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  • 1. Ariza, A.
    et al.
    Eklöf, Jens
    KTH, School of Biotechnology (BIO), Glycoscience.
    Spadiut, Oliver
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Offen, W.A.
    Roberts, S.M.
    Wilson, K.S.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Davies, G.J.
    Structure and Activity of a Paenibacillus polymyxa Xyloglucanase from Glycoside Hydrolase Family 442011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 39, p. 33890-33900Article in journal (Refereed)
    Abstract [en]

    The enzymatic degradation of plant polysaccharides is emerging as one of the key environmental goals of the early 21st century, impacting on many processes in the textile and detergent industries as well as biomass conversion to biofuels. One of the well known problems with the use of nonstarch (nonfood)-based substrates such as the plant cell wall is that the cellulose fibers are embedded in a network of diverse polysaccharides, including xyloglucan, that renders access difficult. There is therefore increasing interest in the "accessory enzymes," including xyloglucanases, that may aid biomass degradation through removal of "hemicellulose" polysaccharides. Here, we report the biochemical characterization of the endo-beta-1,4-(xylo)glucan hydrolase from Paenibacillus polymyxa with polymeric, oligomeric, and defined chromogenic aryl-oligosaccharide substrates. The enzyme displays an unusual specificity on defined xyloglucan oligosaccharides, cleaving the XXXG-XXXG repeat into XXX and GXXXG. Kinetic analysis on defined oligosaccharides and on aryl-glycosides suggests that both the -4 and +1 subsites show discrimination against xylose-appended glucosides. The three-dimensional structures of PpXG44 have been solved both in apo-form and as a series of ligand complexes that map the -3 to -1 and +1 to +5 subsites of the extended ligand binding cleft. Complex structures are consistent with partial intolerance of xylosides in the -4' subsites. The atypical specificity of PpXG44 may thus find use in industrial processes involving xyloglucan degradation, such as biomass conversion, or in the emerging exciting applications of defined xyloglucans in food, pharmaceuticals, and cellulose fiber modification.

  • 2. Azarias, Guillaume
    et al.
    Kruusmagi, Markus
    Connor, Siobhan
    Akkuratov, Evgeny E.
    Liu, Xiao-Li
    Lyons, David
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Broberger, Christian
    Aperia, Anita
    A Specific and Essential Role for Na,K-ATPase alpha 3 in Neurons Co-expressing alpha 1 and alpha 32013In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 4, p. 2734-2743Article in journal (Refereed)
    Abstract [en]

    Most neurons co-express two catalytic isoforms of Na,K-ATPase, the ubiquitous alpha 1, and the more selectively expressed alpha 3. Although neurological syndromes are associated with alpha 3 mutations, the specific role of this isoform is not completely understood. Here, we used electrophysiological and Na+ imaging techniques to study the role of alpha 3 in central nervous system neurons expressing both isoforms. Under basal conditions, selective inhibition of alpha 3 using a low concentration of the cardiac glycoside, ouabain, resulted in a modest increase in intracellular Na+ concentration ([Na+](i)) accompanied by membrane potential depolarization. When neurons were challenged with a large rapid increase in [Na+](i), similar to what could be expected following suprathreshold neuronal activity, selective inhibition of alpha 3 almost completely abolished the capacity to restore [Na+](i) in soma and dendrite. Recordings of Na, K-ATPase specific current supported the notion that when [Na+](i) is elevated in the neuron, alpha 3 is the predominant isoform responsible for rapid extrusion of Na+. Low concentrations of ouabain were also found to disrupt cortical network oscillations, providing further support for the importance of alpha 3 function in the central nervous system. The alpha isoforms express a well conserved protein kinase A consensus site, which is structurally associated with an Na+ binding site. Following activation of protein kinase A, both the alpha 3-dependent current and restoration of dendritic [Na+](i) were significantly attenuated, indicating that alpha 3 is a target for phosphorylation and may participate in short term regulation of neuronal function.

  • 3. Brouns, Stan J. J.
    et al.
    Walther, Jasper
    Snijders, Ambrosius P. L.
    de Werken, Harmen J. G. van
    Willemen, Hanneke L. D. M.
    Worm, Petra
    de Vos, Marjon G. J.
    Andersson, Anders
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundgren, Magnus
    Mazon, Hortense F. M.
    van den Heuvel, Robert H. H.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics.
    Salmon, Laurent
    de Vos, Willem M.
    Wright, Phillip C.
    Bernander, Rolf
    van der Oost, John
    Identification of the missing links in prokaryotic pentose oxidation pathways - Evidence for enzyme recruitment2006In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 37, p. 27378-27388Article in journal (Refereed)
    Abstract [en]

    The pentose metabolism of Archaea is largely unknown. Here, we have employed an integrated genomics approach including DNA microarray and proteomics analyses to elucidate the catabolic pathway for D-arabinose in Sulfolobus solfataricus. During growth on this sugar, a small set of genes appeared to be differentially expressed compared with growth on D-glucose. These genes were heterologously overexpressed in Escherichia coli, and the recombinant proteins were purified and biochemically studied. This showed that D-arabinose is oxidized to 2-oxoglutarate by the consecutive action of a number of previously uncharacterized enzymes, including a D-arabinose dehydrogenase, a D-arabinonate dehydratase, a novel 2-keto-3-deoxy-D-arabinonate dehydratase, and a 2,5-dioxopentanoate dehydrogenase. Promoter analysis of these genes revealed a palindromic sequence upstream of the TATA box, which is likely to be involved in their concerted transcriptional control. Integration of the obtained biochemical data with genomic context analysis strongly suggests the occurrence of pentose oxidation pathways in both Archaea and Bacteria, and predicts the involvement of additional enzyme components. Moreover, it revealed striking genetic similarities between the catabolic pathways for pentoses, hexaric acids, and hydroxyproline degradation, which support the theory of metabolic pathway genesis by enzyme recruitment.

  • 4. Cartmell, Alan
    et al.
    McKee, Lauren
    KTH, School of Biotechnology (BIO). University of Georgia, United States.
    Pena, Maria J.
    Larsbrink, Johan
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Kaneko, Satoshi
    Ichinose, Hitomi
    Lewis, Richard J.
    Vikso-Nielsen, Anders
    Gilbert, Harry J.
    Marles-Wright, Jon
    The structure and function of an arabinan-specific alpha-1,2-arabinofuranosidase identified from screening the activities of bacterial GH43 glycoside hydrolases2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 17Article in journal (Refereed)
    Abstract [en]

    Reflecting the diverse chemistry of plant cell walls, microorganisms that degrade these composite structures synthesize an array of glycoside hydrolases. These enzymes are organized into sequence-, mechanism-, and structure-based families. Genomic data have shown that several organisms that degrade the plant cell wall contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. Here we report the biochemical properties of the GH43 enzymes of a saprophytic soil bacterium, Cellvibrio japonicus, and a human colonic symbiont, Bacteroides thetaiotaomicron. The data show that C. japonicus uses predominantly exo-acting enzymes to degrade arabinan into arabinose, whereas B. thetaiotaomicron deploys a combination of endo-and side chain-cleaving glycoside hydrolases. Both organisms, however, utilize an arabinan-specific alpha-1,2-arabinofuranosidase in the degradative process, an activity that has not previously been reported. The enzyme can cleave alpha-1,2-arabinofuranose decorations in single or double substitutions, the latter being recalcitrant to the action of other arabinofuranosidases. The crystal structure of the C. japonicus arabinan-specific alpha-1,2-arabinofuranosidase, CjAbf43A, displays a five-bladed beta-propeller fold. The specificity of the enzyme for arabinan is conferred by a surface cleft that is complementary to the helical backbone of the polysaccharide. The specificity of CjAbf43A for alpha-1,2-L-arabinofuranose side chains is conferred by a polar residue that orientates the arabinan backbone such that O2 arabinose decorations are directed into the active site pocket. A shelflike structure adjacent to the active site pocket accommodates O3 arabinose side chains, explaining how the enzyme can target O2 linkages that are components of single or double substitutions.

  • 5.
    Eklöf, Jens M.
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Shojania, S.
    Okon, M.
    McIntosh, L. P.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Structure-function analysis of a broad specificity Populus trichocarpa endo-β-glucanase reveals an evolutionary link between bacterial licheninases and plant XTH gene products2013In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 22, p. 15786-15799Article in journal (Refereed)
    Abstract [en]

    The large xyloglucan endotransglycosylase/hydrolase (XTH) gene family continues to be the focus of much attention in studies of plant cell wall morphogenesis due to the unique catalytic functions of the enzymes it encodes. The XTH gene products compose a subfamily of glycoside hydrolase family 16 (GH16), which also comprises a broad range of microbial endoglucanases and endogalactanases, as well as yeast cell wall chitin/β-glucan transglycosylases. Previous whole-family phylogenetic analyses have suggested that the closest relatives to the XTH gene products are the bacterial licheninases (EC 3.2.1.73), which specifically hydrolyze linear mixed linkage β(1→3)/β(1→4)-glucans. In addition to their specificity for the highly branched xyloglucan polysaccharide, XTH gene products are distinguished from the licheninases and other GH16 enzyme subfamilies by significant active site loop alterations and a large C-terminal extension. Given these differences, the molecular evolution of the XTH gene products in GH16 has remained enigmatic. Here, we present the biochemical and structural analysis of a unique, mixed function endoglucanase from black cottonwood (Populus trichocarpa), which reveals a small, newly recognized subfamily of GH16 members intermediate between the bacterial licheninases and plant XTH gene products. We postulate that this clade comprises an important link in the evolution of the large plant XTH gene families from a putative microbial ancestor. As such, this analysis provides new insights into the diversification of GH16 and further unites the apparently disparate members of this important family of proteins. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

  • 6. Gloster, Tracey M.
    et al.
    Ibatullin, Farid M.
    Macauley, Katherine
    Eklöf, Jens
    KTH, School of Biotechnology (BIO), Glycoscience.
    Roberts, Shirley
    Turkenburg, Johan P.
    Bjornvad, Mads E.
    Jorgensen, Per Lina
    Danielsen, Steffen
    Johansen, Katja S.
    Borchert, Torben V.
    Wilson, Keith S.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Davies, Gideon J.
    Characterization and three-dimensional structures of two distinct bacterial xyloglucanases from families GH5 and GH122007In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 26, p. 19177-19189Article in journal (Refereed)
    Abstract [en]

    The plant cell wall is a complex material in which the cellulose microfibrils are embedded within a mesh of other polysaccharides, some of which are loosely termed hemicellulose. One such hemicellulose is xyloglucan, which displays a beta-1,4-linked D-glucose backbone substituted with xylose, galactose, and occasionally fucose moieties. Both xyloglucan and the enzymes responsible for its modification and degradation are finding increasing prominence, reflecting both the drive for enzymatic biomass conversion, their role in detergent applications, and the utility of modified xyloglucans for cellulose fiber modification. Here we present the enzymatic characterization and three-dimensional structures in ligand free and xyloglucan- oligosaccharide complexed forms of two distinct xyloglucanases from glycoside hydrolase families GH5 and GH12. The enzymes, Paenibacillus pabuli XG5 and Bacillus licheniformis XG12, both display open active center grooves grafted upon their respective (beta/alpha)(8) and beta-jelly roll folds, in which the side chain decorations of xyloglucan may be accommodated. For the beta-jelly roll enzyme topology of GH12, binding of xylosyl and pendant galactosyl moieties is tolerated, but the enzymeis similarly competent in the degradation of unbranched glucans. In the case of the (beta/alpha)(8) GH5 enzyme, kinetically productive interactions are made with both xylose and galactose substituents, as reflected in both a high specific activity on xyloglucan and the kinetics of a series of aryl glycosides. The differential strategies for the accommodation of the side chains of xyloglucan presumably facilitate the action of these microbial hydrolases in milieus where diverse and differently substituted substrates may be encountered.

  • 7. Goldstein, S.
    et al.
    Czapski, G.
    Lind, Johan
    KTH, Superseded Departments, Chemistry.
    Merenyi, Gabor
    KTH, Superseded Departments, Chemistry.
    Tyrosine nitration by simultaneous generation of (NO)-N-center dot and O-2(center dot) under physiological conditions - How the radicals do the job2000In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, no 5, p. 3031-3036Article in journal (Refereed)
    Abstract [en]

    Radiation chemical experiments demonstrate that the reaction of tyrosyl radical (TyrO(.)) with (NO2)-N-. yields 45 +/- 3% 3-nitrotyrosine and that a major product of the reaction of TyrO(.) with (NO)-N-. is 3,3'-dityrosine. Radiolysis was used to generate (NO)-N-. and O-2(-.) in the presence of tyrosine and bicarbonate at pH 7.5 +/- 0.1. The nitration yield was found to be dose rate-dependent, and the yield per radical produced by pulse radiolysis was identical to that obtained with authentic peroxynitrite, The proposed mechanism that accounts for the data is as follows: (i) In the presence of CO2 the reaction of (NO)-N-. with O-2(-.) yields 33% (NO2)-N-. and CO3-. where the latter reacts rapidly with tyrosine to form TyrO; (ii) The formation of 3-nitrotyrosine takes place via the reaction of (NO2)-N-. with TyrO(.), which is the main process at high dose rates; and (iii) Under continuous generation of (NO)-N-. and O-2(-.) the formation of 3-nitrotyrosine is strongly suppressed because of efficient scavenging of NO2, by tyrosine. The proposed model shows that the highest nitration yield is obtained for similar fluxes of (NO)-N-. and O-2(-.) and is completely inhibited upon excess production of O-2(-.) because of efficient scavenging of TyrO(.) by O-2(-.). The biological implications of these findings are discussed.

  • 8.
    Gräslund, Torbjörn
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Li, X. L.
    Magnenat, L.
    Popkov, M.
    Barbas, C. F.
    Exploring strategies for the design of artificial transcription factors2005In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 5, p. 3707-3714Article in journal (Refereed)
    Abstract [en]

    Artificial transcription factors can be engineered to interact with specific DNA sequences to modulate endogenous gene expression within cells. A significant hurdle to implementation of this approach is the selection of the appropriate DNA sequence for targeting. We reasoned that a good target site should be located in chromatin, where it is accessible to DNA-binding proteins, and it should be, in the close vicinity of known transcriptional regulators of the gene. Here we have explored the efficacy of these criteria to guide our selection of potential regulators of gamma-globin expression. Several zinc finger-based transcriptional activators were designed to target the sites proximal to the -117-position of the gamma-globin promoter. This region is proximal to the binding sites of known and potential natural transcription factors. Design and study of three transcription factors identified the potent transcriptional activator, ggl-VP64-RA. This transcription factor was able to interact directly with the gamma-globin promoter and up-regulate expression of reporter gene constructs as well as the endogenous gene in a selective manner. Transfection of a gg1-VP64-RA expression vector or retroviral delivery of this transcription factor into the erythroleukemia cell line K562 resulted in an increase of fetal hemoglobin. The gamma-globin content of cells expressing gg1-vp64-HA showed up to 16-fold higher levels of fetal hemoglobin than the native K562 cell line. These transcriptional activators constitute a novel class of regulators of the globin locus that may be suitable for treatment of diseases arising from mutations in this locus such as sickle cell disease and thalassemic diseases.

  • 9. Guce, Abigail I.
    et al.
    Clark, Nathaniel E.
    Salgado, Eric N.
    Ivanen, Dina R.
    Kulminskaya, Anna A.
    Brumer, Harry, III
    Garman, Scott C.
    Catalytic Mechanism of Human alpha-Galactosidase2010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 6, p. 3625-3632Article in journal (Refereed)
    Abstract [en]

    The enzyme alpha-galactosidase (alpha-GAL, also known as alpha-GAL A; E.C. 3.2.1.22) is responsible for the breakdown of alpha-galactosides in the lysosome. Defects in human alpha-GAL lead to the development of Fabry disease, a lysosomal storage disorder characterized by the buildup of alpha-galactosylated substrates in the tissues. alpha-GAL is an active target of clinical research: there are currently two treatment options for Fabry disease, recombinant enzyme replacement therapy (approved in the United States in 2003) and pharmacological chaperone therapy (currently in clinical trials). Previously, we have reported the structure of human alpha-GAL, which revealed the overall structure of the enzyme and established the locations of hundreds of mutations that lead to the development of Fabry disease. Here, we describe the catalytic mechanism of the enzyme derived from x-ray crystal structures of each of the four stages of the double displacement reaction mechanism. Use of a difluoro-alpha-galactopyranoside allowed trapping of a covalent intermediate. The ensemble of structures reveals distortion of the ligand into a S-1(3) skew (or twist) boat conformation in the middle of the reaction cycle. The high resolution structures of each step in the catalytic cycle will allow for improved drug design efforts on alpha-GAL and other glycoside hydrolase family 27 enzymes by developing ligands that specifically target different states of the catalytic cycle. Additionally, the structures revealed a second ligand-binding site suitable for targeting by novel pharmacological chaperones.

  • 10. Hallberg, B. M.
    et al.
    Henriksson, Gunnar
    KTH, Superseded Departments, Pulp and Paper Technology.
    Pettersson, G.
    Vasella, A.
    Divne, Christina
    KTH, Superseded Departments, Biotechnology.
    Mechanism of the reductive half-reaction in cellobiose dehydrogenase2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 9, p. 7160-7166Article in journal (Refereed)
    Abstract [en]

    The extracellular flavocytochrome cellobiose dehydrogenase (CDH; EC 1.1.99.18) participates in lignocellulose degradation by white-rot fungi with a proposed role in the early events of wood degradation. The complete hemoflavoenzyme consists of a catalytically active dehydrogenase fragment (DHcdh) connected to a b-type cytochrome domain via a linker peptide. In the reductive half-reaction, DHcdh catalyzes the oxidation of cellobiose to yield cellobiono-1,5-lactone. The active site of DHcdh is structurally similar to that of glucose oxidase and cholesterol oxidase, with a conserved histidine residue positioned at the re face of the flavin ring close to the N5 atom. The mechanisms of oxidation in glucose oxidase and cholesterol oxidase are still poorly understood, partly because of lack of experimental structure data or difficulties in interpreting existing data for enzyme-ligand complexes. Here we report the crystal structure of the Phanerochaete chrysosporium DHcdh with a bound inhibitor, cellobiono-1,5-lactam, at 1.8-Angstrom resolution. The distance between the lactam C1 and the flavin N5 is only 2.9 Angstrom, implying that in an approximately planar transition state, the maximum distance for the axial 1-hydrogen to travel for covalent addition to N5 is 0.8-0.9 Angstrom. The lactam O1 interacts intimately with the side chains of His-689 and Asn-732. Our data lend substantial structural support to a reaction mechanism where His-689 acts as a general base by abstracting the O1 hydroxyl proton in concert with transfer of the C1 hydrogen as hydride to the re face of the flavin N5.

  • 11.
    Hedengren-Olcott, Marika
    et al.
    Oregon State University.
    Olcott, Michael
    Oregon State University.
    Mooney, Duane, T
    Oregon State University.
    Ekengren, Sophia
    Stockholm University.
    Geller, Bruce, L
    Oregon State University.
    Taylor, Barbara, J.
    Oregon State University.
    Differential Activation of the NF-κB-like Factors Relish and Dif in Drosophila melanogaster by Fungi and Gram-positive Bacteria2004In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, p. 21121-21127Article in journal (Refereed)
    Abstract [en]

    The current model of immune activation in Drosophila melanogaster suggests that fungi and Gram-positive (G(+)) bacteria activate the Toll/Dif pathway and that Gram-negative (G(-)) bacteria activate the Imd/Relish pathway. To test this model, we examined the response of Relish and Dif (Dorsal-related immunity factor) mutants to challenge by various fungi and G(+) and G(-) bacteria. In Relish mutants, the Cecropin A gene was induced by the G(+) bacteria Micrococcus luteus and Staphylococcus aureus, but not by other G(+) or G(-) bacteria. This Relish-independent Cecropin A induction was blocked in Dif/Relish double mutant flies. Induction of the Cecropin A1 gene by M. luteus required Relish, whereas induction of the Cecropin A2 gene required Dif. Intact peptidoglycan (PG) was necessary for this differential induction of Cecropin A. PG extracted from M. luteus induced Cecropin A in Relish mutants, whereas PGs from the G(+) bacteria Bacillus megaterium and Bacillus subtilis did not, suggesting that the Drosophila immune system can distinguish PGs from various G(+) bacteria. Various fungi stimulated antimicrobial peptides through at least two different pathways requiring Relish and/or Dif. Induction of Attacin A by Geotrichum candidum required Relish, whereas activation by Beauvaria bassiana required Dif, suggesting that the Drosophila immune system can distinguish between at least these two fungi. We conclude that the Drosophila immune system is more complex than the current model. We propose a new model to account for this immune system complexity, incorporating distinct pattern recognition receptors of the Drosophila immune system, which can distinguish between various fungi and G(+) bacteria, thereby leading to selective induction of antimicrobial peptides via differential activation of Relish and Dif.

  • 12. Hrmova, M.
    et al.
    Imai, T.
    Rutten, S. J.
    Fairweather, J. K.
    Pelosi, L.
    Bulone, Vincent
    KTH, Superseded Departments, Biotechnology.
    Driguez, H.
    Fincher, G. B.
    Mutated barley (1,3)-beta-D-glucan endohydrolases synthesize crystalline (1,3)-beta-D-glucans2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 33, p. 30102-30111Article in journal (Refereed)
    Abstract [en]

    Barley (1,3)-beta-D-glucan endohydrolases (EC 3.2.1.39), inactivated by site-directed mutagenesis of their catalytic nucleophiles, show autocondensation glucosynthetic activity with alpha-laminaribiosyl fluoride and heterocondensation glycosynthetic activity with a-laminaribiosyl fluoride and 4'-nitrophenyl beta-D-glucopyranoside. The native enzyme is a retaining endohydrolase of the family 17 group and catalyzes glycosyl transfer reactions at high substrate concentrations. Catalytic efficiencies (k(cat) K-m(-1)) of mutants E231G, E231S, and E231A as glycosynthases are 28.9, 0.9, and 0.5 x 10(-4) M-1 s(-1), respectively. Glycosynthase reactions appear to be processive and proceed with pH optima of 6-8 and yields of up to 75%. Insoluble products formed during the glycosynthase reaction appear as lamellar, hexagonal crystals when observed by electron microscopy. Methylation, NMR, and matrix-assisted laser desorption ionization time-of-flight analyses show that the reaction products are linear (1,3)-beta-D-glucans with a degree of polymerization of 30-34, whereas electron and x-ray diffraction patterns indicate that these (1,3)-beta-D-glucan chains adopt a parallel, triple helical conformation. The (1,3)-beta-D-glucan triple helices are orientated perpendicularly to the plane of the lamellar crystals. The barley (1,3)-beta-D-glucan glycosynthases have considerable potential for tailored and high efficiency synthesis of (1,3)-beta-D-linked oligo- and polysaccharides, some of which could have immunomodulating activity, or for the coupling of (1,3)-beta-D-linked glucosyl residues onto other oligosaccharides or glycoproteins.

  • 13.
    Jansson, Magnus
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Hallén, D
    Koho, H
    Andersson, G
    Berghard, L
    Heidrich, J
    Nyberg, E
    Uhlén, M
    Kördel, J
    Nilsson, B
    Characterization of ligand binding of a soluble human insulin-like growth factor I receptor variant suggests a ligand-induced conformational change.1997In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 272, no 13, p. 8189-8197Article in journal (Refereed)
    Abstract [en]

    Details of the signal transduction mechanisms of the tyrosine kinase family of growth factor receptors remain elusive. In this work, we describe an extensive study of kinetic and thermodynamic aspects of growth factor binding to a soluble extracellular human insulin-like growth factor-I receptor (sIGF-IR) variant. The extracellular receptor domains were produced fused to an IgG-binding protein domain (Z) in transfected human 293 cells as a correctly processed secreted alpha-beta'-Z dimer. The receptor was purified using IgG affinity chromatography, rendering a pure and homogenous protein in yields from 1 to 5 mg/liter of conditioned cell media. Biosensor technology (BIAcore) was applied to measure the insulin-like growth factor-I (IGF-I), des(1-3)IGF-I, insulin-like growth factor-II, and insulin ligand binding rate constants to the immobilized IGF-IR-Z. The association equilibrium constant, Ka, for the IGF-I interaction is determined to 2.8 x 10(8) M-1 (25 degrees C). Microcalorimetric titrations on IGF-I/IGF-IR-Z were performed at three different temperatures (15, 25, and 37 degrees C) and in two different buffer systems at 25 degrees C. From these measurements, equilibrium constants for the 1:1 (IGF-I:(alpha-beta'-Z)2) receptor complex in solution are deduced to 0.96 x 10(8) M-1 (25 degrees C). The determined heat capacity change for the process is large and negative, -0.51 kcal (K mol)-1. Further, the entropy change (DeltaS) at 25 degrees C is large and negative. Far- and near-UV circular dichroism measurements display significant changes over the entire wavelength range upon binding of IGF-I to IGF-IR-Z. These data are all consistent with a significant change in structure of the system upon IGF-I binding.

  • 14. Johansson, M. U.
    et al.
    Frick, I. M.
    Nilsson, H.
    Kraulis, P. J.
    Hober, Sophia
    KTH, Superseded Departments, Biotechnology.
    Jonasson, P.
    Linhult, M.
    Nygren, Per-Åke
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Bjorck, L.
    Drakenberg, T.
    Forsen, S.
    Wikstrom, M.
    Structure, specificity, and mode of interaction for bacterial albumin-binding modules2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 10, p. 8114-8120Article in journal (Refereed)
    Abstract [en]

    We have determined the solution structure of an albumin binding domain of protein G, a surface protein of group C and G streptococci. We find that it folds into a left handed three-helix bundle similar to the albumin binding domain of protein PAB from Peptostreptococcus magnus. The two domains share 59% sequence identity, are thermally very stable, and bind to the same site on human serum albumin. The albumin binding site, the first determined for this structural motif known as the GA module, comprises residues spanning the first loop to the beginning of the third helix and includes the most conserved region of GA modules. The two GA modules have different affinities for albumin from different species, and their albumin binding patterns correspond directly to the host specificity of C/G streptococci and P. magnus, respectively. These studies of the evolution, structure, and binding properties of the GA module emphasize the power of bacterial adaptation and underline ecological and medical problems connected with the use of antibiotics.

  • 15. Kujawa, Magdalena
    et al.
    Ebner, Heidemarie
    Leitner, Christian
    Hallberg, B. Martin
    Prongjit, Methinee
    Sucharitakul, Jeerus
    Ludwig, Roland
    Rudsander, Ulla
    Peterbauer, Clemens
    Chaiyen, Pimchai
    Haltrich, Dietmar
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Structural basis for substrate binding and regioselective oxidation of monosaccharides at C3 by pyranose 2-oxidase2006In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 46, p. 35104-35115Article in journal (Refereed)
    Abstract [en]

    Pyranose2-oxidase(P2Ox) participates in fungal lignin degradation by producing the H2O2 needed for lignin-degrading peroxidases. The enzyme oxidizes cellulose- and hemicellulose-derived aldopyranoses at C2 preferentially, but also on C3, to the corresponding ketoaldoses. To investigate the structural determinants of catalysis, covalent flavinylation, substrate binding, and regios-electivity, wild-type and mutant P2Ox enzymes were produced and characterized biochemically and structurally. Removal of the histidyl-FAD linkage resulted in a catalytically competent enzyme containing tightly, but noncovalently bound FAD. This mutant (H167A) is characterized by a 5-fold lower k(cat), and a 35-mV lower redox potential, although no significant structural changes were seen in its crystal structure. In previous structures of P2Ox, the substrate loop (residues 452-457) covering the active site has been either disordered or in a conformation incompatible with carbohydrate binding. We present here the crystal structure of H167A in complex with a slow substrate, 2-fluoro-2-deoxy-D-glucose. Based on the details of 2-fluoro-2-deoxy-D-glucose binding in position for oxidation at C3, we also outline a probable binding mode for D-glucose positioned for regioselective oxidation at C2. The tentative determinant for discriminating between the two binding modes is the position of the O6 hydroxyl group, which in the C2-oxidation mode can make favorable interactions with Asp(452) in the substrate loop and, possibly, a nearby arginine residue (Arg(472)). We also substantiate our hypothesis with steady-state kinetics data for the alanine replacements of Asp(452) and Arg(472) as well as the double alanine 452/472 mutant.

  • 16. Lai-Kee-Him, J.
    et al.
    Chanzy, H.
    Muller, M.
    Putaux, J. L.
    Imai, T.
    Bulone, Vincent
    KTH, Superseded Departments, Biotechnology.
    In vitro versus in vivo cellulose microfibrils from plant primary wall synthases: Structural differences2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 40, p. 36931-36939Article in journal (Refereed)
    Abstract [en]

    Detergent extracts of microsomal fractions from suspension cultured cells of Rubus fruticosus (blackberry) were tested for their ability to synthesize in vitro sizable quantities of cellulose from UDP-glucose. Both Brij 58 and taurocholate were effective and yielded a substantial percentage of cellulose microfibrils together with (1-->3)-beta-D-glucan (callose). The taurocholate extracts, which did not require the addition of Mg2+, were the most efficient, yielding roughly 20% of cellulose. This cellulose was characterized after callose removal by methylation analysis, electron microscopy, and electron and x-ray synchrotron diffractions; its resistance toward the acid Updegraff reagent was also evaluated. The cellulose microfibrils synthesized in vitro had the same diameter as the endogenous microfibrils isolated from primary cell walls. Both polymers diffracted as cellulose IVI, a disorganized form of cellulose I. Besides these similarities, the in vitro microfibrils had a higher perfection and crystallinity as well as a better resistance toward the Updegraff reagent. These differences can be attributed to the mode of synthesis of the in vitro microfibrils that are able to grow independently in a neighbor-free environment, as opposed to the cellulose in the parent cell walls where new microfibrils have to interweave with the already laid polymers, with the result of a number of structural defects.

  • 17.
    Larsbrink, Johan
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Izumi, Atsushi
    Hemsworth, Glyn R.
    Davies, Gideon J.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Structural Enzymology of Cellvibrio japonicus Agd31B Protein Reveals alpha-Transglucosylase Activity in Glycoside Hydrolase Family 312012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 52, p. 43288-43299Article in journal (Refereed)
    Abstract [en]

    The metabolism of the storage polysaccharides glycogen and starch is of vital importance to organisms from all domains of life. In bacteria, utilization of these alpha-glucans requires the concerted action of a variety of enzymes, including glycoside hydrolases, glycoside phosphorylases, and transglycosylases. In particular, transglycosylases from glycoside hydrolase family 13 (GH13) and GH77 play well established roles in alpha-glucan side chain (de) branching, regulation of oligo-and polysaccharide chain length, and formation of cyclic dextrans. Here, we present the biochemical and tertiary structural characterization of a new type of bacterial 1,4-alpha-glucan 4-alpha-glucosyltransferase from GH31. Distinct from 1,4-alpha-glucan 6-alpha-glucosyltransferases (EC 2.4.1.24) and 4-alpha-glucanotransferases (EC 2.4.1.25), this enzyme strictly transferred one glucosyl residue from alpha(1 -> 4)-glucans in disproportionation reactions. Substrate hydrolysis was undetectable for a series of malto-oligosaccharides except maltose for which transglycosylation nonetheless dominated across a range of substrate concentrations. Crystallographic analysis of the enzyme in free, acarbose-complexed, and trapped 5-fluoro-beta-glucosyl-enzyme intermediate forms revealed extended substrate interactions across one negative and up to three positive subsites, thus providing structural rationalization for the unique, single monosaccharide transferase activity of the enzyme.

  • 18. Liu, Xiao
    et al.
    Spicarova, Zuzana
    Rydholm, Susanna
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Li, Juan
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Aperia, Anita
    Ankyrin B Modulates the Function of Na,K-ATPase/Inositol 1,4,5-Trisphosphate Receptor Signaling Microdomain2008In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 17, p. 11461-11468Article in journal (Refereed)
    Abstract [en]

    Na, K-ATPase and inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) can form a signaling microdomain that in the presence of ouabain triggers highly regular calcium oscillations. Downstream effects include NF-kappa B activation. Here we report that ankyrin B (Ank-B), expressed in most mammalian cells, plays a pivotal role in the function of the Na, K-ATPase/ IP3R signaling microdomain. In studies performed on a monkey kidney cell line, we show that Ank-B co-precipitates with both Na, K-ATPase and IP3R. We identify the N terminus tail of the Na, K-ATPase catalytic subunit and the N-terminal portion 1-604 of the IP3R as novel binding sites for Ank-B. Knockdown of Ank-B with small interfering RNA reduced the expression of Ank-B to 15-30%. This down-regulation of Ank-B attenuated the interaction between Na, K-ATPase and IP3R, reduced the number of cells responding to pM doses of ouabain with calcium oscillations, altered the calcium oscillatory pattern, and abolished the ouabain effect on NF-kappa B. In contrast, Ank-B down-regulation had no effect on the ion transporting function of Na, K-ATPase and no effect on the distribution and apparent mobility of Na, K-ATPase in the plasma membrane.

  • 19.
    Lundin, Daniel
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Poole, Anthony M.
    Sjöberg, Britt-Marie
    Högbom, Martin
    Use of Structural Phylogenetic Networks for Classification of the Ferritin-like Superfamily2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 24, p. 20565-20575Article in journal (Refereed)
    Abstract [en]

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

  • 20. Martinez-Fleites, Carlos
    et al.
    Guerreiro, Catarina I. P. D.
    Baumann, Martin J.
    KTH, School of Biotechnology (BIO).
    Taylor, Edward J.
    Prates, Jose A. M.
    Ferreira, Luis M. A.
    Fontes, Carlos M. G. A.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Davies, Gideon J.
    Crystal structures of Clostridium thermocellum xyloglucanase, XGH74A, reveal the structural basis for xyloglucan recognition and degradation2006In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 34, p. 24922-24933Article in journal (Refereed)
    Abstract [en]

    The enzymatic degradation of the plant cell wall is central both to the natural carbon cycle and, increasingly, to environmentally friendly routes to biomass conversion, including the production of biofuels. The plant cell wall is a complex composite of cellulose microfibrils embedded in diverse polysaccharides collectively termed hemicelluloses. Xyloglucan is one such polysaccharide whose hydrolysis is catalyzed by diverse xyloglucanases. Here we present the structure of the Clostridium thermocellum xyloglucanase Xgh74A in both apo and ligand-complexed forms. The structures, in combination with mutagenesis data on the catalytic residues and the kinetics and specificity of xyloglucan hydrolysis reveal a complex subsite specificity accommodating seventeen monosaccharide moieties of the multibranched substrate in an open substrate binding terrain.

  • 21.
    Martins, Antonio
    et al.
    Stockholm Univ, Wenner Gren Inst, Dept Mol Biosci, SE-10691 Stockholm, Sweden..
    Pfirrmann, Thorsten
    Stockholm Univ, Wenner Gren Inst, Dept Mol Biosci, SE-10691 Stockholm, Sweden.;Martin Luther Univ Halle Wittenberg, Inst Physiol Chem, D-06114 Halle, Germany..
    Heessen, Stijn
    Stockholm Univ, Wenner Gren Inst, Dept Mol Biosci, SE-10691 Stockholm, Sweden.;Sanofi Aventis Deutschland GmbH, Global Business Dev & Licensing Consumer Healthca, D-65926 Frankfurt, Germany..
    Sundqvist, Gustav
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Bulone, Vincent
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Andreasson, Claes
    Stockholm Univ, Wenner Gren Inst, Dept Mol Biosci, SE-10691 Stockholm, Sweden..
    Ljungdahl, Per O.
    Stockholm Univ, Wenner Gren Inst, Dept Mol Biosci, SE-10691 Stockholm, Sweden..
    Ssy5 is a signaling serine protease that exhibits atypical biogenesis and marked S1 specificity2018In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 293, no 22, p. 8362-8378Article in journal (Refereed)
    Abstract [en]

    Ssy5 is a signaling endoprotease that plays a key role in regulating central metabolism, cellular aging, and morphological transitions important for growth and survival of yeast (Saccharomyces cerevisiae) cells. In response to extracellular amino acids, Ssy5 proteolytically activates the transcription factors Stp1 and Stp2, leading to enhanced Ssy1-Ptr3-Ssy5 (SPS) sensor-regulated gene expression. Ssy5 comprises a catalytic (Cat) domain and an extensive regulatory prodomain. Ssy5 is refractory to both broad-spectrum and serine protease-specific inhibitors, confounding its classification as a protease, and no information about Ssy5's cleavage-site preferences and its mechanism of substrate selection is available. Here, using mutational and inhibition experiments, we investigated the biogenesis and catalytic properties of Ssy5 and conclusively show that it is a serine protease. Atypical for the majority of serine proteases, Ssy5's prodomain was obligatorily required in cis during biogenesis for the maturation of the proteolytic activity of the Cat domain. Autolysis and Stp1 and Stp2 cleavage occurred between a cysteine (at the P1 site) and a serine or alanine (at the P1 site) and required residues with short side chains at the P1 site. Substitutions in the Cat domain affecting substrate specificity revealed that residues Phe-634, His-661, and Gly-671 in the S1-binding pocket of this domain are important for Ssy5 catalytic function. This study confirms that the signaling protease Ssy5 is a serine protease and provides a detailed understanding of the biogenesis and intrinsic properties of this key enzyme in yeast.

  • 22. Mellroth, Peter
    et al.
    Daniels, Robert
    Eberhardt, Alice
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Normark, Staffan
    Henriques-Normark, Birgitta
    LytA, Major Autolysin of Streptococcus pneumoniae, Requires Access to Nascent Peptidoglycan2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 14, p. 11018-11029Article in journal (Refereed)
    Abstract [en]

    Background: The regulation of cell wall hydrolysis by the pneumococcal autolysin LytA is poorly understood. Results: The cell wall is susceptible to extracellular LytA only during the stationary phase or after cell wall synthesis inhibition. Conclusion: LytA is regulated on the substrate level, where peptidoglycan modifications likely prevent LytA hydrolysis. Significance: The control of amidases is essential for bacterial survival, cell-wall synthesis, and division.

  • 23. Miyakawa-Naito, A.
    et al.
    Uhlen, P.
    Lal, M.
    Aizman, O.
    Mikoshiba, K.
    Brismar, Hjalmar
    Zelenin, S.
    Aperia, A.
    Cell signaling Microdomain with Na,K-ATPase and inositol 1,4,5-trisphosphate receptor generates calcium oscillations2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 50, p. 50355-50361Article in journal (Refereed)
    Abstract [en]

    Recent studies indicate novel roles for the ubiquitous ion pump, Na,K-ATPase, in addition to its function as a key regulator of intracellular sodium and potassium concentration. We have previously demonstrated that ouabain, the endogenous ligand of Na, K-ATPase, can trigger intracellular Ca2+ oscillations, a versatile intracellular signal controlling a diverse range of cellular processes. Here we report that Na, K-ATPase and inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) form a cell signaling microdomain that, in the presence of ouabain, generates slow Ca2+ oscillations in renal cells. Using fluorescent resonance energy transfer ( FRET) measurements, we detected a close spatial proximity between Na, K-ATPase and InsP(3)R. Ouabain significantly enhanced FRET between Na, K-ATPase and InsP(3)R. The FRET effect and ouabain-induced Ca2+ oscillations were not observed following disruption of the actin cytoskeleton. Partial truncation of the NH2 terminus of Na, K-ATPase catalytic alpha1-subunit abolished Ca2+ oscillations and downstream activation of NF-kappaB. Ouabain-induced Ca2+ oscillations occurred in cells expressing an InsP3 sponge and were hence independent of InsP3 generation. Thus, we present a novel principle for a cell signaling microdomain where an ion pump serves as a receptor.

  • 24. Montanier, Cedric Y.
    et al.
    Correia, Marcia A. S.
    Flint, James E.
    Zhu, Yanping
    Basle, Arnaud
    McKee, Lauren
    Newcastle University, United Kingdom; University of Georgia, United States.
    Prates, Jose A. M.
    Polizzi, Samuel J.
    Coutinho, Pedro M.
    Lewis, Richard J.
    Henrissat, Bernard
    Fontes, Carlos M. G. A.
    Gilbert, Harry J.
    A novel, noncatalytic carbohydrate-binding module displays specificity for galactose-containing polysaccharides through calcium-mediated oligomerization2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 25Article in journal (Refereed)
    Abstract [en]

    The enzymic degradation of plant cell walls plays a central role in the carbon cycle and is of increasing environmental and industrial significance. The catalytic modules of enzymes that catalyze this process are generally appended to noncatalytic carbohydrate-binding modules (CBMs). CBMs potentiate the rate of catalysis by bringing their cognate enzymes into intimate contact with the target substrate. A powerful plant cell wall-degrading system is the Clostridium thermocellum multienzyme complex, termed the "cellulosome." Here, we identify a novel CBM (CtCBM62) within the large C. thermocellum cellulosomal protein Cthe_2193 (defined as CtXyl5A), which establishes a new CBM family. Phylogenetic analysis of CBM62 members indicates that a circular permutation occurred within the family. CtCBM62 binds to D-galactose and L-arabinopyranose in either anomeric configuration. The crystal structures of CtCBM62, in complex with oligosaccharides containing alpha- and beta-galactose residues, show that the ligand-binding site in the beta-sandwich protein is located in the loops that connect the two beta-sheets. Specificity is conferred through numerous interactions with the axial O4 of the target sugars, a feature that distinguishes galactose and arabinose from the other major sugars located in plant cell walls. CtCBM62 displays tighter affinity for multivalent ligands compared with molecules containing single galactose residues, which is associated with precipitation of these complex carbohydrates. These avidity effects, which confer the targeting of polysaccharides, are mediated by calcium-dependent oligomerization of the CBM.

  • 25. Pawelzik, Sven-Christian
    et al.
    Uda, Narasimha Rao
    Spahiu, Linda
    Jegerschöld, Caroline
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Stenberg, Patric
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Morgenstern, Ralf
    Jakobsson, Per-Johan
    Identification of Key Residues Determining Species Differences in Inhibitor Binding of Microsomal Prostaglandin E Synthase-12010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 38, p. 29254-29261Article in journal (Refereed)
    Abstract [en]

    Microsomal prostaglandin E synthase-1 (MPGES1) is induced during an inflammatory reaction from low basal levels by pro-inflammatory cytokines and subsequently involved in the production of the important mediator of inflammation, prostaglandin E-2. Nonsteroidal anti-inflammatory drugs prevent prostaglandin E-2 production by inhibiting the upstream enzymes cyclooxygenases 1 and 2. In contrast to these conventional drugs, a new generation of NSAIDs targets the terminal enzyme MPGES1. Some of these compounds potently inhibit human MPGES1 but do not have an effect on the rat orthologue. We investigated this interspecies difference in a rat/human chimeric form of the enzyme as well as in several mutants and identified key residues Thr-131, Leu-135, and Ala-138 in human MPGES1, which play a crucial role as gate keepers for the active site of MPGES1. These residues are situated in transmembrane helix 4, lining the entrance to the cleft between two subunits in the protein trimer, and regulate access of the inhibitor in the rat enzyme. Exchange toward the human residues in rat MPGES1 was accompanied with a gain of inhibitor activity, whereas exchange in human MPGES1 toward the residues found in rat abrogated inhibitor activity. Our data give evidence for the location of the active site at the interface between subunits in the homotrimeric enzyme and suggest a model of how the natural substratePGH(2), or competitive inhibitors of MPGES1, enter the active site via the phospholipid bilayer of the membrane.

  • 26.
    Piens, Kathleen
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Fauré, Régis
    Centre de Recherche Sur Les Macromolécules Végétales, CNRS.
    Sundqvist, Gustav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Baumann, Martin J.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Saura-Valls, Marc
    Laboratory of Biochemistry, Institut Químic de Sarrià, Universitat Ramon Llull.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Cottaz, Sylvain
    Centre de Recherche Sur Les Macromolécules Végétales, CNRS.
    Planas, Antoni
    Laboratory of Biochemistry, Institut Químic de Sarrià, Universitat Ramon Llull.
    Driquez, Hugues
    Centre de Recherche Sur Les Macromolécules Végétales, CNRS.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Mechanism-based labeling defines the free energy change for formation of the covalent glycosyl-enzyme intermediate in a xyloglucan endo-transglycosylase2008In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 32, p. 21864-21872Article in journal (Refereed)
    Abstract [en]

    Xyloglucan endo-transglycosylases (XETs) are key enzymes involved in the restructuring of plant cell walls during morphogenesis. As members of glycoside hydrolase family 16 (GH16), XETs are predicted to employ the canonical retaining mechanism of glycosyl transfer involving a covalent glycosyl-enzyme intermediate. Here, we report the accumulation and direct observation of such intermediates of PttXET16-34 from hybrid aspen by electrospray mass spectrometry in combination with synthetic "blocked" substrates, which function as glycosyl donors but are incapable of acting as glycosyl acceptors. Thus, GalGXXXGGG and GalGXXXGXXXG react with the wild-type enzyme to yield relatively stable, kinetically competent, covalent GalG-enzyme and GalGXXXG-enzyme complexes, respectively (Gal = Gal beta(1 -> 4), G = Glc beta(1 -> 4), and X = Xyl alpha(1 -> 6) Glc beta(1 -> 4)). Quantitation of ratios of protein and saccharide species at pseudo-equilibrium allowed us to estimate the free energy change (Delta G(0)) for the formation of the covalent GalGXXXG-enzyme as 6.3-8.5 kJ/mol (1.5-2.0 kcal/mol). The data indicate that the free energy of the beta(1 -> 4) glucosidic bond in xyloglucans is preserved in the glycosyl-enzyme intermediate and harnessed for religation of the polysaccharide in vivo.

  • 27.
    Piguet, Joachim
    et al.
    Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland; 24IP Law Group, Germany.
    Schreiter, Christoph
    Segura, Jean-Manuel
    Vogel, Horst
    Hovius, Ruud
    Acetylcholine receptor organization in membrane domains in muscle cells: evidence for rapsyn-independent and rapsyn-dependent mechanisms2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 1Article in journal (Refereed)
    Abstract [en]

    Nicotinic acetylcholine receptors (nAChR) in muscle fibers are densely packed in the postsynaptic region at the neuromuscular junction. Rapsyn plays a central role in directing and clustering nAChR during cellular differentiation and neuromuscular junction formation; however, it has not been demonstrated whether rapsyn is the only cause of receptor immobilization. Here, we used single-molecule tracking methods to investigate nAChR mobility in plasma membranes of myoblast cells during their differentiation to myotubes in the presence and absence of rapsyn. We found that in myoblasts the majority of nAChR were immobile and that ∼20% of the receptors showed restricted diffusion in small domains of ∼50 nm. In myoblasts devoid of rapsyn, the fraction of mobile nAChR was considerably increased, accompanied by a 3-fold decrease in the immobile population of nAChR with respect to rapsyn-expressing cells. Half of the mobile receptors were confined to domains of ∼120 nm. Measurements performed in heterologously transfected HEK cells confirmed the direct immobilization of nAChR by rapsyn. However, irrespective of the presence of rapsyn, about one-third of nAChR were confined in 300-nm domains. Our results show (i) that rapsyn efficiently immobilizes nAChR independently of other postsynaptic scaffold components; (ii) nAChR is constrained in confined membrane domains independently of rapsyn; and (iii) in the presence of rapsyn, the size of these domains is strongly reduced.

  • 28. Pitsawong, Warintra
    et al.
    Sucharitakul, Jeerus
    Prongjit, Methinee
    Tan, Tien-Chye
    KTH, School of Biotechnology (BIO), Glycoscience.
    Spadiut, Oliver
    KTH, School of Biotechnology (BIO), Glycoscience.
    Haltrich, Dietmar
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Chaiyen, Pimchai
    A Conserved Active-site Threonine Is Important for Both Sugar and Flavin Oxidations of Pyranose 2-Oxidase2010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 13, p. 9697-9705Article in journal (Refereed)
    Abstract [en]

    Pyranose 2-oxidase (P2O) catalyzes the oxidation by O-2 of D-glucose and several aldopyranoses to yield the 2-ketoaldoses and H2O2. Based on crystal structures, in one rotamer conformation, the threonine hydroxyl of Thr(169) forms H-bonds to the flavin-N5/O4 locus, whereas, in a different rotamer, it may interact with either sugar or other parts of the P2O center dot sugar complex. Transient kinetics of wild-type (WT) and Thr(169)-> S/N/G/A replacement variants show that D-Glc binds to T169S, T169N, and WT with the same K-d (45-47 mM), and the hydride transfer rate constants (k(red)) are similar (15.3-9.7 s(-1) at 4 degrees C). k(red) of T169G with D-glucose (0.7 s(-1), 4 degrees C) is significantly less than that of WT but not as severely affected as in T169A (k(red) of 0.03 s(-1) at 25 degrees C). Transient kinetics of WT and mutants using D-galactose show that P2O binds D-galactose with a one-step binding process, different from binding of D- glucose. In T169S, T169N, and T169G, the overall turnover with D- Gal is faster than that of WT due to an increase of kred. In the crystal structure of T169S, Ser(169) O gamma assumes a position identical to that of O gamma 1 in Thr(169); in T169G, solvent molecules may be able to rescue H-bonding. Our data suggest that a competent reductive half-reaction requires a side chain at position 169 that is able to form an H-bond within the ES complex. During the oxidative half-reaction, all mutants failed to stabilize a C4a-hydroperoxyflavin intermediate, thus suggesting that the precise position and geometry of the Thr(169) side chain are required for intermediate stabilization.

  • 29. Razavi, Asghar M.
    et al.
    Delemotte, Lucie
    KTH, School of Engineering Sciences (SCI), Physics, Theoretical & Computational Biophysics.
    Berlin, Joshua R.
    Carnevale, Vincenzo
    Voelz, Vincent A.
    Molecular simulations and free-energy calculations suggest conformation-dependent anion binding to a cytoplasmic site as a mechanism for Na+/K+-ATPase ion selectivity2017In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 292, no 30, p. 12412-12423Article in journal (Refereed)
    Abstract [en]

    Na+/K+-ATPase transports Na+ and K+ ions across the cell membrane via an ion-binding site becoming alternatively accessible to the intra- and extracellular milieu by conformational transitions that confer marked changes in ion-binding stoichiometry and selectivity. To probe the mechanism of these changes, we used molecular simulation and free-energy perturbation approaches to identify probable protonation states of Na+- and K+-coordinating residues in E1P and E2P conformations of Na+/K+-ATPase. Analysis of these simulations revealed a molecular mechanism responsible for the change in protonation state: the conformation-dependent binding of an anion (a chloride ion in our simulations) to a previously unrecognized cytoplasmic site in the loop between transmembrane helices 8 and 9, which influences the electrostatic potential of the crucial Na+-coordinating residue Asp(926). This mechanistic model is consistent with experimental observations and provides a molecular-level picture of how E1P to E2P enzyme conformational transitions are coupled to changes in ion-binding stoichiometry and selectivity.

  • 30. Rothwell, Paul J.
    et al.
    Allen, William J.
    Sisamakis, Evangelos
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Kalinin, Stanislav
    Felekyan, Suren
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Waksman, Gabriel
    Seidel, Claus A. M.
    dNTP-dependent Conformational Transitions in the Fingers Subdomain of Klentaq1 DNA Polymerase INSIGHTS INTO THE ROLE OF THE "NUCLEOTIDE-BINDING" STATE2013In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 19, p. 13575-13591Article in journal (Refereed)
    Abstract [en]

    DNA polymerases are responsible for the accurate replication of DNA. Kinetic, single-molecule, and x-ray studies show that multiple conformational states are important for DNA polymerase fidelity. Using high precision FRET measurements, we show that Klentaq1 (the Klenow fragment of Thermus aquaticus DNA polymerase 1) is in equilibrium between three structurally distinct states. In the absence of nucleotide, the enzyme is mostly open, whereas in the presence of DNA and a correctly base-pairing dNTP, it re-equilibrates to a closed state. In the presence of a dNTP alone, with DNA and an incorrect dNTP, or in elevated MgCl2 concentrations, an intermediate state termed the "nucleotide-binding" state predominates. Photon distribution and hidden Markov modeling revealed fast dynamic and slow conformational processes occurring between all three states in a complex energy landscape suggesting a mechanism in which dNTP delivery is mediated by the nucleotide-binding state. After nucleotide binding, correct dNTPs are transported to the closed state, whereas incorrect dNTPs are delivered to the open state.

  • 31. Rotsaert, F. A. J.
    et al.
    Hallberg, B. M.
    de Vries, S.
    Moenne-Loccoz, P.
    Divne, Christina
    KTH, Superseded Departments, Biotechnology.
    Renganathan, V.
    Gold, M. H.
    Biophysical and structural analysis of a novel heme b iron ligation in the flavocytochrome cellobiose dehydrogenase2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 35, p. 33224-33231Article in journal (Refereed)
    Abstract [en]

    The fungal extracellular flavocytochrome cellobiose dehydrogenase (CDH) participates in lignocellulose degradation. The enzyme has a cytochrome domain connected to a flavin-binding domain by a peptide linker. The cytochrome domain contains a 6-coordinate low spin b-type heme with unusual iron ligands and coordination geometry. Wild type CDH is only the second example of a b-type heme with Met-His ligation, and it is the first example of a Met-His ligation of heme b where the ligands are arranged in a nearly perpendicular orientation. To investigate the ligation further, Met(65) was replaced with a histidine to create a bis-histidyl ligated iron typical of b-type cytochromes. The variant is expressed as a stable 90-kDa protein that retains the flavin domain catalytic reactivity. However, the ability of the mutant to reduce external one-electron acceptors such as cytochrome c is impaired. Electrochemical measurements demonstrate a decrease in the redox midpoint potential of the heme by 210 mV. In contrast to the wild type enzyme, the ferric state of the protoheme displays a mixed low spin/high spin state at room temperature and low spin character at 90 K, as determined by resonance Raman spectroscopy. The wild type cytochrome does not bind CO, but the ferrous state of the variant forms a CO complex, although the association rate is very low. The crystal structure of the M65H cytochrome domain has been determined at 1.9 Angstrom resolution. The variant structure confirms a bis-histidyl ligation but reveals unusual features. As for the wild type enzyme, the ligands have a nearly perpendicular arrangement. Furthermore, the iron is bound by imidazole N-delta1 and N-epsilon2 nitrogen atoms, rather than the typical N-epsilon2/N-epsilon2 coordination encountered in bis-histidyl ligated heme proteins. To our knowledge, this is the first example of a bis-histidyl N-delta1/N-epsilon2-coordinated protoporphyrin IX iron.

  • 32. Rutsdottir, Gudrun
    et al.
    Härmark, Johan
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine. Karolinska Institutet, Sverige.
    Weide, Yoran
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sverige.
    Rasmussen, Morten I.
    Wernersson, Sven
    Respondek, Michal
    Akke, Mikael
    Højrup, Peter
    Köck, Philip J. B.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sverige.
    Söderberg, Christopher A. G.
    Emanuelsson, Cecilia
    Structural model of dodecameric heat-shock protein Hsp21: Flexible N-terminal arms interact with client proteins while C-terminal tails maintain the dodecamer and chaperone activity2017In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 292, no 19, p. 8103-8121Article in journal (Refereed)
    Abstract [en]

    Small heat-shock proteins (sHsps) prevent aggregation of thermosensitive client proteins in a first line of defense against cellular stress. The mechanisms by which they perform this function have been hard to define due to limited structural information; currently, there is only one high-resolution structure of a plant sHsp published, that of the cytosolic Hsp16.9. We took interest in Hsp21, a chloroplast-localized sHsp crucial for plant stress resistance, which has even longer N-terminal arms than Hsp16.9, with a functionally important and conserved methionine-rich motif. To provide a framework for investigating structure-function relationships of Hsp21 and understanding these sequence variations, we developed a structural model of Hsp21 based on homology modeling, cryo-EM, cross-linking mass spectrometry, NMR, and small-angle X-ray scattering. Our data suggest a dodecameric arrangement of two trimer-of-dimer discs stabilized by the C-terminal tails, possibly through tail-to-tail interactions between the discs, mediated through extended IXVXI motifs. Our model further suggests that six N-terminal arms are located on the outside of the dodecamer, accessible for interaction with client proteins, and distinct from previous undefined or inwardly facing arms. To test the importance of the IXVXI motif, we created the point mutant V181A, which, as expected, disrupts the Hsp21 dodecamer and decreases chaperone activity. Finally, our data emphasize that sHsp chaperone efficiency depends on oligomerization and that client interactions can occur both with and without oligomer dissociation. These results provide a generalizable workflow to explore sHsps, expand our understanding of sHsp structural motifs, and provide a testable Hsp21 structure model to inform future investigations.

  • 33. Sandalova, T.
    et al.
    Michaelsson, J.
    Harris, R. A.
    Odeberg, Jacob
    KTH, School of Biotechnology (BIO), Gene Technology.
    Schneider, G.
    Karres, K.
    Achour, A.
    A structural basis for CD8(+) T cell-dependent recognition of non-homologous peptide ligands - Implications for molecular mimicry in autoreactivity2005In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 29, p. 27069-27075Article in journal (Refereed)
    Abstract [en]

    Molecular mimicry of self-epitopes by viral antigens is one possible pathogenic mechanism underlying induction of autoimmunity. A self-epitope, mDBM, derived from mouse dopamine beta-mono-oxygenase (KALYDYAPI) sharing 44% sequence identity with the lymphocytic choriomeningitis virus-derived immunodominant epitope gp33 (KAVYNFATC/ M), has previously been identified as a cross-reactive self-ligand, presentation of which results in autoimmunity. A rat peptide homologue, rDBM (KALYNYAPI, 56% identity to gp33), which displayed similar properties to mDBM, has also been identified. We herein report the crystal structure of H-2D(b)center dot rDBM and a comparison with the crystal structures of the cross-reactive H-2D(b)center dot gp33 and non- cross- reactive H-2D(b)center dot gp33 (V3L) escape variant (KALYNFATM, 88% identity to gp33). Despite the large sequence disparity, rDBM and gp33 peptides are presented in nearly identical manners by H-2D(b), with a striking juxtaposition of the central sections of both peptides from residues p3 to p7. The structural similarity provides H-2D(b) in complex with either a virus-derived or a dopamine beta-mono-oxygenase-derived peptide with a shared antigenic identity that conserves the positioning of the heavy chain and peptide residues that interact with the T cell receptor (TCR). This stands in contrast to the structure of H-2D(b) center dot gp33 (V3L), in which a single conserved mutation, also present in rDBM, induces large movements of both the peptide backbone and the side chains that interact with the TCR. The TCR-interacting surfaces of the H-2D(b) center dot rDBM and H-2D(b) center dot gp33 major histocompatibility complexes are very similar with regard to shape, topology, and charge distribution, providing a structural basis for CD8 T cell activation by molecular mimicry and potential subsequent development of autoreactivity.

  • 34. Saura-Valls, Marc
    et al.
    Faure, Regis
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Cottaz, Sylvain
    Driguez, Hugues
    Planas, Antoni
    Active-site mapping of a Populus xyloglucan endo-transglycosylase with a library of xylogluco-oligosaccharides2008In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 32, p. 21853-21863Article in journal (Refereed)
    Abstract [en]

    Restructuring the network of xyloglucan (XG) and cellulose during plant cell wall morphogenesis involves the action of xyloglucan endo-transglycosylases (XETs). They cleave the XG chains and transfer the enzyme-bound XG fragment to another XG molecule, thus allowing transient loosening of the cell wall and also incorporation of nascent XG during expansion. The substrate specificity of a XET from Populus (PttXET16-34) has been analyzed by mapping the enzyme binding site with a library of xylogluco-oligosaccharides as donor substrates using a labeled heptasaccharide as acceptor. The extended binding cleft of the enzyme is composed of four negative and three positive subsites (with the catalytic residues between subsites -1 and + 1). Donor binding is dominated by the higher affinity of the XXXGmoiety (G = Glc beta(1 -> 4) and X = Xyl alpha(1 -> 6)Glc beta(1 -> 4)) of the substrate for positive subsites, whereas negative subsites have a more relaxed specificity, able to bind (and transfer to the acceptor) a cello-oligosaccharyl moiety of hybrid substrates such as GGGGXXXG. Subsite mapping with k(cat)/K-m values for the donor substrates showed that a GG-unit on negative and-XXG on positive subsites are the minimal requirements for activity. Subsites -2 and -3 (for backbone Glc residues) and +2' (for Xyl substitution at Glc in subsite +2) have the largest contribution to transition state stabilization. GalGXXXGXXXG (Gal = Gal beta(1 -> 4)) is the best donor substrate with a "blocked" nonreducing end that prevents polymerization reactions and yields a single transglycosylation product. Its kinetics have unambiguously established that the enzyme operates by a ping-pong mechanism with competitive inhibition by the acceptor.

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

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

  • 36.
    Trillo-Muyo, Sergio
    et al.
    Univ Gothenburg, Dept Med Biochem, Box 440, S-40530 Gothenburg, Sweden..
    Nilsson, Harriet
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology. Univ Gothenburg, Dept Med Biochem, Box 440, S-40530 Gothenburg, Sweden.;Karolinska Inst, Dept Biosci & Nutr, S-14157 Huddinge, Sweden.
    Recktenwald, Christian V.
    Univ Gothenburg, Dept Med Biochem, Box 440, S-40530 Gothenburg, Sweden..
    Ermund, Anna
    Univ Gothenburg, Dept Med Biochem, Box 440, S-40530 Gothenburg, Sweden..
    Ridley, Caroline
    Univ Manchester, Manchester Acad Hlth Sci Ctr, Wellcome Trust Ctr Cell Matrix Res, Fac Biol Med & Hlth, Manchester M13 9PT, Lancs, England..
    Meiss, Lauren N.
    Univ Gothenburg, Dept Med Biochem, Box 440, S-40530 Gothenburg, Sweden..
    Baehr, Andrea
    Ludwig Maximilians Univ Munchen, Inst Mol Anim Breeding & Biotechnol, Gene Ctr, Hackerstr 27, D-85764 Oberschleissheim, Germany..
    Klymiuk, Nikolai
    Ludwig Maximilians Univ Munchen, Inst Mol Anim Breeding & Biotechnol, Gene Ctr, Hackerstr 27, D-85764 Oberschleissheim, Germany..
    Wine, Jeffrey J.
    Stanford Univ, Cyst Fibrosis Res Lab, Stanford, CA 94305 USA..
    Köck, Philip J. B.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology. Karolinska Inst, Dept Biosci & Nutr, S-14157 Huddinge, Sweden.
    Thornton, David J.
    Univ Manchester, Manchester Acad Hlth Sci Ctr, Wellcome Trust Ctr Cell Matrix Res, Fac Biol Med & Hlth, Manchester M13 9PT, Lancs, England..
    Hebert, Hans
    Karolinska Inst, Dept Biosci & Nutr, S-14157 Huddinge, Sweden.;KTH Royal Inst Technol, Sch Technol & Hlth, S-14157 Huddinge, Sweden..
    Hansson, Gunnar C.
    Univ Gothenburg, Dept Med Biochem, Box 440, S-40530 Gothenburg, Sweden..
    Granule-stored MUC5B mucins are packed by the non-covalent formation of N-terminal head-to-head tetramers2018In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 293, no 15, p. 5746-5754Article in journal (Refereed)
    Abstract [en]

    Most MUC5B mucin polymers in the upper airways of humans and pigs are produced by submucosal glands. MUC5B forms N-terminal covalent dimers that are further packed into larger assemblies because of low pH and high Ca2+ in the secretory granule of the mucin-producing cell. We purified the recombinant MUC5B N-terminal covalent dimer and used single-particle electron microscopy to study its structure under intracellular conditions. We found that, at intragranular pH, the dimeric MUC5B organized into head-to-head noncovalent tetramers where the von Willebrand D1-D2 domains hooked into each other. These N-terminal tetramers further formed long linear complexes from which, we suggest, the mucin domains and their C termini project radially outwards. Using conventional and video microscopy, we observed that, upon secretion into the submucosal gland ducts, a flow of bicarbonate-rich fluid passes the mucin-secreting cells. We suggest that this unfolds and pulls out the MUC5B assemblies into long linear threads. These further assemble into thicker mucin bundles in the glandular ducts before emerging at the gland duct opening. We conclude that the combination of intracellular packing of the MUC5B mucin and the submucosal gland morphology creates an efficient machine for producing linear mucin bundles.

  • 37. Veya, Luc
    et al.
    Piguet, Joachim
    Ecole Polytech Fed Lausanne.
    Vogel, Horst
    Single Molecule Imaging Deciphers the Relation between Mobility and Signaling of a Prototypical G Protein-coupled Receptor in Living Cells.2015In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 46, p. 27723-27735Article in journal (Refereed)
    Abstract [en]

    Lateral diffusion enables efficient interactions between membrane proteins, leading to signal transmission across the plasma membrane. An open question is how the spatiotemporal distribution of cell surface receptors influences the transmembrane signaling network. Here we addressed this issue by studying the mobility of a prototypical G protein-coupled receptor, the neurokinin-1 receptor, during its different phases of cellular signaling. Attaching a single quantum dot to individual neurokinin-1 receptors enabled us to follow with high spatial and temporal resolution over long time regimes the fate of individual receptors at the plasma membrane. Single receptor trajectories revealed a very heterogeneous mobility distribution pattern with diffusion constants ranging from 0.0005 to 0.1 μm(2)/s comprising receptors freely diffusing and others confined in 100-600-nm-sized membrane domains as well as immobile receptors. A two-dimensional representation of mobility and confinement resolved two major, broadly distributed receptor populations, one showing high mobility and low lateral restriction and the other showing low mobility and high restriction. We found that about 40% of the receptors in the basal state are already confined in membrane domains and are associated with clathrin. After stimulation with an agonist, an additional 30% of receptors became further confined. Using inhibitors of clathrin-mediated endocytosis, we found that the fraction of confined receptors at the basal state depends on the quantity of membrane-associated clathrin and is correlated to a significant decrease of the canonical pathway activity of the receptors. This shows that the high plasticity of receptor mobility is of central importance for receptor homeostasis and fine regulation of receptor activity.

  • 38.
    Zelenina, Marina
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Bondar, Alexander A
    Zelenin, Sergey
    Aperia, Anita
    Nickel and extracellular acidification inhibit the water permeability of human aquaporin-3 in lung epithelial cells.2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 32, p. 30037-43Article in journal (Refereed)
    Abstract [en]

    Nickel is a common cause of pneumoconiosis. Here, we show that nickel inactivates aquaporin (AQP)-3, the water channel expressed apically in epithelial cells of human terminal airways. Human AQP3 was transiently transfected into human lung cells, and water permeability was measured in transfected and neighboring untransfected cells. Incubation with NiCl2 rapidly, dose-dependently, and reversibly decreased water permeability in AQP3-expressing cells. Acidification of the extracellular medium also caused rapid, dose-dependent, and reversible inhibition of AQP3. Sensitivity of AQP3 to nickel was lower at alkaline pH than at neutral and acidic pH. Cells transfected with human AQP4 and AQP5, which are also expressed in airway epithelia, were insensitive to nickel and extracellular acidification. Zinc and cadmium, other common causes of pneumoconiosis, had no effect on the water permeability of AQP3. Three extracellular residues, Trp128, Ser152, and His241, were responsible for the blocking effect of nickel on human AQP3. Ser152 was identified as a common site for nickel and pH sensitivity. His53, Tyr124, and His154 were also involved in regulation of AQP3 by extracellular pH. In addition, the aromatic side chain of His154 was shown to be important for the water permeability of AQP3. Our results imply that nickel and extracellular pH may modulate lung water clearance and that defective water clearance may be an early component of nickel-induced lung disease.

  • 39.
    Zelenina, Marina
    et al.
    Department of Woman and Child Health, Karolinska Institutet.
    Tritto, Simona
    Bondar, Alexander A.
    Zelenin, Sergey
    Aperia, Anita
    Copper inhibits the water and glycerol permeability of aquaporin-32004In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 50, p. 51939-51943Article in journal (Refereed)
    Abstract [en]

    Aquaporin-3 (AQP3) is an aquaglyceroporin expressed in erythrocytes and several other tissues. Erythrocytes are, together with kidney and liver, the main targets for copper toxicity. Here we report that both water and glycerol permeability of human AQP3 is inhibited by copper. Inhibition is fast, dose-dependent, and reversible. If copper is dissolved in carbonic acid-bicarbonate buffer, the natural buffer system in our body, doses in the range of those observed in Wilson disease and in copper poisoning caused significant inhibition. AQP7, another aquaglyceroporin, was insensitive to copper. Three extracellular amino acid residues, Trp128, Ser152, and His241, were identified as responsible for the effect of copper on AQP3. We have previously shown that Ser152 is involved in regulation of AQP3 by pH. The fact that Ser152 mediates regulation of AQP3 by copper may explain the phenomenon of exquisite sensitivity of human erythrocytes to copper at acidic pH. When AQP3 was co-expressed with another AQP, only glycerol but not water permeability was inhibited by copper. Our results provide a better understanding of processes that occur in severe copper metabolism defects such as Wilson disease and in copper poisoning.

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