FMP Publications

Our publications are recorded in a searchable database since 2010, updates will be added regularly.

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Structure of the competence pilus major pilin ComGC in Streptococcus pneumoniae
Muschiol(*), S., Erlendsson(*), S., Aschtgen(*), M. S., Oliveira(*), V., Schmieder, P., de Lichtenberg(*), C., Teilum(*), K., Boesen(*), T., Akbey(*), Ü.; Henriques-Normark(*), B.
J Biol Chem, 292:14134-14146

Tags: Solution NMR (Schmieder)

Abstract: Type IV pili are important virulence factors on the surface of many pathogenic bacteria and have been implicated in a wide range of diverse functions, including attachment, twitching motility, biofilm formation, and horizontal gene transfer. The respiratory pathogen Streptococcus pneumoniae deploys type IV pili to take up DNA during transformation. These "competence pili" are composed of the major pilin protein ComGC and exclusively assembled during bacterial competence, but their biogenesis remains unclear. Here, we report the high resolution NMR structure of N-terminal truncated ComGC revealing a highly flexible and structurally divergent type IV pilin. It consists of only three alpha-helical segments forming a well-defined electronegative cavity and confined electronegative and hydrophobic patches. The structure is particularly flexible between the first and second alpha-helix with the first helical part exhibiting slightly slower dynamics than the rest of the pilin, suggesting that the first helix is involved in forming the pilus structure core and that parts of helices two and three are primarily surface-exposed. Taken together, our results provide the first structure of a type IV pilin protein involved in the formation of competence-induced pili in Gram-positive bacteria and corroborate the remarkable structural diversity among type IV pilin proteins.

Sapofectosid - Ensuring non-toxic and effective DNA and RNA delivery
Sama(*), S., Jerz(*), G., Schmieder, P., Woith(*), E., Melzig(*), M. F.; Weng(*), A.
International journal of pharmaceutics, 534:195-205

Tags: Solution NMR (Schmieder)

Abstract: Different methods are being deployed for non-viral DNA/RNA delivery. However non-viral formulations for DNA/RNA-delivery are often accompanied by severe toxicity and thus low efficiency. Particular costly cell culture media are required as well. Here we introduce sapofection as a valuable enhancing method for non-viral DNA/RNA delivery. Sapofection is based on the application of DNA/RNA nanoplexes and sapofectosid, a plant derived natural transfection reagent. Sapofectosid was produced from plant raw material by chromatographic methods and characterized by tandem mass spectrometry and intensive one and two dimensional NMR-spectroscopy. Sapofectosid did enhance the transfection efficiency of different DNA- and RNA-nanoplexes formulated with liposomes, polyethylenimine (PEI) or targeted and non-targeted oligo-lysine peptides. All nanoplexes were characterized physicochemically and the influence of sapofectosid on the nanoplex integrity was determined by DNA complexation assays. The nanoplexes and sapofectosid were administered to a variety of cancer cell lines and the transfection efficiency was investigated by flow cytometry and confocal microscopy. Dependent on the cell line the transfection efficiencies varied from 6 to 76%. The saponin- and receptor-mediated endocytosis of nanoplexes was investigated by flow cytometry. As demonstrated by impedance based live cell imaging sapofection was non-toxic. The findings show the great potential of sapofection to be used as an effective and non-toxic transfection enhancing method.

Direct Experimental Evidence for Halogen-Aryl pi Interactions in Solution from Molecular Torsion Balances
Sun, H., Horatscheck, A., Martos, V., Bartetzko, M., Uhrig, U., Lentz, D., Schmieder, P.; Nazare, M.
Angew Chem Int Ed Engl, 56:6454-6458

Tags: Medicinal Chemistry (Nazare), Solution NMR (Schmieder), Computational Chemistry/ Drug Design (Kühne)

Abstract: We dissected halogen-aryl pi interactions experimentally using a bicyclic N-arylimide based molecular torsion balances system, which is based on the influence of the non-bonded interaction on the equilibria between folded and unfolded states. Through comparison of balances modulated by higher halogens with fluorine balances, we determined the magnitude of the halogen-aryl pi interactions in our unimolecular systems to be larger than -5.0 kJ mol-1 , which is comparable with the magnitude estimated in the biomolecular systems. Our study provides direct experimental evidence of halogen-aryl pi interactions in solution, which until now have only been revealed in the solid state and evaluated theoretically by quantum-mechanical calculations.

Structural Biology outside the box-inside the cell
Plitzko(*), J. M., Schuler(*), B.; Selenko, P.
Curr Opin Struct Biol, 46:110-121

Tags: In-Cell NMR (Selenko)

Abstract: Recent developments in cellular cryo-electron tomography, in-cell single-molecule Forster resonance energy transfer-spectroscopy, nuclear magnetic resonance-spectroscopy and electron paramagnetic resonance-spectroscopy delivered unprecedented insights into the inner workings of cells. Here, we review complementary aspects of these methods and provide an outlook toward joint applications in the future.

Membrane-traversing mechanism of thyroid hormone transport by monocarboxylate transporter 8
Protze, J., Braun(*), D., Hinz, K. M., Bayer-Kusch(*), D., Schweizer(*), U.; Krause, G.
Cellular and molecular life sciences : CMLS,

Tags: Structural Bioinformatics and Protein Design (Krause, G.)

Abstract: Monocarboxylate transporter 8 (MCT8) mediates thyroid hormone (TH) transport across the plasma membrane in many cell types. In order to better understand its mechanism, we have generated three new MCT8 homology models based on sugar transporters XylE in the intracellular opened (PDB ID: 4aj4) and the extracellular partly occluded (PDB ID: 4gby) conformations as well as FucP (PDB ID: 3o7q) and GLUT3 (PDB ID: 4zwc) in the fully extracellular opened conformation. T3-docking studies from both sides revealed interactions with His192, His415, Arg445 and Asp498 as previously identified. Selected mutations revealed further transport-sensitive positions mainly at the discontinuous transmembrane helices TMH7 and 10. Lys418 is potentially involved in neutralising the charge of the TH substrate because it can be replaced by charged, but not by uncharged, amino acids. The side chain of Thr503 was hypothesised to stabilise a helix break at TMH10 that undergoes a prominent local shift during the transport cycle. A T503V mutation accordingly affected transport. The aromatic Tyr419, the polar Ser313 and Ser314 as well as the charged Glu422 and Glu423 lining the transport channel have been studied. Based on related sugar transporters, we suggest an alternating access mechanism for MCT8 involving a series of amino acid positions previously and newly identified as critical for transport.

Targeting G-protein-coupled receptors by Capture Compound Mass Spectrometry (CCMS) - a case study with sertindole
Blex(*), C., Michaelis(*), S., Schrey(*), A. K., Furkert, J., Eichhorst, J., Bartho(*), K., Quast(*), F. G., Marais(*), A., Hakelberg(*), M., Gruber(*), U., Niquet(*), S., Popp(*), O., Kroll(*), F., Sefkow(*), M., Schülein, R., Mathias(*), D.; Koster(*), H.
Chembiochem, 18:1639-1649

Tags: Protein Trafficking (Schülein), Cellular Imaging (Wiesner/Puchkov)

Abstract: Unbiased chemoproteomic profiling of small molecule interactions with endogenous proteins is important for drug discovery. For meaningful results, all protein classes have to be tractable, including G-protein coupled receptors (GPCRs). These are hardly tractable by affinity pulldown from lysates. We report a Capture Compound (CC)-based strategy to target and identify GPCRs directly from living cells. We synthesized CCs with sertindole attached to the CC scaffold in different orientations to target the dopamine D2 receptor (DRD2) heterologously expressed in HEK293 cells. The structure-activity relationship of sertindole for DRD2 binding is reflected in the activities of the sertindole CCs in radioligand displacement, cell-based assays, and CCMS. The activity pattern was rationalized by molecular modelling. The most active CC showed activities very similar to unmodifed sertindole. Well below 100 fmol of DRD2 in living cells used as experiment input were sufficient for unambiguous identification of captured DRD2 by mass spectrometry. Our new CCMS workflow broadens the arsenal of chemoproteomic technologies to close a critical gap for the comprehensive characterization of drug-protein interactions.

Molecular features of the L-type amino acid transporter 2 determine different import and export profiles for thyroid hormones and amino acids
Hinz, K. M., Neef, D., Rutz, C., Furkert, J., Köhrle(*), J., Schülein, R.; Krause, G.
Mol Cell Endocrinol, 443:163-174

Tags: Structural Bioinformatics and Protein Design (Krause, G.), Protein Trafficking (Schülein)

Abstract: The L-type amino acid transporter 2 (LAT2) imports amino acids (AA) and also certain thyroid hormones (TH), e.g. 3,3'-T2 and T3, but not rT3 and T4. We utilized LAT2 mutations (Y130A, N133S, F242W) that increase 3,3'-T2 import and focus here on import and export capacity for AA, T4, T3, BCH and derivatives thereof to delineate molecular features. Transport studies and analysis of competitive inhibition of import by radiolabelled TH and AA were performed in Xenopus laevis oocytes. Only Y130A, a pocket widening mutation, enabled import for T4 and increased it for T3. Mutant F242W showed increased 3,3'-T2 import but no import rates for other TH derivatives. No export was detected for any TH by LAT2-wild type (WT). Mutations Y130A and N133S enabled only the export of 3,3'-T2, while N133S also increased AA export. Thus, distinct molecular LAT2-features determine bidirectional AA transport but only an unidirectional 3,3'-T2 and T3 import.

Evidence for Heterodimerization and Functional Interaction of the Angiotensin Type 2 Receptor and the Receptor MAS
Leonhardt(*), J., Villela(*), D. C., Teichmann, A., Munter(*), L. M., Mayer(*), M. C., Mardahl(*), M., Kirsch(*), S., Namsolleck(*), P., Lucht(*), K., Benz(*), V., Alenina(*), N., Daniell(*), N., Horiuchi(*), M., Iwai(*), M., Multhaup(*), G., Schülein, R., Bader(*), M., Santos(*), R. A., Unger(*), T.; Steckelings(*), U. M.

Tags: Protein Trafficking (Schülein), Cellular Imaging (Wiesner)

Abstract: The angiotensin type 2 receptor (AT2R) and the receptor MAS are receptors of the protective arm of the renin-angiotensin system. They mediate strikingly similar actions. Moreover, in various studies, AT2R antagonists blocked the effects of MAS agonists and vice versa. Such cross-inhibition may indicate heterodimerization of these receptors. Therefore, this study investigated the molecular and functional interplay between MAS and the AT2R. Molecular interactions were assessed by fluorescence resonance energy transfer and by cross correlation spectroscopy in human embryonic kidney-293 cells transfected with vectors encoding fluorophore-tagged MAS or AT2R. Functional interaction of AT2R and MAS was studied in astrocytes with CX3C chemokine receptor-1 messenger RNA expression as readout. Coexpression of fluorophore-tagged AT2R and MAS resulted in a fluorescence resonance energy transfer efficiency of 10.8 +/- 0.8%, indicating that AT2R and MAS are capable to form heterodimers. Heterodimerization was verified by competition experiments using untagged AT2R and MAS. Specificity of dimerization of AT2R and MAS was supported by lack of dimerization with the transient receptor potential cation channel, subfamily C-member 6. Dimerization of the AT2R was abolished when it was mutated at cysteine residue 35. AT2R and MAS stimulation with the respective agonists, Compound 21 or angiotensin-(1-7), significantly induced CX3C chemokine receptor-1 messenger RNA expression. Effects of each agonist were blocked by an AT2R antagonist (PD123319) and also by a MAS antagonist (A-779). Knockout of a single of these receptors made astrocytes unresponsive for both agonists. Our results suggest that MAS and the AT2R form heterodimers and that-at least in astrocytes-both receptors functionally depend on each other.

Functional Significance of the Signal Peptides of Corticotropin-Releasing Factor Receptors
Schülein, R., Gibert, A.; Rutz, C.
Current molecular pharmacology,

Tags: Protein Trafficking (Schülein)

Abstract: The corticotropin releasing factor (CRF) receptors belong to the large family of G protein-coupled receptors (GPCRs) and must be transported to the plasma membrane to function properly. The first step of the intracellular transport of GPCRs is their insertion into the membrane of the endoplasmic reticulum (ER). This process is mediated by the translocon complex of the ER membrane and the signal sequences of the receptors. Most GPCRs possess signal sequences which form part of the mature proteins, the so called signal anchor sequences (usually transmembrane domain 1). The CRF receptors possess instead signal sequences at their extreme N tails which were thought to be cleaved off following integration of the receptors into the ER membrane (signal peptides, SPs, also called cleaved signal sequences). Recent work, however, showed that not all subtypes of CRF receptors stick to this rule. Whereas the corticotropin-releasing factor receptor type 1 (CRF1R) and the corticotropin-releasing factor receptor type 2b (CRF2(b)R) possess conventional SPs which are indeed cleaved off following ER insertion, the SP of the cortictropin-releasing factor receptor type 2a (CRF2(a)R) remains uncleaved. It forms a unique N-terminal domain (pseudo signal peptide, PSP) which has surprising functions beyond the ER level. Its presence not only influences expression levels at the plasma membrane but also receptor homodimerisation and, as a consequence, G protein selectivity. In this mini-review, we summarize the progress in understanding the functions of SPs of CRF receptors. Recent data also allow deriving hypotheses for a physiological significance of these sequences.

Oxidative inactivation of the endogenous antioxidant protein DJ-1 by the food contaminants 3-MCPD and 2-MCPD
Buhrke(*), T., Voss(*), L., Briese(*), A., Stephanowitz, H., Krause, E., Braeuning(*), A.; Lampen(*), A.
Archives of toxicology,

Tags: Mass Spectrometry (Krause, E.)

Abstract: 3-Chloro-1,2-propanediol (3-MCPD) and 2-chloro-1,3-propanediol (2-MCPD) are heat-induced food contaminants being present either as free substances or as fatty acid esters in numerous foods. 3-MCPD was classified to be possibly carcinogenic to humans (category 2B) with kidney and testis being the primary target organs according to animal studies. A previous 28-day oral feeding study with rats revealed that the endogenous antioxidant protein DJ-1 was strongly deregulated at the protein level in kidney, liver, and testis of the experimental animals that had been treated either with 3-MCPD, 2-MCPD or their dipalmitate esters. Here we show that this deregulation is due to the oxidation of a conserved, redox-active cysteine residue (Cys106) of DJ-1 to a cysteine sulfonic acid which is equivalent to loss of function of DJ-1. Irreversible oxidation of DJ-1 is associated with a number of oxidative stress-related diseases such as Parkinson, cancer, and type II diabetes. It is assumed that 3-MCPD or 2-MCPD do not directly oxidize DJ-1, but that these substances induce the formation of reactive oxygen species (ROS) which in turn trigger DJ-1 oxidation. The implications of 3-MCPD/2-MCPD-mediated ROS formation in vivo for the ongoing risk assessment of these compounds as well as the potential of oxidized DJ-1 to serve as a novel effect biomarker for 3-MCPD/2-MCPD toxicity are being discussed.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
Robert-Roessle-Str. 10
13125 Berlin, Germany
+4930 94793 - 100 
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