XMG : eXtending MetaGrammars to MCTAG

In this paper, we introduce an extension of the XMG system (eXtensibleMeta-Grammar) in order to allow for the description of Multi-Component Tree Adjoining Grammars. In particular, we introduce the XMG formalism and its implementation, and show how the latter makes it possible to extend the system relatively easily to different target formalisms, thus opening the way towards multi-formalism.Dans cet article, nous présentons une extension du système XMG (eXtensible MetaGrammar) afin de permettre la description de grammaires darbres adjoints à composantes multiples. Nous présentons en particulier le formalisme XMG et son implantation et montrons comment celle-ci permet relativement aisément détendre le système à différents formalismes grammaticaux cibles, ouvrant ainsi la voie au multi-formalisme

TuLiPA : a syntax-semantics parsing environment for mildly context-sensitive formalisms

In this paper we present a parsing architecture that allows processing of different mildly context-sensitive formalisms, in particular Tree-Adjoining Grammar (TAG), Multi-Component Tree-Adjoining Grammar with Tree Tuples (TT-MCTAG) and simple Range Concatenation Grammar (RCG). Furthermore, for tree-based grammars, the parser computes not only syntactic analyses but also the corresponding semantic representations

The dynamic organization of fungal acetyl-CoA carboxylase

Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control

Developing a TT-MCTAG for German with an RCG-based parser

Developing linguistic resources, in particular grammars, is known to be a complex task in itself, because of (amongst others) redundancy and consistency issues. Furthermore some languages can reveal themselves hard to describe because of specific characteristics, e.g. the free word order in German. In this context, we present (i) a framework allowing to describe tree-based grammars, and (ii) an actual fragment of a core multicomponent tree-adjoining grammar with tree tuples (TT-MCTAG) for German developed using this framework. This framework combines a metagrammar compiler and a parser based on range concatenation grammar (RCG) to respectively check the consistency and the correction of the grammar. The German grammar being developed within this framework already deals with a wide range of scrambling and extraction phenomena

Crystal Structure of Thermotoga maritima α-Glucosidase AglA Defines a New Clan of NAD+-dependent Glycosidases

Glycoside hydrolase family 4 represents an unusual group of glucosidases with a requirement for NAD(+), divalent metal cations, and reducing conditions. The family is also unique in its inclusion of both alpha- and beta-specific enzymes. The alpha-glucosidase A, AglA, from Thermotoga maritima is a typical glycoside hydrolase family 4 enzyme, requiring NAD(+) and Mn2+ as well as strongly reducing conditions for activity. Here we present the crystal structure of the protein complexed with NAD(+) and maltose, refined at a resolution of 1.9 Angstrom. The NAD(+) is bound to a typical Rossman fold NAD(+)-binding site, and the nicotinamide moiety is localized close to the maltose substrate. Within the active site the conserved Cys-174 and surrounding histidines are positioned to play a role in the hydrolysis reaction. The electron density maps indicate that Cys-174 is oxidized to a sulfinic acid. Most likely, the strongly reducing conditions are necessary to reduce the oxidized cysteine side chain. Notably, the canonical set of catalytic acidic residues common to other glucosidases is not present in the active site. This, combined with a high structural homology to NAD-dependent dehydrogenases, suggests an unusual and possibly unique mechanism of action for a glycoside-hydrolyzing enzyme

TuLiPA : towards a multi-formalism parsing environment for grammar engineering

In this paper, we present an open-source parsing environment (Tübingen Linguistic Parsing Architecture, TuLiPA) which uses Range Concatenation Grammar (RCG) as a pivot formalism, thus opening the way to the parsing of several mildly context-sensitive formalisms. This environment currently supports tree-based grammars (namely Tree-Adjoining Grammars (TAG) and Multi-Component Tree-Adjoining Grammars with Tree Tuples (TT-MCTAG)) and allows computation not only of syntactic structures, but also of the corresponding semantic representations. It is used for the development of a tree-based grammar for German

TuLiPA : towards a multi-formalism parsing environment for grammar engineering

In this paper, we present an open-source parsing environment (Tübingen Linguistic Parsing Architecture, TuLiPA) which uses Range Concatenation Grammar (RCG) as a pivot formalism, thus opening the way to the parsing of several mildly context-sensitive formalisms. This environment currently supports tree-based grammars (namely Tree-Adjoining Grammars (TAG) and Multi-Component Tree-Adjoining Grammars with Tree Tuples (TT-MCTAG)) and allows computation not only of syntactic structures, but also of the corresponding semantic representations. It is used for the development of a tree-based grammar for German

β-Hexosaminidase B and Sphingolipid Activator Proteins

Titelblatt Inhaltsverzeichnis Zusammenfassung Danksagung Abkürzungen 1 Einleitung 1 1.1 Erbliche Stoffwechselstörungen 1 1.2 Glykosphingolipide 3 1.3 Glykosphingolipid-Speicherkrankheiten 4 1.4 β-Hexosaminidase und GM2-Gangliosidosen 5 1.5 Sphingolipid-Aktivatorproteine 9 1.6 Aufgabenstellung 10 1.7 Gliederung dieser Arbeit 11 2 Materialien und Methoden 12 2.1 Materialien 12 2.2 Geräte 14 2.3 Expression, Reinigung und Kristallisation von HexB 14 2.4 Expression, Reinigung und Kristallisation von SapC und SapD 16 2.5 Kristallographische Methoden 18 2.6 Strukturbestimmung von HexB nach der MIRAS-Methode 21 2.7 Strukturbestimmung von SapD durch molekularen Ersatz 29 2.8 Modellverfeinerung 30 2.9 Modellanalyse 32 2.10 Kleinwinkel-Röntgenstreuung 32 3 Ergebnisse zur β-Hexosaminidase B 38 3.1 Kristallisation von HexB 38 3.2 Schweratomderivatisierung von HexB-Kristallen 40 3.3 Strukturbestimmung von HexB mit der MIRAS-Methode 42 3.4 Modellbau und Verfeinerung 45 3.5 Faltung von HexB 47 3.6 Strukturverwandte von HexB 49 3.7 Der katalytische Mechanismus von HexB 50 3.8 Oligomerstruktur von HexB im Kristall 55 3.9 Dimere Struktur von HexB 61 3.10 Krankheitsassoziierte Mutationen in HexB 63 3.11 Röntgen-Kleinwinkelstreuung: Oligomerstruktur von HexB in Lösung 71 3.12 Ansätze zu weiteren Arbeiten zu HexB 86 4 Ergebnisse zu SapC und SapD 89 4.1 Kristallisation von SapC 89 4.2 Kristallisation und Derivatisierung von SapD 92 4.3 Strukturaufklärung von SapD 95 4.4 Die Faltung von SapD 98 4.5 Vergleich der Strukturen von SapD und Iodo-SapD 101 4.6 Strukturvergleich von SapD mit homologen Proteinen 107 4.7 Ladungsverteilung und Lipid-Interaktion Sap-ähnlicher Proteine 112 4.8 Ansätze zu weiteren Arbeiten zum Mechanismus Sap-ähnlicher Proteine 118 5 Resümee und Ausblick 120 Bibliographie Abbildungsverzeichnis Tabellenverzeichnis AnhangDie Dynamik der eukaryotischen Zellmembranen verlangt einen ständigen Abbau komplexer Membranlipide in ihre Grundbausteine und den Wiederaufbau neuer Membranbestandteile. Der Abbau komplexer Lipide erfolgt sequentiell durch lösliche Hydrolasen in Lysosomen, den sauren katabolischen Zellkompartimenten. Viele dieser Enzyme benötigen für die effiziente Umsetzung ihrer membranständigen Substrate kleine, enzymatisch nicht aktive Aktivatorproteine. Von besonderem Interesse ist der Abbauweg der Glykosphingolipide, da Unterbrechungen dieses Stoffwechselweges durch genetische Veränderungen zu angeborenen Stoffwechselstörungen führen, die durch eine Anhäufung von Abbauzwischenprodukten zu tödlicher Neurodegenration führen. Das Thema der vorliegenden Arbeit ist die strukturelle Charakterisierung von Proteinen des menschlichen Glykosphingolipidabbaus, nämlich des Enzyms β-Hexosaminindase und der Sphingolipidaktivatorproteine SapC und SapD. β-Hexosaminindase ist eine lysosomale Hydrolase mit breiter Substratspezifität. Sie ist jedoch nur für den Glykosphingolipidabbau essentiell, wie ihre Assoziation mit den Sphingolipid-Speicherkrankheiten Tay-Sachs- und Sandhoff-Krankheit zeigt. Das dimere Enzym kommt in drei Isoformen, HexA (αβ), HexB (ββ) und HexS (αα), vor, die durch die Kombination zweier nahe verwandter Untereinheiten, α und β, entstehen. In dieser Arbeit wurde die Röntgenkristallstruktur von HexB mit der MIRAS-Methode bei 2,3Å Auflösung bestimmt. Auf der Grundlage früherer Studien und der hier bestimmten Struktur im Komplex mit einem Übergangszustands- analogen Inhibitor, wird der konfigurationserhaltende doppelte Verdrängungsmechanismus von HexB im Detail erklärt. Die Analyse der Protein- Protein-Wechselwirkungen im Kristall offenbart die dimere Struktur von HexB bei lysosomalem pH. Im Dimer ist vermutlich Tyrosin 456 an der wechselseitigen Beeinflussung der Substratspezifität der Untereinheiten beteiligt. Aus der Lage der mit der Sandhoff-Krankheit assozierten Mutationen läßt sich unerwarteter Weise darauf schließen, dass diese eher die notwendige Dimerisierung als direkt das katalytische Zenturm beeinträchtigen. Kleinwinkel-Röntgenstreuexperimente zeigen, dass HexB bei annähernd neutralem pH als Tetramer vorliegt. Im Gegensatz zur Dimerkontaktfläche ist die Tetramerisierungsfläche nicht zwischen den α\- und β-Untereinheiten konserviert. Die auftretende Tetramerisierung bietet daher die erste Erklärung für die gegenüber der Heterodimerisierung mit der α-Untereinheit bevorzugte Homooligomerisierung der β-Untereinheit. Von den Sphingolipidaktivatorproteinen SapC und SapD wurden für Röntgenexperimente taugliche Kristalle erhalten. Die Kristallstruktur von SapD konnte nach einer durch Iodierung der Kristalle ausgelösten Raumgruppenänderung durch Molekularen Ersatz gelöst werden. SapD kristallisiert in einer ligandenfreien Konformation, die sich deutlich von der Kristallstruktur des verwandten SapB unterscheidet. Im Kristall bindet SapD Sulfat aus der Kristallisationslösung. Die Verteilung positiv geladener Reste und in SapD und die beobachteten Bindungsstellen des Phosphat-analogen Sulfat zeigen an, das SapD in einer anderen Art mit seinen primären Bindungspartnern, anionischen Phospholipidmembranen, interagiert als verwandte Proteine, z.B. Granulysin. Die gewonnenen Erkenntnisse erlauben ein neues Verständnis bestimmter erblicher Sphingolipid- Speicherkrankheiten und werfen neue Fragen zum Mechanismus der Katalyse an der Grenzfläche zwischen wässrigem Milieu und Lipidmembranen auf.The dynamic nature of eukaryotic cell membranes requires constant degradation of complex membrane lipids to basic building blocks and the re-synthesis of new membrane constituents. The breakdown of complex lipids is carried out by the sequential action of soluble hydrolases in lysosomes, the acidic, catabolic compartments of the cell. Many of these enzymes require the presence of small nonenzymatic activator proteins for efficient processing of membrane- bound lipid substrates. The catabolism of glycosphingolipids, an important class of outer layer membrane lipids, is of special interest because the disruption of the sequential degradation pathway by genetic alterations is the cause of multiple inborn errors of metabolism, which are all characterised by an accumulation of catabolic intermediates resulting in fatal neurodegeneration. This work focuses on the structural characterisation of proteins from human glycosphingolipid degradation, the enzyme β-hexosaminidase and the sphingolipid activator proteins SapC and SapD. β-Hexosaminidase is a lysosomal glycosidase with a broad substrate specificity. However, it is indispensable only for glycosphingolipid degradation, as demonstrated by its association with the fatal sphingolipid storage diseases Tay-Sachs and Sandhoff disease. The dimeric enzyme exists in three isoforms, HexA (αβ), HexB (ββ) and HexS (αα), generated by combinations of two closely related monomers, α and β. In this work the X-ray crystal structure of HexB has been determined by the MIRAS method to 2.3Å resolution. On the basis of prior work on related bacterial enzymes and the crystal structure in complex with a transition-state analogue inhibitor determined here, the configurationretaining double displacement mechanism of HexB is explained in detail. The analysis of protein-protein interactions in the crystal reveals the dimeric structure of HexB at lysosomal pH and identifies Tyrosine 456 as pointing inwards the active site of the companion subunit, potentially mediating an intersubunit crosstalk affecting substrate specificity. The locations of mutations associated with Sandhoff disease indicate that unexpectedly many of these are destabilizing the dimerisation contact rather than affecting the active site directly. Near neutral pH HexB is a tetramer in solution as demonstrated by small-angle X-ray scattering. In contrast to the dimerisation interface the tetramerisation site is not conserved between the α\- and β\- subunits, thus tetramerisation provides the first explanation for the observed preference of β-chains for homooligomerisation over heterodimerisation with α-chains. Diffraction-quality crystals have been obtained for the sphingolipid activator proteins SapC and SapD. The crystal structure of SapD has been determined by molecular replacement to 2.1Å resolution upon a change to a higher symmetry space group provoked by the iodination of SapD crystals. SapD crystallises in a closed, non-liganded conformation significantly different from those observed for the related SapB. In the crystal SapD binds sulfate from the crystallisation solution. The distribution of positively charged residues in SapD and the observed binding sites of sulfate, a structural analogue of phosphate, suggest a novel mode of binding of SapD to their primary interaction partners, anionic phospholipid-containing membranes, different from those decribed for related proteins, e.g. granulysin. The results obtained provide a new understanding of certain genetic sphingolipid stroage diseases and raise new questions regarding the mechanisms of catalysis at the interface of aqueous phase and lipid membranes

Structure and function of eukaryotic fatty acid synthases

In all organisms, fatty acid synthesis is achieved in variations of a common cyclic reaction pathway by stepwise, iterative elongation of precursors with two-carbon extender units. In bacteria, all individual reaction steps are carried out by monofunctional dissociated enzymes, whereas in eukaryotes the fatty acid synthases (FASs) have evolved into large multifunctional enzymes that integrate the whole process of fatty acid synthesis. During the last few years, important advances in understanding the structural and functional organization of eukaryotic FASs have been made through a combination of biochemical, electron microscopic and X-ray crystallographic approaches. They have revealed the strikingly different architectures of the two distinct types of eukaryotic FASs, the fungal and the animal enzyme system. Fungal FAS is a 2·6 MDa α6β6 heterododecamer with a barrel shape enclosing two large chambers, each containing three sets of active sites separated by a central wheel-like structure. It represents a highly specialized micro-compartment strictly optimized for the production of saturated fatty acids. In contrast, the animal FAS is a 540 kDa X-shaped homodimer with two lateral reaction clefts characterized by a modular domain architecture and large extent of conformational flexibility that appears to contribute to catalytic efficienc

Mycocerosic acid synthase exemplifies the architecture of reducing polyketide synthases

Polyketide synthases (PKSs) are biosynthetic factories that produce natural products with important biological and pharmacological activities1, 2, 3. Their exceptional product diversity is encoded in a modular architecture. Modular PKSs (modPKSs) catalyse reactions colinear to the order of modules in an assembly line3, whereas iterative PKSs (iPKSs) use a single module iteratively as exemplified by fungal iPKSs (fiPKSs)3. However, in some cases non-colinear iterative action is also observed for modPKSs modules and is controlled by the assembly line environment4, 5. PKSs feature a structural and functional separation into a condensing and a modifying region as observed for fatty acid synthases6. Despite the outstanding relevance of PKSs, the detailed organization of PKSs with complete fully reducing modifying regions remains elusive. Here we report a hybrid crystal structure of Mycobacterium smegmatis mycocerosic acid synthase based on structures of its condensing and modifying regions. Mycocerosic acid synthase is a fully reducing iPKS, closely related to modPKSs, and the prototype of mycobacterial mycocerosic acid synthase-like7, 8 PKSs. It is involved in the biosynthesis of C20–C28 branched-chain fatty acids, which are important virulence factors of mycobacteria9. Our structural data reveal a dimeric linker-based organization of the modifying region and visualize dynamics and conformational coupling in PKSs. On the basis of comparative small-angle X-ray scattering, the observed modifying region architecture may be common also in modPKSs. The linker-based organization provides a rationale for the characteristic variability of PKS modules as a main contributor to product diversity. The comprehensive architectural model enables functional dissection and re-engineering of PKSs