• Crystal Structure of Dihydro-Heme d Dehydrogenase NirN from Pseudomonas aeruginosa Reveals Amino Acid Residues Essential for Catalysis.

      Klünemann, Thomas; Preuß, Arne; Adamczack, Julia; Rosa, Luis F M; Harnisch, Falk; Layer, Gunhild; Blankenfeldt, Wulf; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier, 2019-06-04)
      Many bacteria can switch from oxygen to nitrogen oxides, such as nitrate or nitrite, as terminal electron acceptors in their respiratory chain. This process is called "denitrification" and enables biofilm formation of the opportunistic human pathogen Pseudomonas aeruginosa, making it more resilient to antibiotics and highly adaptable to different habitats. The reduction of nitrite to nitric oxide is a crucial step during denitrification. It is catalyzed by the homodimeric cytochrome cd1 nitrite reductase (NirS), which utilizes the unique isobacteriochlorin heme d1 as its reaction center. Although the reaction mechanism of nitrite reduction is well understood, far less is known about the biosynthesis of heme d1. The last step of its biosynthesis introduces a double bond in a propionate group of the tetrapyrrole to form an acrylate group. This conversion is catalyzed by the dehydrogenase NirN via a unique reaction mechanism. To get a more detailed insight into this reaction, the crystal structures of NirN with and without bound substrate have been determined. Similar to the homodimeric NirS, the monomeric NirN consists of an eight-bladed heme d1-binding β-propeller and a cytochrome c domain, but their relative orientation differs with respect to NirS. His147 coordinates heme d1 at the proximal side, whereas His323, which belongs to a flexible loop, binds at the distal position. Tyr461 and His417 are located next to the hydrogen atoms removed during dehydrogenation, suggesting an important role in catalysis. Activity assays with NirN variants revealed the essentiality of His147, His323 and Tyr461, but not of His417.
    • Position 123 of halohydrin dehalogenase HheG plays an important role in stability, activity, and enantioselectivity.

      Solarczek, Jennifer; Klünemann, Thomas; Brandt, Felix; Schrepfer, Patrick; Wolter, Mario; Jacob, Christoph R; Blankenfeldt, Wulf; Schallmey, Anett; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Nature publishing group, 2019-03-25)
      HheG from Ilumatobacter coccineus is a halohydrin dehalogenase with synthetically useful activity in the ring opening of cyclic epoxides with various small anionic nucleophiles. This enzyme provides access to chiral β-substituted alcohols that serve as building blocks in the pharmaceutical industry. Wild-type HheG suffers from low thermostability, which poses a significant drawback for potential applications. In an attempt to thermostabilize HheG by protein engineering, several single mutants at position 123 were identified which displayed up to 14 °C increased apparent melting temperatures and up to three-fold higher activity. Aromatic amino acids at position 123 resulted even in a slightly higher enantioselectivity. Crystal structures of variants T123W and T123G revealed a flexible loop opposite to amino acid 123. In variant T123G, this loop adopted two different positions resulting in an open or partially closed active site. Classical molecular dynamics simulations confirmed a high mobility of this loop. Moreover, in variant T123G this loop adopted a position much closer to residue 123 resulting in denser packing and increased buried surface area. Our results indicate an important role for position 123 in HheG and give first structural and mechanistic insight into the thermostabilizing effect of mutations T123W and T123G.
    • ER intrabody-mediated inhibition of interferon α secretion by mouse macrophages and dendritic cells.

      Büssow, Konrad; Themann, Philipp; Luu, Sabine; Pentrowski, Paul; Harting, Claudia; Majewski, Mira; Vollmer, Veith; Köster, Mario; Grashoff, Martina; Zawatzky, Rainer; et al. (Plos, 2019-01-01)
      Interferon α (IFNα) counteracts viral infections by activating various IFNα-stimulated genes (ISGs). These genes encode proteins that block viral transport into the host cell and inhibit viral replication, gene transcription and translation. Due to the existence of 14 different, highly homologous isoforms of mouse IFNα, an IFNα knockout mouse has not yet been established by genetic knockout strategies. An scFv intrabody for holding back IFNα isoforms in the endoplasmic reticulum (ER) and thus counteracting IFNα secretion is reported. The intrabody was constructed from the variable domains of the anti-mouse IFNα rat monoclonal antibody 4EA1 recognizing the 5 isoforms IFNα1, IFNα2, IFNα4, IFNα5, IFNα6. A soluble form of the intrabody had a KD of 39 nM to IFNα4. It could be demonstrated that the anti-IFNα intrabody inhibits clearly recombinant IFNα4 secretion by HEK293T cells. In addition, the secretion of IFNα4 was effectively inhibited in stably transfected intrabody expressing RAW 264.7 macrophages and dendritic D1 cells. Colocalization of the intrabody with IFNα4 and the ER marker calnexin in HEK293T cells indicated complex formation of intrabody and IFNα4 inside the ER. Intracellular binding of intrabody and antigen was confirmed by co-immunoprecipitation. Complexes of endogenous IFNα and intrabody could be visualized in the ER of Poly (I:C) stimulated RAW 264.7 macrophages and D1 dendritic cells. Infection of macrophages and dendritic cells with the vesicular stomatitis virus VSV-AV2 is attenuated by IFNα and IFNβ. The intrabody increased virus proliferation in RAW 264.7 macrophages and D1 dendritic cells under IFNβ-neutralizing conditions. To analyze if all IFNα isoforms are recognized by the intrabody was not in the focus of this study. Provided that binding of the intrabody to all isoforms was confirmed, the establishment of transgenic intrabody mice would be promising for studying the function of IFNα during viral infection and autoimmune diseases.
    • Molecular Mechanisms of Vaspin Action - From Adipose Tissue to Skin and Bone, from Blood Vessels to the Brain.

      Weiner, Juliane; Zieger, Konstanze; Pippel, Jan; Heiker, John T; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer, 2019-01-01)
      Visceral adipose tissue-derived serine protease inhibitor (vaspin) or SERPINA12 according to the serpin nomenclature was identified together with other genes and gene products that were specifically expressed or overexpressed in the intra-abdominal or visceral adipose tissue (AT) of the Otsuka Long-Evans Tokushima fatty rat. These rats spontaneously develop visceral obesity, insulin resistance, hyperinsulinemia and -glycemia, as well as hypertension and thus represent a well suited animal model of obesity and related metabolic disorders such as type 2 diabetes.The follow-up study reporting the cloning, expression and functional characterization of vaspin suggested the great and promising potential of this molecule to counteract obesity induced insulin resistance and inflammation and has since initiated over 300 publications, clinical and experimental, that have contributed to uncover the multifaceted functions and molecular mechanisms of vaspin action not only in the adipose, but in many different cells, tissues and organs. This review will give an update on mechanistic and structural aspects of vaspin with a focus on its serpin function, the physiology and regulation of vaspin expression, and will summarize the latest on vaspin function in various tissues such as the different adipose tissue depots as well as the vasculature, skin, bone and the brain.
    • Distinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Non-redundant Functions in Vivo.

      Schaks, Matthias; Singh, Shashi P; Kage, Frieda; Thomason, Peter; Klünemann, Thomas; Steffen, Anika; Blankenfeldt, Wulf; Stradal, Theresia E; Insall, Robert H; Rottner, Klemens; et al. (2018-10-25)
      Cell migration often involves the formation of sheet-like lamellipodia generated by branched actin filaments. The branches are initiated when Arp2/3 complex [1] is activated by WAVE regulatory complex (WRC) downstream of small GTPases of the Rac family [2]. Recent structural studies defined two independent Rac binding sites on WRC within the Sra-1/PIR121 subunit of the pentameric WRC [3, 4], but the functions of these sites in vivo have remained unknown. Here we dissect the mechanism of WRC activation and the in vivo relevance of distinct Rac binding sites on Sra-1, using CRISPR/Cas9-mediated gene disruption of Sra-1 and its paralog PIR121 in murine B16-F1 cells combined with Sra-1 mutant rescue. We show that the A site, positioned adjacent to the binding region of WAVE-WCA mediating actin and Arp2/3 complex binding, is the main site for allosteric activation of WRC. In contrast, the D site toward the C terminus is dispensable for WRC activation but required for optimal lamellipodium morphology and function. These results were confirmed in evolutionarily distant Dictyostelium cells. Moreover, the phenotype seen in D site mutants was recapitulated in Rac1 E31 and F37 mutants; we conclude these residues are important for Rac-D site interaction. Finally, constitutively activated WRC was able to induce lamellipodia even after both Rac interaction sites were lost, showing that Rac interaction is not essential for membrane recruitment. Our data establish that physical interaction with Rac is required for WRC activation, in particular through the A site, but is not mandatory for WRC accumulation in the lamellipodium.
    • Pseudomonas aeruginosa pyoverdine maturation enzyme PvdP has a noncanonical domain architecture and affords insight into a new subclass of tyrosinases

      Poppe, Juliane; Reichelt, Joachim; Blankenfeldt, Wulf; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2018-09-21)
      Pyoverdines (PVDs) are important chromophore-containing siderophores of fluorescent pseudomonad bacteria such as the opportunistic human pathogen
    • Structural insights into antigen recognition of an anti-β-(1,6)-β-(1,3)-D-glucan antibody.

      Sung, Kwang Hoon; Josewski, Jörn; Dübel, Stefan; Blankenfeldt, Wulf; Rau, Udo; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2018-09-12)
      Schizophyllan (SCH) is a high molecular weight homopolysaccharide composed of a β-(1,3)-D-glucan main chain with branching β-(1,6)-bound D-glucose residues. It forms triple helices that are highly stable towards heat and extreme pH, which provides SCH with interesting properties for industrial and medical applications. The recombinant anti-SCH antibody JoJ48C11 recognizes SCH and related β-(1,6)-branched β-(1,3)-D-glucans, but details governing its specificity are not known. Here, we fill this gap by determining crystal structures of the antigen binding fragment (Fab) of JoJ48C11 in the apo form and in complex with the unbranched β-(1,3)-D-glucose hexamer laminarihexaose 3.0 and 2.4 Å resolution, respectively. Together with docking studies, this allowed construction of a JoJ48C11/triple-helical SCH complex, leading to the identification of eight amino acid residues of JoJ48C11 (Tyr27
    • TMPRSS11A activates the influenza A virus hemagglutinin and the MERS coronavirus spike protein and is insensitive against blockade by HAI-1.

      Zmora, Pawel; Hoffmann, Markus; Kollmus, Heike; Moldenhauer, Anna-Sophie; Danov, Olga; Braun, Armin; Winkler, Michael; Schughart, Klaus; Pöhlmann, Stefan; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-09-07)
      The influenza virus hemagglutinin (HA) facilitates viral entry into target cells. Cleavage of HA by host cell proteases is essential for viral infectivity, and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease (TTSP) TMPRSS2 has been identified as an HA activator in cell culture and in the infected host. However, it is less clear whether TMPRSS2-related enzymes can also activate HA for spread in target cells. Moreover, the activity of cellular serine protease inhibitors against HA-activating TTSPs is poorly understood. Here, we show that TMPRSS11A, another member of the TTSP family, cleaves and activates the influenza A virus (FLUAV) HA and the Middle East respiratory syndrome coronavirus spike protein (MERS-S). Moreover, we demonstrate that TMPRSS11A is expressed in murine tracheal epithelium, which is a target of FLUAV infection, and in human trachea, suggesting that the protease could support FLUAV spread in patients. Finally, we show that HA activation by the TMPRSS11A-related enzymes human airway tryptase and DESC1, but not TMPRSS11A itself, is blocked by the cellular serine protease inhibitor hepatocyte growth factor activator inhibitor type-1 (HAI-1). Our results suggest that TMPRSS11A could promote FLUAV spread in target cells and that HA-activating TTSPs exhibit differential sensitivity to blockade by cellular serine protease inhibitors.
    • Exchange of amino acids in the H1-haemagglutinin to H3 residues is required for efficient influenza A virus replication and pathology in Tmprss2 knock-out mice.

      Lambertz, Ruth L O; Pippel, Jan; Gerhauser, Ingo; Kollmus, Heike; Anhlan, Darisuren; Hrincius, Eike R; Krausze, Joern; Kühn, Nora; Schughart, Klaus; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-09-01)
      The haemagglutinin (HA) of H1N1 and H3N2 influenza A virus (IAV) subtypes has to be activated by host proteases. Previous studies showed that H1N1 virus cannot replicate efficiently in Tmprss2/ knock-out mice whereas H3N2 viruses are able to replicate to the same levels in Tmprss2/ as in wild type (WT) mice. Here, we investigated the sequence requirements for the HA molecule that allow IAV to replicate efficiently in the absence of TMPRSS2. We showed that replacement of the H3 for the H1-loop sequence (amino acids 320 to 329, at the C-terminus of HA1) was not sufficient for equal levels of virus replication or severe pathology in Tmprss2/ knock-out mice compared to WT mice. However, exchange of a distant amino acid from H1 to H3 sequence (E31D) in addition to the HA-loop substitution resulted in virus replication in Tmprss2/ knockout mice that was comparable to WT mice. The higher virus replication and lung damage was associated with increased epithelial damage and higher mortality. Our results provide further evidence and insights into host proteases as a promising target for therapeutic intervention of IAV infections.
    • The Alkylquinolone Repertoire of Pseudomonas aeruginosa is Linked to Structural Flexibility of the FabH-like 2-Heptyl-3-hydroxy-4(1H)-quinolone (PQS) Biosynthesis Enzyme PqsBC.

      Witzgall, Florian; Depke, Tobias; Hoffmann, Michael; Empting, Martin; Brönstrup, Mark; Müller, Rolf; Blankenfeldt, Wulf; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley-VCH, 2018-07-16)
      Pseudomonas aeruginosa is a bacterial pathogen that causes life-threatening infections in immunocompromised patients. It produces a large armory of saturated and mono-unsaturated 2-alkyl-4(1H)-quinolones (AQs) and AQ N-oxides (AQNOs) that serve as signaling molecules to control the production of virulence factors and that are involved in membrane vesicle formation and iron chelation; furthermore, they also have, for example, antibiotic properties. It has been shown that the β-ketoacyl-acyl-carrier protein synthase III (FabH)-like heterodimeric enzyme PqsBC catalyzes the last step in the biosynthesis of the most abundant AQ congener, 2-heptyl-4(1H)-quinolone (HHQ), by condensing octanoyl-coenzyme A (CoA) with 2-aminobenzoylacetate (2-ABA), but the basis for the large number of other AQs/AQNOs produced by P. aeruginosa is not known. Here, we demonstrate that PqsBC uses different medium-chain acyl-CoAs to produce various saturated AQs/AQNOs and that it also biosynthesizes mono-unsaturated congeners. Further, we determined the structures of PqsBC in four different crystal forms at 1.5 to 2.7 Å resolution. Together with a previous report, the data reveal that PqsBC adopts open, intermediate, and closed conformations that alter the shape of the acyl-binding cavity and explain the promiscuity of PqsBC. The different conformations also allow us to propose a model for structural transitions that accompany the catalytic cycle of PqsBC that might have broader implications for other FabH-enzymes, for which such structural transitions have been postulated but have never been observed.
    • Investigations on the mode of action of gephyronic acid, an inhibitor of eukaryotic protein translation from myxobacteria.

      Muthukumar, Yazh; Münkemer, Johanna; Mathieu, Daniel; Richter, Christian; Schwalbe, Harald; Steinmetz, Heinrich; Kessler, Wolfgang; Reichelt, Joachim; Beutling, Ulrike; Frank, Ronald; et al. (PLOS, 2018-01-01)
      The identification of inhibitors of eukaryotic protein biosynthesis, which are targeting single translation factors, is highly demanded. Here we report on a small molecule inhibitor, gephyronic acid, isolated from the myxobacterium Archangium gephyra that inhibits growth of transformed mammalian cell lines in the nM range. In direct comparison, primary human fibroblasts were shown to be less sensitive to toxic effects of gephyronic acid than cancer-derived cells. Gephyronic acid is targeting the protein translation system. Experiments with IRES dual luciferase reporter assays identified it as an inhibitor of the translation initiation. DARTs approaches, co-localization studies and pull-down assays indicate that the binding partner could be the eukaryotic initiation factor 2 subunit alpha (eIF2α). Gephyronic acid seems to have a different mode of action than the structurally related polyketides tedanolide, myriaporone, and pederin and is a valuable tool for investigating the eukaryotic translation system. Because cancer derived cells were found to be especially sensitive, gephyronic acid could potentially find use as a drug candidate.
    • Crystal Structures of R-Type Bacteriocin Sheath and Tube Proteins CD1363 and CD1364 From in the Pre-assembled State.

      Schwemmlein, Nina; Pippel, Jan; Gazdag, Emerich-Mihai; Blankenfeldt, Wulf (2018-01-01)
      iffocins are high-molecular-weight phage tail-like bacteriocins (PTLBs) that some Clostridium difficile strains produce in response to SOS induction. Similar to the related R-type pyocins from Pseudomonas aeruginosa, R-type diffocins act as molecular puncture devices that specifically penetrate the cell envelope of other C. difficile strains to dissipate the membrane potential and kill the attacked bacterium. Thus, R-type diffocins constitute potential therapeutic agents to counter C. difficile-associated infections. PTLBs consist of rigid and contractile protein complexes. They are composed of a baseplate, receptor-binding tail fibers and an inner needle-like tube surrounded by a contractile sheath. In the mature particle, the sheath and tube structure form a complex network comprising up to 200 copies of a sheath and a tube protein each. Here, we report the crystal structures together with small angle X-ray scattering data of the sheath and tube proteins CD1363 (39 kDa) and CD1364 (16 kDa) from C. difficile strain CD630 in a monomeric pre-assembly form at 1.9 and 1.5 Å resolution, respectively. The tube protein CD1364 displays a compact fold and shares highest structural similarity with a tube protein from Bacillus subtilis but is remarkably different from that of the R-type pyocin from P. aeruginosa. The structure of the R-type diffocin sheath protein, on the other hand, is highly conserved. It contains two domains, whereas related members such as bacteriophage tail sheath proteins comprise up to four, indicating that R-type PTLBs may represent the minimal protein required for formation of a complete sheath structure. Comparison of CD1363 and CD1364 with structures of PTLBs and related assemblies suggests that several conformational changes are required to form complete assemblies. In the sheath, rearrangement of the flexible N- and C-terminus enables extensive interactions between the other subunits, whereas for the tube, such contacts are primarily established by mobile α-helices. Together, our results combined with information from structures of homologous assemblies allow constructing a preliminary model of the sheath and tube assembly from R-type diffocin.
    • Recent developments in the isolation, biological function, biosynthesis, and synthesis of phenazine natural products.

      Guttenberger, Nikolaus; Blankenfeldt, Wulf; Breinbauer, Rolf; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-11-15)
      Phenazines are natural products which are produced by bacteria or by archaeal Methanosarcina species. The tricyclic ring system enables redox processes, which producing organisms use for oxidation of NADH or for the generation of reactive oxygen species (ROS), giving them advantages over other microorganisms. In this review we summarize the progress in the field since 2005 regarding the isolation of new phenazine natural products, new insights in their biological function, and particularly the now almost completely understood biosynthesis. The review is complemented by a description of new synthetic methods and total syntheses of phenazines.
    • Biosynthesis of methyl-proline containing griselimycins, natural products with anti-tuberculosis activity.

      Lukat, Peer; Katsuyama, Yohei; Wenzel, Silke; Binz, Tina; König, Claudia; Blankenfeldt, Wulf; Brönstrup, Mark; Müller, Rolf; Helmholtz-Institut für pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2017-11-01)
      Griselimycins (GMs) are depsidecapeptides with superb anti-tuberculosis activity. They contain up to three (2S,4R)-4-methyl-prolines (4-MePro), of which one blocks oxidative degradation and increases metabolic stability in animal models. The natural congener with this substitution is only a minor component in fermentation cultures. We showed that this product can be significantly increased by feeding the reaction with 4-MePro and we investigated the molecular basis of 4-MePro biosynthesis and incorporation. We identified the GM biosynthetic gene cluster as encoding a nonribosomal peptide synthetase and a sub-operon for 4-MePro formation. Using heterologous expression, gene inactivation, and in vitro experiments, we showed that 4-MePro is generated by leucine hydroxylation, oxidation to an aldehyde, and ring closure with subsequent reduction. The crystal structures of the leucine hydroxylase GriE have been determined in complex with substrates and products, providing insight into the stereospecificity of the reaction.
    • ER-targeted intrabodies mediating specific in vivo knockdown of transitory proteins in comparison to RNAi

      Backhaus, Oliver; Böldicke, Thomas; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-10-25)
      In animals and mammalian cells, protein function can be analyzed by nucleotide sequence-based methods such as gene knockout, targeted gene disruption, CRISPR/Cas, TALEN, zinc finger nucleases, or the RNAi technique. Alternatively, protein knockdown approaches are available based on direct interference of the target protein with the inhibitor.Among protein knockdown techniques, the endoplasmic reticulum (ER) intrabodies arepotent molecules for protein knockdown in vitro and in vivo. These molecules are increasingly used for protein knockdown in living cells and transgenic mice. ER intrabody knockdown technique is based on the retention of membrane proteins and secretory proteins inside the ER, mediated by recombinant antibody fragments. In contrast to nucleotide sequence-based methods, the intrabody-mediated knockdown actsonly on the posttranslational level. In this review, the ER intrabody technology has been compared with the RNAi technique on the molecular level. The generation of intrabodies and RNAi has also been discussed. Specificity and off-target effects (OTE) of these molecules as well as the therapeutic potential of ER intrabodies and RNAi have been compared.
    • Characterization and structural determination of a new anti-MET function-blocking antibody with binding epitope distinct from the ligand binding domain.

      DiCara, Danielle M; Chirgadze, Dimitri Y; Pope, Anthony R; Karatt-Vellatt, Aneesh; Winter, Anja; Slavny, Peter; van den Heuvel, Joop; Parthiban, Kothai; Holland, Jane; Packman, Len C; et al. (2017-08-21)
      The growth and motility factor Hepatocyte Growth Factor/Scatter Factor (HGF/SF) and its receptor, the product of the MET proto-oncogene, promote invasion and metastasis of tumor cells and have been considered potential targets for cancer therapy. We generated a new Met-blocking antibody which binds outside the ligand-binding site, and determined the crystal structure of the Fab in complex with its target, which identifies the binding site as the Met Ig1 domain. The antibody, 107_A07, inhibited HGF/SF-induced cell migration and proliferation in vitro and inhibited growth of tumor xenografts in vivo. In biochemical assays, 107_A07 competes with both HGF/SF and its truncated splice variant NK1 for MET binding, despite the location of the antibody epitope on a domain (Ig1) not reported to bind NK1 or HGF/SF. Overlay of the Fab-MET crystal structure with the InternalinB-MET crystal structure shows that the 107_A07 Fab comes into close proximity with the HGF/SF-binding SEMA domain when MET is in the "compact", InternalinB-bound conformation, but not when MET is in the "open" conformation. These findings provide further support for the importance of the "compact" conformation of the MET extracellular domain, and the relevance of this conformation to HGF/SF binding and signaling.
    • Mechanisms and Specificity of Phenazine Biosynthesis Protein PhzF.

      Diederich, Christina; Leypold, Mario; Culka, Martin; Weber, Hansjörg; Breinbauer, Rolf; Ullmann, G Matthias; Blankenfeldt, Wulf; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-07-24)
      Phenazines are bacterial virulence and survival factors with important roles in infectious disease. PhzF catalyzes a key reaction in their biosynthesis by isomerizing (2 S,3 S)-2,3-dihydro-3-hydroxy anthranilate (DHHA) in two steps, a [1,5]-hydrogen shift followed by tautomerization to an aminoketone. While the [1,5]-hydrogen shift requires the conserved glutamate E45, suggesting acid/base catalysis, it also shows hallmarks of a sigmatropic rearrangement, namely the suprafacial migration of a non-acidic proton. To discriminate these mechanistic alternatives, we employed enzyme kinetic measurements and computational methods. Quantum mechanics/molecular mechanics (QM/MM) calculations revealed that the activation barrier of a proton shuttle mechanism involving E45 is significantly lower than that of a sigmatropic [1,5]-hydrogen shift. QM/MM also predicted a large kinetic isotope effect, which was indeed observed with deuterated substrate. For the tautomerization, QM/MM calculations suggested involvement of E45 and an active site water molecule, explaining the observed stereochemistry. Because these findings imply that PhzF can act only on a limited substrate spectrum, we also investigated the turnover of DHHA derivatives, of which only O-methyl and O-ethyl DHHA were converted. Together, these data reveal how PhzF orchestrates a water-free with a water-dependent step. Its unique mechanism, specificity and essential role in phenazine biosynthesis may offer opportunities for inhibitor development.
    • Intrabodies against the Polysialyltransferases ST8SiaII and ST8SiaIV inhibit Polysialylation of NCAM in rhabdomyosarcoma tumor cells.

      Somplatzki, Stefan; Mühlenhoff, Martina; Kröger, Andrea; Gerardy-Schahn, Rita; Böldicke, Thomas; Helmholtz Centr for infection research (2017-05-12)
      Polysialic acid (polySia) is a carbohydrate modification of the neural cell adhesion molecule (NCAM), which is implicated in neural differentiation and plays an important role in tumor development and metastasis. Polysialylation of NCAM is mediated by two Golgi-resident polysialyltransferases (polyST) ST8SiaII and ST8SiaIV. Intracellular antibodies (intrabodies; IB) expressed inside the ER and retaining proteins passing the ER such as cell surface receptors or secretory proteins provide an efficient means of protein knockdown. To inhibit the function of ST8SiaII and ST8SiaIV specific ER IBs were generated starting from two corresponding hybridoma clones. Both IBs αST8SiaII-IB and αST8SiaIV-IB were constructed in the scFv format and their functions characterized in vitro and in vivo.
    • Structural, mechanistic and functional insight into gliotoxin bis-thiomethylation in Aspergillus fumigatus.

      Dolan, Stephen K; Bock, Tobias; Hering, Vanessa; Owens, Rebecca A; Jones, Gary W; Blankenfeldt, Wulf; Doyle, Sean; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-02)
      Gliotoxin is an epipolythiodioxopiperazine (ETP) class toxin, contains a disulfide bridge that mediates its toxic effects via redox cycling and is produced by the opportunistic fungal pathogen Aspergillus fumigatus Self-resistance against gliotoxin is effected by the gliotoxin oxidase GliT, and attenuation of gliotoxin biosynthesis is catalysed by gliotoxin S-methyltransferase GtmA. Here we describe the X-ray crystal structures of GtmA-apo (1.66 Å), GtmA complexed to S-adenosylhomocysteine (1.33 Å) and GtmA complexed to S-adenosylmethionine (2.28 Å), providing mechanistic insights into this important biotransformation. We further reveal that simultaneous elimination of the ability of A. fumigatus to dissipate highly reactive dithiol gliotoxin, via deletion of GliT and GtmA, results in the most significant hypersensitivity to exogenous gliotoxin observed to date. Indeed, quantitative proteomic analysis of ΔgliT::ΔgtmA reveals an uncontrolled over-activation of the gli-cluster upon gliotoxin exposure. The data presented herein reveal, for the first time, the extreme risk associated with intracellular dithiol gliotoxin biosynthesis-in the absence of an efficient dismutation capacity. Significantly, a previously concealed protective role for GtmA and functionality of ETP bis-thiomethylation as an ancestral protection strategy against dithiol compounds is now evident.
    • Single domain antibodies for the knockdown of cytosolic and nuclear proteins.

      Böldicke, Thomas; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley-Blackwell, 2017-01-01)
      Single domain antibodies (sdAbs) from camels or sharks comprise only the variable heavy chain domain. Human sdAbs comprise the variable domain of the heavy chain (VH) or light chain (VL) and can be selected from human antibodies. SdAbs are stable, nonaggregating molecules in vitro and in vivo compared to complete antibodies and scFv fragments. They are excellent novel inhibitors of cytosolic/nuclear proteins because they are correctly folded inside the cytosol in contrast to scFv fragments. SdAbs are unique because of their excellent specificity and possibility to target posttranslational modifications such as phosphorylation sites, conformers or interaction regions of proteins that cannot be targeted with genetic knockout techniques and are impossible to knockdown with RNAi. The number of inhibiting cytosolic/nuclear sdAbs is increasing and usage of synthetic single pot single domain antibody libraries will boost the generation of these fascinating molecules without the need of immunization. The most frequently selected antigenic epitopes belong to viral and oncogenic proteins, followed by toxins, proteins of the nervous system as well as plant‐ and drosophila proteins. It is now possible to select functional sdAbs against virtually every cytosolic/nuclear protein and desired epitope. The development of new endosomal escape protein domains and cell‐penetrating peptides for efficient transfection broaden the application of inhibiting sdAbs. Last but not least, the generation of relatively new cell‐specific nanoparticles such as polymersomes and polyplexes carrying cytosolic/nuclear sdAb‐DNA or –protein will pave the way to apply cytosolic/nuclear sdAbs for inhibition of viral infection and cancer in the clinic. Keywords: intrabodies, single domain antibodies, scFv fragment, cytosolic/nuclear intrabodies, camelid VHHs, shark vNARs, human VH, human VL