• 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.
    • Juxtanodin is an intrinsically disordered F-actin-binding protein.

      Ruskamo, Salla; Chukhlieb, Maryna; Vahokoski, Juha; Bhargav, Saligram Prabhakar; Liang, Fengyi; Kursula, Inari; Kursula, Petri (2012)
      Juxtanodin, also called ermin, is an F-actin-binding protein expressed by oligodendrocytes, the myelin-forming cells of the central nervous system. While juxtanodin carries a short conserved F-actin-binding segment at its C terminus, it otherwise shares no similarity with known protein sequences. We carried out a structural characterization of recombinant juxtanodin in solution. Juxtanodin turned out to be intrinsically disordered, as evidenced by conventional and synchrotron radiation CD spectroscopy. Small-angle X-ray scattering indicated that juxtanodin is a monomeric, highly elongated, unfolded molecule. Ensemble optimization analysis of the data suggested also the presence of more compact forms of juxtanodin. The C terminus was a strict requirement for co-sedimentation of juxtanodin with microfilaments, but juxtanodin had only mild effects on actin polymerization. The disordered nature of juxtanodin may predict functions as a protein interaction hub, although F-actin is its only currently known binding partner.
    • The lasso segment is required for functional dimerization of the Plasmodium formin 1 FH2 domain.

      Ignatev, Alexander; Bhargav, Saligram Prabhakar; Vahokoski, Juha; Kursula, Petri; Kursula, Inari; Helmholtz Centre for Infection Research, University of Hamburg, and German Electron Synchrotron (DESY), Hamburg, Germany. (2012)
      Apicomplexan parasites, such as the malaria-causing Plasmodium species, utilize a unique way of locomotion and host cell invasion. This substrate-dependent gliding motility requires rapid cycling of actin between the monomeric state and very short, unbranched filaments. Despite the crucial role of actin polymerization for the survival of the malaria parasite, the majority of Plasmodium cellular actin is present in the monomeric form. Plasmodium lacks most of the canonical actin nucleators, and formins are essentially the only candidates for this function in all Apicomplexa. The malaria parasite has two formins, containing conserved formin homology (FH) 2 and rudimentary FH1 domains. Here, we show that Plasmodium falciparum formin 1 associates with and nucleates both mammalian and Plasmodium actin filaments. Although Plasmodium profilin alone sequesters actin monomers, thus inhibiting polymerization, its monomer-sequestering activity does not compete with the nucleating activity of formin 1 at an equimolar profilin-actin ratio. We have determined solution structures of P. falciparum formin 1 FH2 domain both in the presence and absence of the lasso segment and the FH1 domain, and show that the lasso is required for the assembly of functional dimers.
    • 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.
    • Preliminary crystallographic analysis of the N-terminal PDZ-like domain of periaxin, an abundant peripheral nerve protein linked to human neuropathies.

      Han, Huijong; Kursula, Petri (2013-07)
      Periaxin (PRX) is an abundant protein in peripheral nerves and contains a predicted PDZ-like domain at its N-terminus. The large isoform, L-PRX, is required for the maintenance of myelin in the peripheral nervous system and its defects cause neurological disease. Here, the human periaxin PDZ-like domain was crystallized and X-ray diffraction data were collected to 2.85 Å resolution using synchrotron radiation. The crystal belonged to the primitive hexagonal space group P3121 or P3221, with unit-cell parameters a = b = 80.6, c = 81.0 Å, γ = 120° and either two or three molecules in the asymmetric unit. The structure of PRX will shed light on its poorly characterized function in the nervous system.
    • Purification, crystallization and preliminary X-ray crystallographic analysis of MIL, a glycosylated jacalin-related lectin from mulberry (Morus indica) latex.

      Patel, Ashok K; Singh, Vijay K; Bergmann, Ulrich; Jagannadham, Medicherla V; Kursula, Petri (2011-05-01)
      A quantitatively major protein has been purified from the latex of Morus indica. The purified previously uncharacterized protein, M. indica lectin (MIL), was further shown to be a glycosylated tetramer and belongs to the family of jacalin-related lectins. Crystallization of MIL was also accomplished and the tetragonal crystals diffracted synchrotron X-rays to a resolution of 2.8 Å.
    • 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 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.