Recent Submissions

  • Transcriptional and mutational profiling of an aminoglycoside resistant Pseudomonas aeruginosa small colony variant.

    Schniederjans, Monika; Koska, Michal; Häussler, Susanne; Helmholtz Centre for infection researchGmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-09-05)
    Pseudomonas aeruginosa is a major causative agent of both acute and chronic infections. Although aminoglycoside antibiotics are very potent drugs to fight such infections, antibiotic failure is steadily increasing mainly due to increasing resistance of the bacteria. Many molecular mechanisms that determine resistance such as acquisition of genes encoding for aminoglycoside-inactivating enzymes or overexpression of efflux pumps have been elucidated. However, there are additional, less-well described mechanisms of aminoglycoside resistance. In this study we have profiled a clinical tobramycin resistant P. aeruginosa strain that exhibited a small colony variant (SCV) phenotype. Both, the resistance and the colony morphology phenotypes were lost upon passaging the isolate under rich medium conditions. Transcriptional and mutational profiling revealed that the SCV harbored activating mutations in the two two-component systems AmgRS and PmrAB. Introduction of these mutations singularly into the type strain PA14 conferred tobramycin and colistin resistance, respectively. However, their combined introduction had an additive effect on the tobramycin resistance phenotype. Activation of the AmgRS system slightly reduced the colony size of the PA14 wild-type, whereas the simultaneous overexpression of gacA, the response regulator of the GacSA two component system, further reduced colony size. In conclusion, we uncovered combinatorial influences of two-component systems on clinically relevant phenotypes, such as resistance and the expression of the SCV phenotype. Our results clearly demonstrate that combined activation of P. aeruginosa two-component systems exhibit pleiotropic effects with unforeseen consequences.
  • Human lung tissue explants reveal novel interactions during Legionella pneumophila infections.

    Jäger, Jens; Marwitz, Sebastian; Tiefenau, Jana; Rasch, Janine; Shevchuk, Olga; Kugler, Christian; Goldmann, Torsten; Steinert, Michael; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2014-01)
    Histological and clinical investigations describe late stages of Legionnaires' disease but cannot characterize early events of human infection. Cellular or rodent infection models lack the complexity of tissue or have nonhuman backgrounds. Therefore, we developed and applied a novel model for Legionella pneumophila infection comprising living human lung tissue. We stimulated lung explants with L. pneumophila strains and outer membrane vesicles (OMVs) to analyze tissue damage, bacterial replication, and localization as well as the transcriptional response of infected tissue. Interestingly, we found that extracellular adhesion of L. pneumophila to the entire alveolar lining precedes bacterial invasion and replication in recruited macrophages. In contrast, OMVs predominantly bound to alveolar macrophages. Specific damage to septa and epithelia increased over 48 h and was stronger in wild-type-infected and OMV-treated samples than in samples infected with the replication-deficient, type IVB secretion-deficient DotA(-) strain. Transcriptome analysis of lung tissue explants revealed a differential regulation of 2,499 genes after infection. The transcriptional response included the upregulation of uteroglobin and the downregulation of the macrophage receptor with collagenous structure (MARCO). Immunohistochemistry confirmed the downregulation of MARCO at sites of pathogen-induced tissue destruction. Neither host factor has ever been described in the context of L. pneumophila infections. This work demonstrates that the tissue explant model reproduces realistic features of Legionnaires' disease and reveals new functions for bacterial OMVs during infection. Our model allows us to characterize early steps of human infection which otherwise are not feasible for investigations.
  • Structures of two bacterial resistance factors mediating tRNA-dependent aminoacylation of phosphatidylglycerol with lysine or alanine.

    Hebecker, Stefanie; Krausze, Joern; Hasenkampf, Tatjana; Schneider, Julia; Groenewold, Maike; Reichelt, Joachim; Jahn, Dieter; Heinz, Dirk W; Moser, Jürgen; Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2015-08-25)
    The cytoplasmic membrane is probably the most important physical barrier between microbes and the surrounding habitat. Aminoacylation of the polar head group of the phospholipid phosphatidylglycerol (PG) catalyzed by Ala-tRNA(Ala)-dependent alanyl-phosphatidylglycerol synthase (A-PGS) or by Lys-tRNA(Lys)-dependent lysyl-phosphatidylglycerol synthase (L-PGS) enables bacteria to cope with cationic peptides that are harmful to the integrity of the cell membrane. Accordingly, these synthases also have been designated as multiple peptide resistance factors (MprF). They consist of a separable C-terminal catalytic domain and an N-terminal transmembrane flippase domain. Here we present the X-ray crystallographic structure of the catalytic domain of A-PGS from the opportunistic human pathogen Pseudomonas aeruginosa. In parallel, the structure of the related lysyl-phosphatidylglycerol-specific L-PGS domain from Bacillus licheniformis in complex with the substrate analog L-lysine amide is presented. Both proteins reveal a continuous tunnel that allows the hydrophobic lipid substrate PG and the polar aminoacyl-tRNA substrate to access the catalytic site from opposite directions. Substrate recognition of A-PGS versus L-PGS was investigated using misacylated tRNA variants. The structural work presented here in combination with biochemical experiments using artificial tRNA or artificial lipid substrates reveals the tRNA acceptor stem, the aminoacyl moiety, and the polar head group of PG as the main determinants for substrate recognition. A mutagenesis approach yielded the complementary amino acid determinants of tRNA interaction. These results have broad implications for the design of L-PGS and A-PGS inhibitors that could render microbial pathogens more susceptible to antimicrobial compounds.
  • Metabolic peculiarities of Aspergillus niger disclosed by comparative metabolic genomics.

    Sun, Jibin; Lu, Xin; Rinas, Ursula; Zeng, An Ping; Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2007)
    Aspergillus niger is an important industrial microorganism for the production of both metabolites, such as citric acid, and proteins, such as fungal enzymes or heterologous proteins. Despite its extensive industrial applications, the genetic inventory of this fungus is only partially understood. The recently released genome sequence opens a new horizon for both scientific studies and biotechnological applications.
  • Recombinant production of Yersinia enterocolitica pyruvate kinase isoenzymes PykA and PykF.

    Hofmann, Julia; Heider, Christine; Li, Wei; Krausze, Joern; Roessle, Manfred; Wilharm, Gottfried; Robert Koch-Institute, Wernigerode Branch, Burgstr. 37, D-38855 Wernigerode, Germany. (2013-04)
    The glycolytic enzyme pyruvate kinase (PK) generates ATP from ADP through substrate-level phosphorylation powered by the conversion of phosphoenolpyruvate to pyruvate. In contrast to other bacteria, Enterobacteriaceae, such as pathogenic yersiniae, harbour two pyruvate kinases encoded by pykA and pykF. The individual roles of these isoenzymes are poorly understood. In an attempt to make the Yersinia enterocolitica pyruvate kinases PykA and PykF amenable to structural and functional characterisation, we produced them untagged in Escherichia coli and purified them to near homogeneity through a combination of ion exchange and size exclusion chromatography, yielding more than 180 mg per litre of batch culture. The solution structure of PykA and PykF was analysed through small angle X-ray scattering which revealed the formation of PykA and PykF tetramers and confirmed the binding of the allosteric effector fructose-1,6-bisphosphate (FBP) to PykF but not to PykA.
  • In situ structural analysis of the Yersinia enterocolitica injectisome

    Kudryashev, M.; Stenta, M.; Schmelz, S.; Amstutz, M.; Wiesand, U.; Castano-Diez, D.; Degiacomi, M. T.; Munnich, S.; Bleck, C. K.; Kowal, J.; et al. (2013-07-30)
  • PfaH2: A novel hydrophobin from the ascomycete Paecilomyces farinosus.

    Zelena, Katerina; Takenberg, Meike; Lunkenbein, Stefan; Woche, Susanne K; Nimtz, Manfred; Berger, Ralf G; Naturwissenschaftliche Fakultät der Leibniz Universität Hannover, Institut für Lebensmittelchemie, Hannover, Germany. (2013-03)
    The pfah2 gene coding for a novel hydrophobin PfaH2 from the ascomycete Paecilomyces farinosus was identified during sequencing of random clones from a cDNA library. The corresponding protein sequence of PfaH2 deduced from the cDNA comprised 134 amino acids (aa). A 16 aa signal sequence preceded the N-terminus of the mature protein. PfaH2 belonged to the class Ia hydrophobins. The protein was isolated using trifluoroacetic acid extraction and purified via SDS-PAGE and high-performance liquid chromatography. The surface activity of the recently described PfaH1 and of PfaH2 was compared by the determination of contact angles (CAs) on glass slides and Teflon tape, and the CA of distilled water droplets was measured on glass slides coated with hydrophobin PfaH1 or PfaH2. Surprisingly, both hydrophobins adsorbed to hydrophilic surfaces and changed their physicochemical properties to a similar quantitative extent, although little aa sequence homology was found.
  • Structural characterization of Spinacia oleracea trypsin inhibitor III (SOTI-III).

    Glotzbach, Bernhard; Schmelz, Stefan; Reinwarth, Michael; Christmann, Andreas; Heinz, Dirk W; Kolmar, Harald; Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Germany. (2013-01)
    In recent decades, several canonical serine protease inhibitor families have been classified and characterized. In contrast to most trypsin inhibitors, those from garden four o'clock (Mirabilis jalapa) and spinach (Spinacia oleracea) do not share sequence similarity and have been proposed to form the new Mirabilis serine protease inhibitor family. These 30-40-amino-acid inhibitors possess a defined disulfide-bridge topology and belong to the cystine-knot miniproteins (knottins). To date, no atomic structure of this inhibitor family has been solved. Here, the first structure of S. oleracea trypsin inhibitor III (SOTI-III), in complex with bovine pancreatic trypsin, is reported. The inhibitor was synthesized by solid-phase peptide synthesis on a multi-milligram scale and was assayed to test its inhibitory activity and binding properties. The structure confirmed the proposed cystine-bridge topology. The structural features of SOTI-III suggest that it belongs to a new canonical serine protease inhibitor family with promising properties for use in protein-engineering and medical applications.
  • Structural basis for complex formation between human IRSp53 and the translocated intimin receptor Tir of enterohemorrhagic E. coli.

    de Groot, Jens C; Schlüter, Kai; Carius, Yvonne; Quedenau, Claudia; Vingadassalom, Didier; Faix, Jan; Weiss, Stefanie M; Reichelt, Joachim; Standfuss-Gabisch, Christine; Lesser, Cammie F; et al. (2011-09-07)
    Actin assembly beneath enterohemorrhagic E. coli (EHEC) attached to its host cell is triggered by the intracellular interaction of its translocated effector proteins Tir and EspF(U) with human IRSp53 family proteins and N-WASP. Here, we report the structure of the N-terminal I-BAR domain of IRSp53 in complex with a Tir-derived peptide, in which the homodimeric I-BAR domain binds two Tir molecules aligned in parallel. This arrangement provides a protein scaffold linking the bacterium to the host cell's actin polymerization machinery. The structure uncovers a specific peptide-binding site on the I-BAR surface, conserved between IRSp53 and IRTKS. The Tir Asn-Pro-Tyr (NPY) motif, essential for pedestal formation, is specifically recognized by this binding site. The site was confirmed by mutagenesis and in vivo-binding assays. It is possible that IRSp53 utilizes the NPY-binding site for additional interactions with as yet unknown partners within the host cell.
  • Biosynthesis of the repeating units of the exopolysaccharides amylovoran from Erwinia amylovora and stewartan from Pantoea stewartii

    Langlotz, Christine; Schollmeyer, Martin; Coplin, David L.; Nimtz, Manfred; Geider, Klaus; Max-Planck-Institute for Cell Biology, Ladenburg, Germany (2012-06-27)
  • Kinome analysis of receptor-induced phosphorylation in human natural killer cells.

    König, Sebastian; Nimtz, Manfred; Scheiter, Maxi; Ljunggren, Hans-Gustaf; Bryceson, Yenan T; Jänsch, Lothar; Department of Molecular Structural Biology, Helmholtz-Zentrum für Infektionsforschung, Braunschweig, Germany. (2012)
    Natural killer (NK) cells contribute to the defense against infected and transformed cells through the engagement of multiple germline-encoded activation receptors. Stimulation of the Fc receptor CD16 alone is sufficient for NK cell activation, whereas other receptors, such as 2B4 (CD244) and DNAM-1 (CD226), act synergistically. After receptor engagement, protein kinases play a major role in signaling networks controlling NK cell effector functions. However, it has not been characterized systematically which of all kinases encoded by the human genome (kinome) are involved in NK cell activation.
  • Streamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development.

    Wilke, Sonja; Groebe, Lothar; Maffenbeier, Vitali; Jäger, Volker; Gossen, Manfred; Josewski, Jörn; Duda, Agathe; Polle, Lilia; Owens, Raymond J; Wirth, Dagmar; et al. (2011)
    Studying the biophysical characteristics of glycosylated proteins and solving their three-dimensional structures requires homogeneous recombinant protein of high quality.We introduce here a new approach to produce glycoproteins in homogenous form with the well-established, glycosylation mutant CHO Lec3.2.8.1 cells. Using preparative cell sorting, stable, high-expressing GFP 'master' cell lines were generated that can be converted fast and reliably by targeted integration via Flp recombinase-mediated cassette exchange (RMCE) to produce any glycoprotein. Small-scale transient transfection of HEK293 cells was used to identify genetically engineered constructs suitable for constructing stable cell lines. Stable cell lines expressing 10 different proteins were established. The system was validated by expression, purification, deglycosylation and crystallization of the heavily glycosylated luminal domains of lysosome-associated membrane proteins (LAMP).
  • Crystal structure of the heme d1 biosynthesis enzyme NirE in complex with its substrate reveals new insights into the catalytic mechanism of S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferases.

    Storbeck, Sonja; Saha, Sayantan; Krausze, Joern; Klink, Björn U; Heinz, Dirk W; Layer, Gunhild; Institute of Microbiology, Technische Universität Braunschweig, 38106 Braunschweig, Germany. (2011-07-29)
    During the biosynthesis of heme d(1), the essential cofactor of cytochrome cd(1) nitrite reductase, the NirE protein catalyzes the methylation of uroporphyrinogen III to precorrin-2 using S-adenosyl-L-methionine (SAM) as the methyl group donor. The crystal structure of Pseudomonas aeruginosa NirE in complex with its substrate uroporphyrinogen III and the reaction by-product S-adenosyl-L-homocysteine (SAH) was solved to 2.0 Å resolution. This represents the first enzyme-substrate complex structure for a SAM-dependent uroporphyrinogen III methyltransferase. The large substrate binds on top of the SAH in a "puckered" conformation in which the two pyrrole rings facing each other point into the same direction either upward or downward. Three arginine residues, a histidine, and a methionine are involved in the coordination of uroporphyrinogen III. Through site-directed mutagenesis of the nirE gene and biochemical characterization of the corresponding NirE variants the amino acid residues Arg-111, Glu-114, and Arg-149 were identified to be involved in NirE catalysis. Based on our structural and biochemical findings, we propose a potential catalytic mechanism for NirE in which the methyl transfer reaction is initiated by an arginine catalyzed proton abstraction from the C-20 position of the substrate.
  • Expression of protein complexes using multiple Escherichia coli protein co-expression systems: a benchmarking study.

    Busso, Didier; Peleg, Yoav; Heidebrecht, Tatjana; Romier, Christophe; Jacobovitch, Yossi; Dantes, Ada; Salim, Loubna; Troesch, Edouard; Schuetz, Anja; Heinemann, Udo; et al. (2011-08)
    Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for optimizing production of protein complexes using co-expression strategies. In this study, we focus on the effect of the choice of the expression vector system: by standardizing experimental factors including bacterial strain, cultivation temperature and growth medium composition, we compare the effectiveness of expression technologies used by the partners of the Structural Proteomics in Europe 2 (SPINE2-complexes) consortium. Four different protein complexes, including three binary and one ternary complex, all known to be produced in the soluble form in E. coli, are used as the benchmark targets. The respective genes were cloned by each partner into their preferred set of vectors. The resulting constructs were then used for comparative co-expression analysis done in parallel and under identical conditions at a single site. Our data show that multiple strategies can be applied for the expression of protein complexes in high yield. While there is no 'silver bullet' approach that was infallible even for this small test set, our observations are useful as a guideline to delineate co-expression strategies for particular protein complexes.
  • Polysaccharide synthesis of the levansucrase SacB from Bacillus megaterium is controlled by distinct surface motifs.

    Strube, Christian P; Homann, Arne; Gamer, Martin; Jahn, Dieter; Seibel, Jürgen; Heinz, Dirk W; Department of Molecular Structural Biology, Helmholtz-Centre for Infection Research, Inhoffenstrasse 7B, 38124 Braunschweig, Germany. (2011-05-20)
    Despite the widespread biological function of carbohydrates, the polysaccharide synthesis mechanisms of glycosyltransferases remain largely unexplored. Bacterial levansucrases (glycoside hydrolase family 68) synthesize high molecular weight, β-(2,6)-linked levan from sucrose by transfer of fructosyl units. The kinetic and biochemical characterization of Bacillus megaterium levansucrase SacB variants Y247A, Y247W, N252A, D257A, and K373A reveal novel surface motifs remote from the sucrose binding site with distinct influence on the polysaccharide product spectrum. The wild type activity (k(cat)) and substrate affinity (K(m)) are maintained. The structures of the SacB variants reveal clearly distinguishable subsites for polysaccharide synthesis as well as an intact active site architecture. These results lead to a new understanding of polysaccharide synthesis mechanisms. The identified surface motifs are discussed in the context of related glycosyltransferases.
  • Structure of the effector-binding domain of the LysR-type transcription factor RovM from Yersinia pseudotuberculosis.

    Quade, Nick; Dieckmann, Marieke; Haffke, Matthias; Heroven, Ann Kathrin; Dersch, Petra; Heinz, Dirk W; Department of Molecular Structural Biology, Helmholtz Centre for Infection Research, D-38124 Braunschweig, Germany. (2011-02)
    In enteropathogenic Yersinia, the expression of several early-phase virulence factors such as invasin is tightly regulated in response to environmental cues. The responsible regulatory network is complex, involving several regulatory RNAs and proteins such as the LysR-type transcription regulator (LTTR) RovM. In this study, the crystal structure of the effector-binding domain (EBD) of RovM, the first LTTR protein described as being involved in virulence regulation, was determined at a resolution of 2.4 Å. Size-exclusion chromatography and comparison with structures of full-length LTTRs show that RovM is most likely to adopt a tetrameric arrangement with two distant DNA-binding domains (DBDs), causing the DNA to bend around it. Additionally, a cavity was detected in RovM which could bind small inducer molecules.
  • Saliva proteins of vector Culicoides modify structure and infectivity of bluetongue virus particles.

    Darpel, Karin E; Langner, Kathrin F A; Nimtz, Manfred; Anthony, Simon J; Brownlie, Joe; Takamatsu, Haru-Hisa; Mellor, Philip S; Mertens, Peter P C; Pirbright Laboratory, Vector-borne Disease Programme, Institute for Animal Health, Woking, United Kingdom. karin.darpel@bbsrc.ac.uk (2011)
    Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are related orbiviruses, transmitted between their ruminant hosts primarily by certain haematophagous midge vectors (Culicoides spp.). The larger of the BTV outer-capsid proteins, 'VP2', can be cleaved by proteases (including trypsin or chymotrypsin), forming infectious subviral particles (ISVP) which have enhanced infectivity for adult Culicoides, or KC cells (a cell-line derived from C. sonorensis). We demonstrate that VP2 present on purified virus particles from 3 different BTV strains can also be cleaved by treatment with saliva from adult Culicoides. The saliva proteins from C. sonorensis (a competent BTV vector), cleaved BTV-VP2 more efficiently than those from C. nubeculosus (a less competent/non-vector species). Electrophoresis and mass spectrometry identified a trypsin-like protease in C. sonorensis saliva, which was significantly reduced or absent from C. nubeculosus saliva. Incubating purified BTV-1 with C. sonorensis saliva proteins also increased their infectivity for KC cells ∼10 fold, while infectivity for BHK cells was reduced by 2-6 fold. Treatment of an 'eastern' strain of EHDV-2 with saliva proteins of either C. sonorensis or C. nubeculosus cleaved VP2, but a 'western' strain of EHDV-2 remained unmodified. These results indicate that temperature, strain of virus and protein composition of Culicoides saliva (particularly its protease content which is dependent upon vector species), can all play a significant role in the efficiency of VP2 cleavage, influencing virus infectivity. Saliva of several other arthropod species has previously been shown to increase transmission, infectivity and virulence of certain arboviruses, by modulating and/or suppressing the mammalian immune response. The findings presented here, however, demonstrate a novel mechanism by which proteases in Culicoides saliva can also directly modify the orbivirus particle structure, leading to increased infectivity specifically for Culicoides cells and, in turn, efficiency of transmission to the insect vector.
  • Structures of the nucleotide-binding domain of the human ABCB6 transporter and its complexes with nucleotides.

    Haffke, Matthias; Menzel, Anja; Carius, Yvonne; Jahn, Dieter; Heinz, Dirk W; Helmholtz Zentrum für Infektionsforschung, Braunschweig, Germany. (2010-09)
    The human ATP-binding cassette (ABC) transporter ABCB6 is involved in haem-precursor transport across the mitochondrial membrane. The crystal structure of its nucleotide-binding domain (NBD) has been determined in the apo form and in complexes with ADP, with ADP and Mg(2+) and with ATP at high resolution. The overall structure is L-shaped and consists of two lobes, consistent with other reported NBD structures. Nucleotide binding is mediated by the highly conserved Tyr599 and the Walker A motif, and induces notable structural changes. Structural comparison with other structurally characterized NBDs and full-length ABC transporters gives the first insight into the possible catalytic mechanism of ABCB6 and the role of the N-terminal helix alpha(1) in full-length ABCB6.
  • Nonomuraea rosea sp. nov.

    Kämpfer, Peter; Busse, Hans-Jürgen; Tindall, Brian J; Nimtz, Manfred; Grün-Wollny, Iris; Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen, D-35392 Giessen, Germany. peter.kaempfer@umwelt.uni-giessen.de (2010-05)
    A Gram-positively staining, aerobic, non-motile actinomycete, strain GW 12687(T), that formed rose-pigmented colonies and branched substrate and aerial mycelia was studied in detail for its taxonomic position. On the basis of 16S rRNA gene sequence similarity studies, strain GW 12687(T) was grouped into the genus Nonomuraea, being most closely related to Nonomuraea dietziae (97.6 %), Nonomuraea africana (97.1 %), and Nonomuraea kuesteri (97.1 %). The 16S rRNA gene sequence similarity to other species of the genus Nonomuraea was < or =97 %. The chemotaxonomic characterization supported allocation of the strain to the genus Nonomuraea. The major menaquinone was MK-9(H(4)) with minor amounts of MK-9(H(2)), MK-9(H(6)), MK-9(H(0)) and MK-8(H(4)). The polar lipid profile contained the major compound diphosphatidylglycerol, moderate amounts of phosphatidylmonomethylethanolamine, phosphatidylethanolamine, hydroxy-phosphatidylethanolamine, hydroxy-phosphatidylmonomethylethanolamine, and an unknown aminophosphoglycolipid. Phosphatidylinositol mannosides and phosphatidylinositol were also present. The major fatty acids were iso- and anteiso- and 10-methyl-branched fatty acids. The results of physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain GW 12687(T) from closely related species. Thus, GW 12687(T) represents a novel species of the genus Nonomuraea, for which the name Nonomuraea rosea sp. nov. is proposed, with GW 12687(T) (=DSM 45177(T) =CCUG 56107(T)) as the type strain.
  • Structure of Shigella IpgB2 in complex with human RhoA: implications for the mechanism of bacterial guanine nucleotide exchange factor mimicry.

    Klink, Björn U; Barden, Stephan; Heidler, Thomas V; Borchers, Christina; Ladwein, Markus; Stradal, Theresia E B; Rottner, Klemens; Heinz, Dirk W; Division of Structural Biology, Helmholtz Zentrum für Infektionsforschung, D-38124 Braunschweig, Germany. (2010-05-28)
    A common theme in bacterial pathogenesis is the manipulation of eukaryotic cells by targeting the cytoskeleton. This is in most cases achieved either by modifying actin, or indirectly via activation of key regulators controlling actin dynamics such as Rho-GTPases. A novel group of bacterial virulence factors termed the WXXXE family has emerged as guanine nucleotide exchange factors (GEFs) for these GTPases. The precise mechanism of nucleotide exchange, however, has remained unclear. Here we report the structure of the WXXXE-protein IpgB2 from Shigella flexneri and its complex with human RhoA. We unambiguously identify IpgB2 as a bacterial RhoA-GEF and dissect the molecular mechanism of GDP release, an essential prerequisite for GTP binding. Our observations uncover that IpgB2 induces conformational changes on RhoA mimicking DbI- but not DOCK family GEFs. We also show that dissociation of the GDP.Mg(2+) complex is preceded by the displacement of the metal ion to the alpha-phosphate of the nucleotide, diminishing its affinity to the GTPase. These data refine our understanding of the mode of action not only of WXXXE GEFs but also of mammalian GEFs of the DH/PH family.

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