• 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.
    • Genome-scale metabolic network analysis of the opportunistic pathogen Pseudomonas aeruginosa PAO1.

      Oberhardt, Matthew A; Puchałka, Jacek; Fryer, Kimberly E; Martins dos Santos, Vítor A P; Papin, Jason A; Department of Biomedical Engineering, University of Virginia Health System, Box 800759, Charlottesville, VA 22908, USA. (2008-04)
      Pseudomonas aeruginosa is a major life-threatening opportunistic pathogen that commonly infects immunocompromised patients. This bacterium owes its success as a pathogen largely to its metabolic versatility and flexibility. A thorough understanding of P. aeruginosa's metabolism is thus pivotal for the design of effective intervention strategies. Here we aim to provide, through systems analysis, a basis for the characterization of the genome-scale properties of this pathogen's versatile metabolic network. To this end, we reconstructed a genome-scale metabolic network of Pseudomonas aeruginosa PAO1. This reconstruction accounts for 1,056 genes (19% of the genome), 1,030 proteins, and 883 reactions. Flux balance analysis was used to identify key features of P. aeruginosa metabolism, such as growth yield, under defined conditions and with defined knowledge gaps within the network. BIOLOG substrate oxidation data were used in model expansion, and a genome-scale transposon knockout set was compared against in silico knockout predictions to validate the model. Ultimately, this genome-scale model provides a basic modeling framework with which to explore the metabolism of P. aeruginosa in the context of its environmental and genetic constraints, thereby contributing to a more thorough understanding of the genotype-phenotype relationships in this resourceful and dangerous pathogen.
    • 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.
    • Structure of the human receptor tyrosine kinase met in complex with the Listeria invasion protein InlB.

      Niemann, Hartmut H; Jäger, Volker; Butler, P Jonathan G; van den Heuvel, Joop; Schmidt, Sabine; Ferraris, Davide; Gherardi, Ermanno; Heinz, Dirk W; Division of Structural Biology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany. (2007-07-27)
      The tyrosine kinase Met, the product of the c-met proto-oncogene and the receptor for hepatocyte growth factor/scatter factor (HGF/SF), mediates signals critical for cell survival and migration. The human pathogen Listeria monocytogenes exploits Met signaling for invasion of host cells via its surface protein InlB. We present the crystal structure of the complex between a large fragment of the human Met ectodomain and the Met-binding domain of InlB. The concave face of the InlB leucine-rich repeat region interacts tightly with the first immunoglobulin-like domain of the Met stalk, a domain which does not bind HGF/SF. A second contact between InlB and the Met Sema domain locks the otherwise flexible receptor in a rigid, signaling competent conformation. Full Met activation requires the additional C-terminal domains of InlB which induce heparin-mediated receptor clustering and potent signaling. Thus, although it elicits a similar cellular response, InlB is not a structural mimic of HGF/SF.
    • Structure of the Yersinia enterocolitica type III secretion translocator chaperone SycD.

      Büttner, Carina R; Sorg, Isabel; Cornelis, Guy R; Heinz, Dirk W; Niemann, Hartmut H; Division of Structural Biology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany. (2008-01-25)
      Many Gram-negative bacteria use a type III secretion (T3S) system to directly inject effector molecules into eucaryotic cells in order to establish a symbiotic or pathogenic relationship with their host. The translocation of many T3S proteins requires specialized chaperones from the bacterial cytosol. SycD belongs to a class of T3S chaperones that assists the secretion of pore-forming translocators and, specifically chaperones the translocators YopB and YopD from enteropathogenic Yersinia enterocolitica. In addition, SycD is involved in the regulation of virulence factor biosynthesis and secretion. In this study, we present two crystal structures of Y. enterocolitica SycD at 1.95 and 2.6 A resolution, the first experimental structures of a T3S class II chaperone specific for translocators. The fold of SycD is entirely alpha-helical and reveals three tetratricopeptide repeat-like motifs that had been predicted from amino acid sequence. In both structures, SycD forms dimers utilizing residues from the first tetratricopeptide repeat motif. Using site-directed mutagenesis and size exclusion chromatography, we verified that SycD forms head-to-head homodimers in solution. Although in both structures, dimerization largely depends on the same residues, the two assemblies represent alternative dimers that exhibit different monomer orientations and overall shape. In these two distinct head-to-head dimers, both the concave and the convex surface of each monomer are accessible for interactions with the SycD binding partners YopB and YopD. A SycD variant carrying two point mutations in the dimerization interface is properly folded but defective in dimerization. Expression of this stable SycD monomer in Yersinia does not rescue the phenotype of a sycD null mutant, suggesting a physiological relevance of the dimerization interface.
    • Structures and diseases.

      Wendt, K Ulrich; Weiss, Manfred S; Cramer, Patrick; Heinz, Dirk W; Department of Chemical and Analytical Sciences at Sanofi-Aventis, D-65926 Frankfurt, Germany. (2008-02)
    • Thermodynamically reengineering the listerial invasion complex InlA/E-cadherin.

      Wollert, Thomas; Heinz, Dirk W; Schubert, Wolf-Dieter; Molecular Host-Pathogen Interactions, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany. (2007-08-28)
      Biological processes essentially all depend on the specific recognition between macromolecules and their interaction partners. Although many such interactions have been characterized both structurally and biophysically, the thermodynamic effects of small atomic changes remain poorly understood. Based on the crystal structure of the bacterial invasion protein internalin (InlA) of Listeria monocytogenes in complex with its human receptor E-cadherin (hEC1), we analyzed the interface to identify single amino acid substitutions in InlA that would potentially improve the overall quality of interaction and hence increase the weak binding affinity of the complex. Dissociation constants of InlA-variant/hEC1 complexes, as well as enthalpy and entropy of binding, were quantified by isothermal titration calorimetry. All single substitutions indeed significantly increase binding affinity. Structural changes were verified crystallographically at < or =2.0-A resolution, allowing thermodynamic characteristics of single substitutions to be rationalized structurally and providing unique insights into atomic contributions to binding enthalpy and entropy. Structural and thermodynamic data of all combinations of individual substitutions result in a thermodynamic network, allowing the source of cooperativity between distant recognition sites to be identified. One such pair of single substitutions improves affinity 5,000-fold. We thus demonstrate that rational reengineering of protein complexes is possible by making use of physically distant hot spots of recognition.
    • X-ray and neutron small-angle scattering analysis of the complex formed by the Met receptor and the Listeria monocytogenes invasion protein InlB.

      Niemann, Hartmut H; Petoukhov, Maxim V; Härtlein, Michael; Moulin, Martine; Gherardi, Ermanno; Timmins, Peter; Heinz, Dirk W; Svergun, Dmitri I; Division of Structural Biology, Helmholtz Center for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany. (2008-03-21)
      The Listeria monocytogenes surface protein InlB binds to the extracellular domain of the human receptor tyrosine kinase Met, the product of the c-met proto-oncogene. InlB binding activates the Met receptor, leading to uptake of Listeria into normally nonphagocytic host cells. The N-terminal half of InlB (InlB(321)) is sufficient for Met binding and activation. The complex between this Met-binding domain of InlB and various constructs of the Met ectodomain was characterized by size exclusion chromatography and dynamic light scattering, and structural models were built using small-angle X-ray scattering and small-angle neutron scattering. Although most receptor tyrosine kinase ligands induce receptor dimerization, InlB(321) consistently binds the Met ectodomain with a 1:1 stoichiometry. A construct comprising the Sema and PSI domains of Met, although sufficient to bind the physiological Met ligand hepatocyte growth factor/scatter factor, does not form a complex with InlB(321) in solution, highlighting the importance of Met Ig domains for InlB binding. Small-angle X-ray scattering and small-angle neutron scattering measurements of ligand and receptor, both free and in complex, reveal an elongated shape for the receptor. The four Ig domains form a bent, rather than a fully extended, conformation, and InlB(321) binds to Sema and the first Ig domain of Met, in agreement with the recent crystal structure of a smaller Met fragment in complex with InlB(321). These results call into question whether receptor dimerization is the basic underlying event in InlB(321)-mediated Met activation and demonstrate differences in the mechanisms by which the physiological ligand hepatocyte growth factor/scatter factor and InlB(321) bind and activate the Met receptor.