent.

  • 26. Stahlberg, J
    et al.
    Divne, Christina
    University of Uppsala, Sweden.
    Koivula, A
    Piens, K
    Claeyssens, M
    Teeri, T T
    Jones, T A
    Activity studies and crystal structures of catalytically deficient mutants of cellobiohydrolase I from Trichoderma reesei1996In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 264, no 2, 337-349 p.Article in journal (Refereed)
  • 27. Stahlberg, J.
    et al.
    Henriksson, H.
    Divne, Christina
    KTH, Superseded Departments, Biotechnology.
    Isaksson, R.
    Pettersson, G.
    Johansson, G.
    Jones, T. A.
    Structural basis for enantiomer binding and separation of a common beta-blocker: Crystal structure of cellobiohydrolase Cel7A with bound (S)-propranolol at 1.9 angstrom resolution2001In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 305, no 1, 79-93 p.Article in journal (Refereed)
    Abstract [en]

    Cellobiohydrolase Cel7A (previously called CBH 1), the major cellulase produced by the mould fungus Trichoderma reesei, has been successfully exploited as a chiral selector for separation of stereo-isomers of some important pharmaceutical compounds, e.g. adrenergic beta -blockers. Previous investigations, including experiments with catalytically deficient mutants of Cel7A, point unanimously to the active site as being responsible for discrimination of enantiomers. In this work the structural basis for enantioselectivity of basic drugs by Cel7A has been studied by X-ray crystallography. The catalytic domain of Cel7A was co-crystallised with the (S)-enantiomer of a common beta -blocker, propranolol, at pH 7, and the structure of the complex was determined and refined at 1.9 Angstrom resolution. Indeed, (S)-propranolol binds at the active site, in glucosyl-binding subsites -1/ + 1. The catalytic residues Glu212 and Glu217 make tight salt links with the secondary amino group of (S)-propranolol. The oxygen atom attached to the chiral centre of (S)-propranolol forms hydrogen bonds to the nucleophile Glu212 O-epsilon1 and to Gln175 N-epsilon2, whereas the aromatic naphthyl moiety stacks with the indole ring of Trp376 in site +1. The bidentate charge interaction with the catalytic glutamate residues is apparently crucial, since no enantioselectivity has been obtained with the catalytically deficient mutants E212Q and E217Q. Activity inhibition experiments with wild-type Cel7A were performed in conditions close to those used for crystallisation. Competitive inhibition constants for (R)- and (S)-propranolol were determined at 220 muM and 44 muM, respectively, corresponding to binding free energies of 20 kJ/ mol and 24 kJ/mol, respectively. The K-i value for (R)-propranolol was 57-fold lower than the highest concentration, 12.5 mM, used in co-crystallisation experiments. Still several attempts to obtain a complex with the (R)-enantiomer have failed. By using cellobiose as a selective competing ligand, the retention of the enantiomers of propranolol on the chiral stationary phase (CSP) based on Cel7A mutant D214N were resolved into enantioselective and non-selective binding. The enantioselective binding was weaker for both enantiomers on D214N-CSP than on wild-type-CSP.

  • 28.
    Tan, Tien-Chye
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Haltrich, Dietmar
    Divne, Christina
    KTH, School of Biotechnology (BIO), Biochemistry.
    Regioselective Control of beta-D-Glucose Oxidation by Pyranose 2-Oxidase Is Intimately Coupled to Conformational Degeneracy2011In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 409, no 4, 588-600 p.Article in journal (Refereed)
    Abstract [en]

    Trametes multicolor pyranose 2-oxidase (P2O) is a flavoprotein oxidase that oxidizes D-glucose at C2 to 2-keto-D-glucose by a highly regioselective mechanism. In this work, fluorinated sugar substrates were used as mechanistic probes to investigate the basis of regioselectivity in P2O. Although frequently used to study the mechanisms of glycoside hydrolases, our work provides the first example of applying these probes to sugar oxidoreductases. Our previous structure of the P2O mutant H167A in complex with the slow substrate 2-deoxy-2-fluoro-D-glucose showed a substrate-binding mode compatible with oxidation at C3. To accommodate the sugar, a gating segment, (FSY456)-F-454, in the substrate recognition loop partly unfolded to create a spacious and more polar active site that is distinct from the closed state of P2O. The crystal structure presented here shows that the preferred C2 oxidation where an ordered complex of P2O H167A with 3-deoxy-3-fluoro-D-glucose at 1.35 angstrom resolution was successfully trapped. In this semi-open C2-oxidation complex, the substrate recognition loop tightens to form an optimized substrate complex stabilized by interactions between Asp452 and glucose O4, as well as Tyr456 and the glucose O6 group, interactions that are not possible when glucose is positioned for oxidation at C3. The different conformations of the (FSY456)-F-454 gating segment in the semiopen and closed states induce backbone and side-chain movements of Thr169 and Asp452 that add further differential stabilization to the individual states. We expect the semi-open state (C2-oxidation state) and closed state to be good approximations of the active-site structure during the reductive half-reaction (sugar oxidation) and oxidative half-reaction (O-2 reduction).

  • 29. Tan, Tien-Chye
    et al.
    Mijts, Benjamin N.
    Swaminathan, Kunchithapadam
    Patel, Bharat K. C.
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Crystal structure of the polyextremophilic alpha-amylase AmyB from Halothermothrix orenii: Details of a productive enzyme-substrate complex and an N domain with a role in binding raw starch2008In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 378, no 4, 852-870 p.Article in journal (Refereed)
    Abstract [en]

    The gene for a membrane-bound, halophilic, and thermostable alpha-amylase, AmyB, from Halothermothrix orenii was cloned and sequenced. The crystal structure shows that, in addition to the typical domain organization of family 13 glycoside hydrolases, AmyB carries an additional N-terminal domain (N domain) that forms a large groove-the N-C groove some 30 angstrom away from the active site. The structure of AmyB with the inhibitor acarbose at 1.35 angstrom resolution shows that a nonasaccharide has been synthesized through successive transglycosylation reactions of acarbose. Unexpectedly, in a complex of wild-type AmyB with alpha-cyclodextrin and maltoheptaose at 2.2 angstrom resolution, a maltotetraose molecule is bound in subsites -1 to +3, spanning the cleavage point at -1 / + 1, with the -1 glucosyl residue present as a S-2(o) skew boat. This wild-type AmyB complex was obtained in the presence of a large excess of substrate, a condition under which it is possible to capture Michaelis complexes, which may explain the observed binding across -1/+ 1 and ring distortion. We observe three methionine side chains that serve as '' binding platforms '' for glucosyl rings in AmyB, a seemingly rare occurrence in carbohydrate-binding proteins. The structures and results from the biochemical characterization of AmyB and AmyB lacking the N domain show that the N domain increases binding of the enzyme to raw starch. Furthermore, theoretical modeling suggests that the N-C groove can accommodate, spatially and chemically, large substrates such as A-starch.

  • 30.
    Tan, Tien-Chye
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Pitsawong, Warintra
    Wongnate, Thanyaporn
    Spadiut, Oliver
    KTH, School of Biotechnology (BIO), Biochemistry. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Haltrich, Dietmar
    Chaiyen, Pimchai
    Divne, Christina
    KTH, School of Biotechnology (BIO), Biochemistry. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    H-Bonding and Positive Charge at the N(5)/O(4) Locus Are Critical for Covalent Flavin Attachment in Trametes Pyranose 2-Oxidase2010In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 402, no 3, 578-594 p.Article in journal (Refereed)
    Abstract [en]

    Flavoenzymes perform a wide range of redox reactions in nature, and a subclass of flavoenzymes carry covalently bound cofactor. The enzyme-flavin bond helps to increase the flavin's redox potential to facilitate substrate oxidation in several oxidases. The formation of the enzyme-flavin covalent bond-the flavinylation reaction-has been studied for the past 40 years. For the most advocated mechanism of autocatalytic flavinylation, the quinone methide mechanism, appropriate stabilization of developing negative charges at the flavin N(1) and N(5) loci is crucial. Whereas the structural basis for stabilization at N(1) is relatively well studied, the structural requisites for charge stabilization at N(5) remain less clear. Here, we show that flavinylation of histidine 167 of pyranose 2-oxidase from Trametes multicolor requires hydrogen bonding at the flavin N(5)/O(4) locus, which is offered by the side chain of Thr169 when the enzyme is in its closed, but not open, state. Moreover, our data show that additional stabilization at N(5) by histidine 548 is required to ensure high occupancy of the histidyl flavin bond. The combination of structural and spectral data on pyranose 2-oxidase mutants supports the quinone methide mechanism. Our results demonstrate an elaborate structural fine-tuning of the active site to complete its own formation that couples efficient holoenzyme synthesis to conformational substates of the substrate-recognition loop and concerted movements of side chains near the flavinylation ligand. (c) 2010 Elsevier Ltd. All rights reserved.

  • 31. von Ossowski, I.
    et al.
    Stahlberg, J.
    Koivula, A.
    Piens, K.
    Becker, D.
    Boer, H.
    Harle, R.
    Harris, M.
    Divne, Christina
    KTH, Superseded Departments, Biotechnology.
    Mahdi, S.
    Zhao, Y. X.
    Driguez, H.
    Claeyssens, M.
    Sinnott, M. L.
    Teeri, Tuula T.
    KTH, Superseded Departments, Biotechnology.
    Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Ce17A. A comparison with Phanerochaete chrysosporium Cel7D2003In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 333, no 4, 817-829 p.Article in journal (Refereed)
    Abstract [en]

    The exo-loop of Trichoderma reesei cellobiohydrolase Cel7A forms the roof of the active site tunnel at the catalytic centre. Mutants were designed to study the role of this loop in crystalline cellulose degradation. A hydrogen bond to substrate made by a tyrosine at the tip of the loop was removed by the Y247F mutation. The mobility of the loop was reduced by introducing a new disulphide bridge in the mutant D241C/D249C. The tip of the loop was deleted in mutant Delta(G245-Y252). No major structural disturbances were observed in the mutant enzymes, nor was the thermostability of the enzyme affected by the mutations. The Y247F mutation caused a slight k(cat) reduction on 4-nitrophenyl lactoside, but only a small effect on cellulose hydrolysis. Deletion of the tip of the loop increased both k(cat) and K-M and gave reduced product inhibition. Increased activity was observed on amorphous cellulose, while only half the original activity remained on crystalline cellulose. Stabilisation of the exo-loop by the disulphide bridge enhanced the activity on both amorphous and crystalline cellulose. The ratio Glc(2)/(Glc(3) + Glc(1)) released from cellulose, which is indicative of processive action, was highest with Tr Cel7A wild-type enzyme and smallest with the deletion mutant on both substrates. Based on these data it seems that the exo-loop of Tr Cel7A has evolved to facilitate processive crystalline cellulose degradation, which does not require significant conformational changes of this loop.

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