• Crystallization, room-temperature X-ray diffraction and preliminary analysis of Kaposi's sarcoma herpesvirus LANA bound to DNA.

      Hellert, Jan; Krausze, Joern; Schulz, Thomas F; Lührs, Thorsten; Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany. (2014-11)
      The latency-associated nuclear antigen (LANA) is the latent origin-binding protein and chromatin anchor of the Kaposi's sarcoma herpesvirus (KSHV/HHV-8) genome. Its C-terminal domain (CTD) binds sequence-specifically to the viral origin of replication, whereas the N-terminal domain links it to nucleosomes of cellular chromatin for long-term persistence in dividing host cells. Here, the crystallization and X-ray data acquisition of a mutant LANA CTD in complex with its wild-type target DNA LBS1 is described. This report describes the rational protein engineering for successful co-crystallization with DNA and X-ray diffraction data collection at room temperature on the high-brilliance third-generation synchrotron PETRA III at DESY, Germany.
    • The structural biology of phenazine biosynthesis.

      Blankenfeldt, Wulf; Parsons, James F; Helmholtz Centre for Infection Research, Structure and Function of Proteins, Inhoffenstr. 7, 38124 Braunschweig, Germany. Electronic address: wulf.blankenfeldt@helmholtz-hzi.de. (2014-09-09)
      The phenazines are a class of over 150 nitrogen-containing aromatic compounds of bacterial and archeal origin. Their redox properties not only explain their activity as broad-specificity antibiotics and virulence factors but also enable them to function as respiratory pigments, thus extending their importance to the primary metabolism of phenazine-producing species. Despite their discovery in the mid-19th century, the molecular mechanisms behind their biosynthesis have only been unraveled in the last decade. Here, we review the contribution of structural biology that has led to our current understanding of phenazine biosynthesis.
    • Oligomerization inhibits Legionella pneumophila PlaB phospholipase A activity.

      Kuhle, Katja; Krausze, Joern; Curth, Ute; Rössle, Manfred; Heuner, Klaus; Lang, Christina; Flieger, Antje (2014-07-04)
      The intracellularly replicating lung pathogen Legionella pneumophila consists of an extraordinary variety of phospholipases, including at least 15 different phospholipases A (PLA). Among them, PlaB, the first characterized member of a novel lipase family, is a hemolytic virulence factor that exhibits the most prominent PLA activity in L. pneumophila. We analyzed here protein oligomerization, the importance of oligomerization for activity, addressed further essential regions for activity within the PlaB C terminus, and the significance of PlaB-derived lipolytic activity for L. pneumophila intracellular replication. We determined by means of analytical ultracentrifugation and small angle x-ray scattering analysis that PlaB forms homodimers and homotetramers. The C-terminal 5, 10, or 15 amino acids, although the individual regions contributed to PLA activity, were not essential for protein tetramerization. Infection of mouse macrophages with L. pneumophila wild type, plaB knock-out mutant, and plaB complementing or various mutated plaB-harboring strains showed that catalytic activity of PlaB promotes intracellular replication. We observed that PlaB was most active in the lower nanomolar concentration range but not at or only at a low level at concentration above 0.1 μm where it exists in a dimer/tetramer equilibrium. We therefore conclude that PlaB is a virulence factor that, on the one hand, assembles in inactive tetramers at micromolar concentrations. On the other hand, oligomer dissociation at nanomolar concentrations activates PLA activity. Our data highlight the first example of concentration-dependent phospholipase inactivation by tetramerization, which may protect the bacterium from internal PLA activity, but enzyme dissociation may allow its activation after export.
    • Crystallization and preliminary X-ray analysis of the ergothioneine-biosynthetic methyltransferase EgtD.

      Vit, Allegra; Misson, Laëtitia; Blankenfeldt, Wulf; Seebeck, Florian Peter; Dept of structure and functions of proteins, Hemholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2014-05)
      Ergothioneine is an amino-acid betaine derivative of histidine that was discovered more than one century ago. Despite significant research pointing to a function in oxidative stress defence, the exact mechanisms of action of ergothioneine remain elusive. Although both humans and bacterial pathogens such as Mycobacterium tuberculosis seem to depend on ergothioneine, humans are devoid of the corresponding biosynthetic enzymes. Therefore, its biosynthesis may emerge as potential drug target in the development of novel therapeutics against tuberculosis. The recent identification of ergothioneine-biosynthetic genes in M. smegmatis enables a more systematic study of its biology. The pathway is initiated by EgtD, a SAM-dependent methyltransferase that catalyzes a trimethylation reaction of histidine to give N(α),N(α),N(α)-trimethylhistidine. Here, the recombinant production, purification and crystallization of EgtD are reported. Crystals of native EgtD diffracted to 2.35 Å resolution at a synchrotron beamline, whereas crystals of seleno-L-methionine-labelled protein diffracted to 1.75 Å resolution and produced a significant anomalous signal to 2.77 Å resolution at the K edge. All of the crystals belonged to space group P212121, with two EgtD monomers in the asymmetric unit.
    • An enzyme from Auricularia auricula-judae combining both benzoyl and cinnamoyl esterase activity

      Haase-Aschoff, Paul; Linke, Diana; Nimtz, Manfred; Popper, Lutz; Berger, Ralf G.; Dept. of structure and function of proteins, Helmhotz Centre for infection research, D-38124 Braunschweig, Germany (2014-01-14)
    • High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells

      Jäger, Volker; Büssow, Konrad; Wagner, Andreas; Weber, Susanne; Hust, Michael; Frenzel, André; Schirrmann, Thomas (2013-06-26)
      Abstract Background The demand of monospecific high affinity binding reagents, particularly monoclonal antibodies, has been steadily increasing over the last years. Enhanced throughput of antibody generation has been addressed by optimizing in vitro selection using phage display which moved the major bottleneck to the production and purification of recombinant antibodies in an end-user friendly format. Single chain (sc)Fv antibody fragments require additional tags for detection and are not as suitable as immunoglobulins (Ig)G in many immunoassays. In contrast, the bivalent scFv-Fc antibody format shares many properties with IgG and has a very high application compatibility. Results In this study transient expression of scFv-Fc antibodies in human embryonic kidney (HEK) 293 cells was optimized. Production levels of 10-20 mg/L scFv-Fc antibody were achieved in adherent HEK293T cells. Employment of HEK293-6E suspension cells expressing a truncated variant of the Epstein Barr virus (EBV) nuclear antigen (EBNA) 1 in combination with production under serum free conditions increased the volumetric yield up to 10-fold to more than 140 mg/L scFv-Fc antibody. After vector optimization and process optimization the yield of an scFv-Fc antibody and a cytotoxic antibody-RNase fusion protein further increased 3-4-fold to more than 450 mg/L. Finally, an entirely new mammalian expression vector was constructed for single step in frame cloning of scFv genes from antibody phage display libraries. Transient expression of more than 20 different scFv-Fc antibodies resulted in volumetric yields of up to 600 mg/L and 400 mg/L in average. Conclusion Transient production of recombinant scFv-Fc antibodies in HEK293-6E in combination with optimized vectors and fed batch shake flasks cultivation is efficient and robust, and integrates well into a high-throughput recombinant antibody generation pipeline.
    • Crystal structure of the conserved domain of the DC lysosomal associated membrane protein: implications for the lysosomal glycocalyx

      Wilke, Sonja; Krausze, Joern; Büssow, Konrad (2012-07-19)
      Abstract Background The family of lysosome-associated membrane proteins (LAMP) comprises the multifunctional, ubiquitous LAMP-1 and LAMP-2, and the cell type-specific proteins DC-LAMP (LAMP-3), BAD-LAMP (UNC-46, C20orf103) and macrosialin (CD68). LAMPs have been implicated in a multitude of cellular processes, including phagocytosis, autophagy, lipid transport and aging. LAMP-2 isoform A acts as a receptor in chaperone-mediated autophagy. LAMP-2 deficiency causes the fatal Danon disease. The abundant proteins LAMP-1 and LAMP-2 are major constituents of the glycoconjugate coat present on the inside of the lysosomal membrane, the 'lysosomal glycocalyx'. The LAMP family is characterized by a conserved domain of 150 to 200 amino acids with two disulfide bonds. Results The crystal structure of the conserved domain of human DC-LAMP was solved. It is the first high-resolution structure of a heavily glycosylated lysosomal membrane protein. The structure represents a novel β-prism fold formed by two β-sheets bent by β-bulges and connected by a disulfide bond. Flexible loops and a hydrophobic pocket represent possible sites of molecular interaction. Computational models of the glycosylated luminal regions of LAMP-1 and LAMP-2 indicate that the proteins adopt a compact conformation in close proximity to the lysosomal membrane. The models correspond to the thickness of the lysosomal glycoprotein coat of only 5 to 12 nm, according to electron microscopy. Conclusion The conserved luminal domain of lysosome-associated membrane proteins forms a previously unknown β-prism fold. Insights into the structure of the lysosomal glycoprotein coat were obtained by computational models of the LAMP-1 and LAMP-2 luminal regions.
    • Side effects of chaperone gene co-expression in recombinant protein production

      Martínez-Alonso, Mónica; García-Fruitós, Elena; Ferrer-Miralles, Neus; Rinas, Ursula; Villaverde, Antonio (2010-09-02)
      Abstract Insufficient availability of molecular chaperones is observed as a major bottleneck for proper protein folding in recombinant protein production. Therefore, co-production of selected sets of cell chaperones along with foreign polypeptides is a common approach to increase the yield of properly folded, recombinant proteins in bacterial cell factories. However, unbalanced amounts of folding modulators handling folding-reluctant protein species might instead trigger undesired proteolytic activities, detrimental regarding recombinant protein stability, quality and yield. This minireview summarizes the most recent observations of chaperone-linked negative side effects, mostly focusing on DnaK and GroEL sets, when using these proteins as folding assistant agents. These events are discussed in the context of the complexity of the cell quality network and the consequent intricacy of the physiological responses triggered by protein misfolding.