• Crystal structure of a non-discriminating glutamyl-tRNA synthetase.

      Schulze, Jörg O; Masoumi, Ava; Nickel, Daniel; Jahn, Martina; Jahn, Dieter; Schubert, Wolf-Dieter; Heinz, Dirk W; Division of Structural Biology, German Research Centre for Biotechnology (GBF), Mascheroder Weg 1, D-38124 Braunschweig, Germany. (2006-09-01)
      Error-free protein biosynthesis is dependent on the reliable charging of each tRNA with its cognate amino acid. Many bacteria, however, lack a glutaminyl-tRNA synthetase. In these organisms, tRNA(Gln) is initially mischarged with glutamate by a non-discriminating glutamyl-tRNA synthetase (ND-GluRS). This enzyme thus charges both tRNA(Glu) and tRNA(Gln) with glutamate. Discriminating GluRS (D-GluRS), found in some bacteria and all eukaryotes, exclusively generates Glu-tRNA(Glu). Here we present the first crystal structure of a non-discriminating GluRS from Thermosynechococcus elongatus (ND-GluRS(Tel)) in complex with glutamate at a resolution of 2.45 A. Structurally, the enzyme shares the overall architecture of the discriminating GluRS from Thermus thermophilus (D-GluRS(Tth)). We confirm experimentally that GluRS(Tel) is non-discriminating and present kinetic parameters for synthesis of Glu-tRNA(Glu) and of Glu-tRNA(Gln). Anticodons of tRNA(Glu) (34C/UUC36) and tRNA(Gln) (34C/UUG36) differ only in base 36. The pyrimidine base of C36 is specifically recognized in D-GluRS(Tth) by the residue Arg358. In ND-GluRS(Tel) this arginine residue is replaced by glycine (Gly366) presumably allowing both cytosine and the bulkier purine base G36 of tRNA(Gln) to be tolerated. Most other ND-GluRS share this structural feature, leading to relaxed substrate specificity.
    • Isolation, characterisation and molecular imaging of a high-molecular-weight insect biliprotein, a member of the hexameric arylphorin protein family.

      Kayser, Hartmut; Mann, Karlheinz; Machaidze, Gia; Nimtz, Manfred; Ringler, Philippe; Müller, Shirley A; Aebi, Ueli; Institut für Allgemeine Zoologie und Endokrinologie, Universität Ulm, Germany. hartmut.kayser@uni-ulm.de (2009-05-29)
      The abundant blue hemolymph protein of the last instar larvae of the moth Cerura vinula was purified and characterized by protein-analytical, spectroscopic and electron microscopic methods. Amino acid sequences obtained from a large number of cleavage peptides revealed a high level of similarity of the blue protein with arylphorins from a number of other moth species. In particular, there is a high abundance of the aromatic amino acids tyrosine and phenylalanine amounting to about 19% of total amino acids and a low content of methionine (0.8%) in the Cerura protein. The mass of the native protein complex was studied by size-exclusion chromatography, analytical ultracentrifugation, dynamic light scattering and scanning transmission electron microscopy and found to be around 500 kDa. Denaturating gel electrophoresis and mass spectrometry suggested the presence of two proteins with masses of about 85 kDa. The native Cerura protein is, therefore, a hexameric complex of two different subunits of similar size, as is known for arylphorins. The protein was further characterized as a weakly acidic (pI approximately 5.5) glycoprotein containing mannose, glucose and N-acetylglucosamine in an approximate ratio of 10:1:1. The structure proposed for the most abundant oligosaccharide of the Cerura arylphorin was the same as already identified in arylphorins from other moths. The intense blue colour of the Cerura protein is due to non-covalent association with a bilin of novel structure at an estimated protein subunit-to-ligand ratio of 3:1. Transmission electron microscopy of the biliprotein showed single particles of cylindrical shape measuring about 13 nm in diameter and 9 nm in height. A small fraction of particles of the same diameter but half the height was likely a trimeric arylphorin dissociation intermediate. Preliminary three-dimensional reconstruction based on averaged transmission electron microscopy projections of the individual particles revealed a double-trimeric structure for the hexameric Cerura biliprotein complex, suggesting it to be a dimer of trimers.
    • Ligand-mediated dimerization of the Met Receptor tyrosine kinase by the bacterial invasion protein InlB.

      Ferraris, Davide M; Gherardi, Ermanno; Di, Ying; Heinz, Dirk W; Niemann, Hartmut H; Division of Structural Biology, Helmholtz Centre for Infection Research (HZI), Inhoffenstrasse 7, 38124 Braunschweig, Germany. yyoshiki@riken.jp (2010-01-22)
      The Listeria monocytogenes surface protein InlB mediates bacterial invasion into host cells by activating the human receptor tyrosine kinase Met. So far, it is unknown how InlB or the physiological Met ligand hepatocyte growth factor/scatter factor causes Met dimerization, which is considered a prerequisite for receptor activation. We determined two new structures of InlB, revealing a recurring, antiparallel, dimeric arrangement, in which the two protomers interact through the convex face of the leucine-rich repeat domain. The same contact is found in one structure of the InlB-Met complex. Mutations disrupting the interprotomeric contact of InlB reduced its ability to activate Met and downstream signaling. Conversely, stabilization of this crystal contact by two intermolecular disulfide bonds generates a constitutively dimeric InlB variant with exceptionally high signaling activity, which can stimulate cell motility and cell division. These data demonstrate that the signaling-competent InlB-Met complex assembles with 2:2 stoichiometry around a back-to-back InlB dimer, enabling the direct contact between the stalk region of two Met molecules.
    • Structure of the type III secretion recognition protein YscU from Yersinia enterocolitica.

      Wiesand, Ulrich; Sorg, Isabel; Amstutz, Marlise; Wagner, Stefanie; van den Heuvel, Joop; Lührs, Thorsten; Cornelis, Guy R; Heinz, Dirk W; Division of Structural Biology, Helmholtz Centre for Infection Research (HZI), Inhoffenstrasse 7, 38124 Braunschweig, Germany. (2009-01-23)
      The inner-membrane protein YscU has an important role during the assembly of the Yersinia enterocolitica type III secretion injectisome. Its cytoplasmic domain (YscU(C)) recognizes translocators as individual substrates in the export hierarchy. Activation of YscU entails autocleavage at a conserved NPTH motif. Modification of this motif markedly changes the properties of YscU, including translocator export cessation and production of longer injectisome needles. We determined the crystal structures of the uncleaved variants N263A and N263D of YscU(C) at 2.05 A and 1.55 A resolution, respectively. The globular domain is found to consist of a central, mixed beta-sheet surrounded by alpha-helices. The NPTH motif forms a type II beta-turn connecting two beta-strands. NMR analysis of cleaved and uncleaved YscU(C) indicates that the global structure of the protein is retained in cleaved YscU(C). The structure of YscU(C) variant N263D reveals that wild type YscU(C) is poised for cleavage due to an optimal reaction geometry for nucleophilic attack of the scissile bond by the side chain of Asn263. In vivo analysis of N263Q and H266A/R314A YscU variants showed a phenotype that combines the absence of translocator secretion with normal needle-length control. Comparing the structure of YscU to those of related proteins reveals that the linker domain between the N-terminal transmembrane domain and the autocleavage domain can switch from an extended to a largely alpha-helical conformation, allowing for optimal positioning of the autocleavage domain during injectisome assembly.
    • 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.
    • 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.