VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

Set-up of databases containing zoonoses-related experts,institutions, research projects, research funding programmes,samples, and cell lines. Providing independent information about zoonotic infectiousdiseases for the general public. Initiation and realization of innovative and interdisciplinary pilotprojects with cross-sectional characters. Support and counselling for the design and implementation ofzoonotic funding schemes.Since 2009 more than 280 scientists joined the National Platform forZoonoses. Three main professional groups are represented within theresearch platform: veterinarians (38%), infectiologists (33%), and physicians(23%). Regarding the diversity of zoonoses, our members are mainly locatedat universities & university hospitals (55%) as well as at federal researchinstitutes (30%). Thereby the National Platform for Zoonoses ensures broadexchange of knowledge and experiences. The platforms’ priority will belinkage of further researchers and research groups to expand intersectionalresearch activities.OTP049Structure and functional studies of the Wolinellasuccinogenes STAS-DomainL. Schneider*, J. Du, O. EinsleInstitute for Biochemistry and Microbiology, Albert-Ludwigs-Unviersity,Freiburg, GermanyAnion transporters of to the Sulp family and the related SLC26 transportershave an N-terminal transmembrane domain that is connected via a linkerregion to the cytoplasmic c-terminal STAS domain (Sulfate TransporterAntagonist of anti-sigma factor). The name of this domain is due to a remotebut significant sequence similarity with bacterial ASA (anti sigma factorantagonist) protein 2. Mutation studies indicate that the STAS domain iscritical for the transporters activity and plays a role in intra- andintermolecular interactions 3 . In human members of the SLC26 transporters,mutations in the transmembrane domain as well as in the STAS domaincause a number of inherited diseases 4 . The structure of E.Coli YchM(SLC26 anion transporter) in complex with Acyl-Carrier protein indicatesthat YchM has a function in Fatty Acid Metabolism 1 .We are working with the STAS domain of Wolinella succinogenes andAquifex aeolicus. In order to crystallize the STAS domain of Wolinellasuccinogenes and Aquifex aeolicus different constructs were created bymutagenesis, a purification strategy was esthablished and the STAS domainwas crystallized to solve its structure by X-Ray diffraction. The other focusof our work is to show the interactions between the STAS domain andputative binding partners.[1] Babu, M. et al (2010): Structure of SLC26 Anion Transporter STAS Domain in Complex withAcyl Carrier Protein:Implications for E.Coli YchM in Fatty Acid Metabolism, Structure 18, 1450-1462.[2] Aravind, L. and E.V. Koonin (2000): The STAS domain - a link between anion transporters andantisigma-factor antagonists. Curr. Biol. 10, R53-R55[3] Shibagaki, N. and A.R. Grossman (2006): The Role of the STAS Domain in the Function andBiogenesis of a Sulfate Transporter as a probed by Random Mutagenesis, Journal of BiologicalChemistry, Volume 281, Number 32.[4] Everett, L. A. and E.D. Green (1999): A family of mammalian anion transporters and theirinvolvement in human genetic diseases. Hum. Mol. Genet. 8, 1883-1891.OTP050In vivo Tat substrate-translocon interactions inEscherichia coliJ. Taubert* 1 , T. Brüser 21 Institute of Biology/Microbiology, Martin-Luther-University Halle-Wittenberg, Halle, Germany2 Institute of Biology/Microbiology, Leibnitz-University, Hannover, GermanyFolded proteins can be translocated across energy-transducing membranesof prokaryotes and plant plastids by the twin-arginine translocation(Tat) system. Tat-dependently translocated proteins (Tat substrates)possess N-terminal signal peptides that contain the eponymoustwin-arginine motif, an amino acid pattern that usually includes twoconsecutive arginines and two consecutive hydrophobic residues,separated by one residue. In this study, we analyzed for the first time in vivoinEscherichia coli the interactions of Tat signal peptides and maturedomains of Tat substrates with the TatABC translocon subunits. Our datareveal the influence of an RR > KK exchange in the twin-arginine motifof translocon binding and indicate that the translocon interaction of matureTat substrate domains strongly depends on specific parameters of thesubstrates. An integrated transport model will be presented that takes allinteraction data into account.OTP051Structural and biochemical characterization of theformate channel FocA from Salmonella typhimuriumW. LüAG Einsle, Albert-Ludwigs-Unviersity, Freiburg, GermanyFormate is a key substrate and regulatory molecule during anaerobicbacterial fermentation. Therefore the transport of formate across thecytoplasmic membrane must be finely regulated in response to the change ofenvironmental parameters such as pH value or the availability of exogenouselectron acceptors. The first protein identified to mediate this transport wasFocA, encoded in the anaerobically induced pfl operon. Previous worksuggests that FocA, a member of the formate and nitrite transporter family,may function as a channel rather than transporter. However, to date little isknown about its regulatory mechanism. In this work we determined thecrystal structure of FocA from Salmonella typhimurium at 2.8 A, whichforms a pentameric assembly. Surprisingly, within the FocA pentamer threedifferent monomer conformations were observed for the very first N-terminal helix preceding the first transmembrane segment. This resulted inthree different states of the formate channel: open, intermediate and closed.With this finding, a working mechanism for the pH-dependent transport ofFocA is proposed.[1] Suppmann, B. and G. Sawers (1994): Isolation and characterization of hypophosphite-resistantmutants of Escherichia coli: identification of the FocA protein, encoded by the pfl operon, as aputative formate transporter. Mol Microbiol. 11, 965-82.[2] Wang, Y. et al (2009): Structure of the formate transporter FocA reveals a pentameric aquaporinlikechannel. Nature 462, 467-472.[3] Waight, A. B. et al (2010): Structure and mechanism of a pentameric formate channel. NatureStructural & Molecular Biology 17, 31-37.OTP052From 2-Oxoglutarate sensing to enzyme control by theSynechococcus elongatus PII signal transduction proteinO. Fokina* 1 , V.-R. Chellamuthu 2 , K. Zeth 2 , K. Forchhammer 11 Institute for Microbiology, Eberhard-Karls-University Tübingen, Tübingen,Germany2 Department of Protein Evolution, Max Planck Institute for DevelopmentalBiology, Tübingen, GermanyP II signal transduction proteins have key functions in coordination of centralmetabolism by integrating signals from carbon, nitrogen and energy status ofthe cell. In the cyanobacterium Synechococcus elongatus PCC7942 P II bindsATP and 2-oxoglutarate (2-OG) in a synergistic manner, with the ATPbindingsites also accepting ADP. Depending on its effector moleculebinding status, P II from this cyanobacterium and other oxygenic phototrophscomplexes regulates the key enzyme of the cyclic ornitine pathway, N-acetyl-L-glutamate kinase (NAGK), to control arginine biosynthesis.In wild type PII E85 forms a salt bridge with R233 of NAGK, andconsequently E85-PII mutants loose the ability to interact with NAGK. Wefound PII variants (I86N and I86T) that are able to bind to a NAGK variant(R233A) that was previously shown to be unable to bind wild type PIIprotein. Analysis of interactions between these P II variants and wild typeNAGK as well as the NAGK R233A variant suggested that the I86N variantin the presence of ATP was a superactive NAGK binder, also indicating thatP II-E85/NAGK-R233 is not essential for the interaction of the two proteins.To reveal the structural basis of this property, the crystal structure of the PIII86N variant was solved at atomic resolution. Based on the data we proposea two-step model for the mechanism of P II-NAGK complex formation: in aninitiating step, a contact between R233 of NAGK and E85 of PII initiates thebending of the extended T-loop of P II, followed by a second step, where abended T-loop deeply inserts into the NAGK clefts to form the tightcomplex.Crystal structures identify the binding site of 2-OG located in the vicinitybetween the subunit clefts and the base of the T-loop showing a novelconformation and explaining the negative effect of 2-OG on PII-NAGKinteraction. Trimers with one or two 2-OG molecules shed light on the intersubunitsignalling mechanism by which P II senses effectors in a wide rangeof concentrations.spektrum | Tagungsband 2011