Research Highlights Archive

  • Sci. Adv. 2019

    Sci. Adv. 2019

    Virion and genome organization of phage P68. (A and B) Structures of P68 virion, (C) genome release intermediate, and (D) empty particle. The whole P68 virion is shown in (A), whereas particles without the front half are shown in (B) to (D). The structures are colored to distinguish individual types of structural proteins and DNA. (E) Schematic diagram of P68 genome organization, with structural proteins color-coded in accordance with the structure diagrams shown in (A) to (D).

    Pavel Plevka Research Group

    Significance

    Phages infecting Staphylococcus aureus can be used as therapeutics against antibiotic-resistant bacterial infections. However, there is limited information about the mechanism of genome delivery of phages that infect Gram-positive bacteria. Here, we present the structures of native S. aureus phage P68, genome ejection intermediate, and empty particle. The P68 head contains 72 subunits of inner core protein, 15 of which bind to and alter the structure of adjacent major capsid proteins and thus specify attachment sites for head fibers. Unlike in the previously studied phages, the head fibers of P68 enable its virion to position itself at the cell surface for genome delivery. The unique interaction of one end of P68 DNA with one of the 12 portal protein subunits is disrupted before the genome ejection. The inner core proteins are released together with the DNA and enable the translocation of phage genome across the bacterial membrane into the cytoplasm.

    Hrebík, D.; Štveráková, D.; Škubník, K.; Füzik, T.; Pantůček, R. & Plevka, P.: Structure and genome ejection mechanism of Staphylococcus aureus phage P68, Sci. Adv. 2019, 5(10), eaaw7414, DOI: 10.1126/sciadv.aaw7414

  • J. Am. Chem. Soc.  2019

    J. Am. Chem. Soc. 2019

    Double-staining (PI/FAM) FCM analysis (A) and confocal microscopy images (B) of cells co-transfected with the (FAM)-MHDNA: netropsin (1:1) complex. In panel (A), the percentages of viable non-transfected cells, viable MH-DNA containing cells, dead/compromised non-transfected cells, and dead/compromised transfected cells are indicated in the bottom-left, bottom-right, top-left, and top-right quadrants, respectively. In panel (B), the green color marks the localization of (FAM)-MH-DNA, while the blue color marks cellc nuclei stained with Hoechst 33342. (C) Deconvoluted imino regions of 1D 1H NMR spectra of MH-DNA in vitro and the 1:1 MHDNA: netropsin complex in vitro and in cells. NMR spectra of extracellular fluid taken from the sample after in-cell NMR spectral acquisition and of non-transfected cells (cellular background) are shown in gray. The vertical green and blue dashed lines mark imino signals specific to the unbound and ligand-bound forms of MH-DNA, respectively.

    Lukáš Trantírek Research Group

    Significance

    Studies on DNA ligand interactions in the cellular environment are problematic due to the lack of suitable biophysical tools. To address this need, we developed an in-cell NMR-based approach for monitoring DNAligand interactions inside the nuclei of living human cells. Our method relies on the acquisition of NMR data from cells electroporated with preformed DNA ligand complexes. The impact of the intracellular environment on the integrity of the complexes is assessed based on in-cell NMR signals from unbound and ligand-bound forms of a given DNA target. This technique was tested on complexes of two model DNA fragments and four ligands, namely, a representative DNA minor-groove binder (netropsin) and ligands binding DNA base-pairing defects (naphthalenophanes). In the latter case, we demonstrate that two of the three in vitro -validated ligands retain their ability to form stable interactions with their model target DNA in cellulo, whereas the third one loses this ability due to off -target interactions with genomic DNA and cellular metabolites. Collectively, our data suggest that direct evaluation of the behavior of druglike molecules in the intracellular environment provides important insights into the development of DNA-binding ligands with desirable biological activity and minimal side effects resulting from off-target binding.

    Krafcikova, M.; Dzatko, S.; Caron, C.; Granzhan, A.; Fiala, R.; Loja, T.; Teulade-Fichou, M.-P.; Fessl, T.; Hänsel-Hertsch, R.; Mergny, J.-L.; Foldynova-Trantirkova, S. & Trantirek, L.: Monitoring DNA−Ligand Interactions in Living Human Cells Using NMR Spectroscopy, J. Am. Chem. Soc.  2019, 141, 13281-13825, DOI: 10.1021/jacs.9b03031

  • PNAS 2019

    PNAS 2019

    (A) Cryo-EM structure of RV-B5 complexed to OBR-5-340 colored radially as indicated by the color bar. Distance from the viral center is 130 Å (white) to 160 Å (dark blue). (B) Example of the quality of the maps of RV-B5 with OBR-5-340 (Left) and without OBR-5-340 (Right). (C) View centered on OBR-5-340 (yellow) in complex with RV-B5 (red). For comparison, the control, i.e., RV-B5 solved in the absence of inhibitor (blue), is overlaid. Residues nearby and contributed by VP1 are labeled. (D) RV-B5 solved in the absence of OBR-5-340. Note the absence of density at the position where the inhibitor is seen in the complex.

    Dieter Blaas and Michaela Schmidtke Research Groups

    Significance

    More than 160 rhinovirus (RV) types cause about a billion respiratory infections annually in the United States alone, contributing to influenza-like illness. This diversity makes vaccination impractical. Existing small-molecule inhibitors target RVs by binding to a hydrophobic pocket in the capsid but exhibit side effects, resistance, and/or mutational escape, impeding registration as drugs. The pyrazolopyrimidine OBR-5-340 acts like other capsid binders by preventing conformational changes required for genome release. However, by using cryo-EM, we show that OBR-5-340 inhibits the naturally pleconaril-resistant RV-B5 by attaching close to the pocket entrance in a binding geometry different from that of most capsid binders. Combinations of inhibitors with disparate binding modes might thus effectively combat RVs while reducing the risk of resistance development.

    Wald, J.; Pasin, M.; Richter, M.; Walther, C.; Mathai, N.; Kirchmair, J.; Makarov, V. M.; Goessweiner-Mohr, N.; Marlovits, T. C.; Zanella, I.; Real-Hohn, A.; Verdaguer, N.; Blaas, D. & Schmidtke, M.: Cryo-EM structure of pleconaril-resistant rhinovirus-B5 complexed to the antiviral OBR-5-340 reveals unexpected binding site, Proc. Natl. Acad. Sci. U.S.A. 2019, 116 (38), 19109-19115. https://doi.org/10.1073/pnas.1904732116

  • Anal. Chem. 2019

    Anal. Chem. 2019

    Utility of phenylhydrazine (PHN) labeling for structural studies of fucosylated N-glycans by tandem MALDI mass spectrometry (MS) in the positive ion mode is proposed. PHN-tag influences the production of specific ion types, and the MS/MS fragmentation pattern provides useful structural information. 

    Zbyněk Zdráhal Research Group

    Significance

    Fucosylation is a common modification, and its site in glycans refers to different normal and pathological processes. Despite intensive research, there is still a lack of methods to discriminate unambiguously the fucose position in one-step. In this work, we propose utility of phenylhydrazine (PHN) labeling for structural studies of fucosylated N-glycans by tandem MALDI mass spectrometry (MS) in the positive ion mode. PHN-tag influences the production of specific ion types, and the MS/MS fragmentation pattern provides useful structural information. All types of core fucosylated N-glycans have produced two abundant ions consistent with B- and C-glycosidic cleavages corresponding to the loss of the FucGlcNAcPHN residue with a mass 457 and 441 Da from the parent ions. These types of fragment ions in N-glycans without a core fucose were associated with the loss of the GlcNAcPHN unit (311 and 295 Da), and fucose cleavage followed the loss of the chitobiose residue. Since diagnostic useful cleavages produce peaks with significant intensities, this approach is also beneficial for rapid recognition of antenna from core fucosylation in glycans detected with low abundances. The practical applicability of the approach is demonstrated on the analysis of multifucosylated N-glycans detected with lower abundances in lung cancer samples.

    Lattová, E.; Skřičková, J. & Zdráhal, Z.: Applicability of Phenylhydrazine Labeling for Structural Studies of Fucosylated N-Glycans, Anal. Chem. 2019 91, 13, 7985-7990. https://doi.org/10.1021/acs.analchem.9b01321

  • Sci. Adv. 2019

    Sci. Adv. 2019

    Potato virus Y (PVY) belongs to the most economically important pathogens. The collaborative research project of the National Institute of Chemistry (Ljublana, Slovenia) and Cryo-Electron Microscopy Core Facility at CEITEC MU reveals the structure of the PVY coat protein (CP) and the PVY virus like particle at near-atomic resolution. The data show a novel luminal interplay between the extended carboxy-terminal CP regions in the virion and describe RNA-CP interactions important for helical conformation and stability of the virus.

    Marjetka Podobnik Research Group

    Significance

    PVY is ranked as fifth in the top 10 most economically important plant viruses and is the most important viral pathogen of potato worldwide. The virus causes potato tuber necrotic ringspot disease, which can result in up to 70% yield reduction, and severely affects other economically important solanaceous crops. Despite extensive availability of data on PVY’s genome and pathogenicity, there has been no high-resolution structuralinformation for this virus. Because of the extreme economic importance of PVY, and the urgent need for structural data to better understand mechanisms of viral infectivity, we have examined in detail the structure of the PVY virion and its CP. We have determined the high-resolution electron cryo-microscopy structures of the PVY virion and a recombinant PVY-based RNA-free virus-like particle (VLP). This provides a new and detailed insight into the RNA-supported helical viral capsid architecture featuring an extended C-terminal region of CP, which is tightly packed in a unique fashion in the virion lumen. In addition, using extensive biochemical, biophysical, and computational characterization, as well as structure-­based mutagenesis, we identified regions of CP that affect VLP filament assembly. Moreover, the biological activities of the CP’s N- and C-terminal regions for virus infectivity were explored by measuring the accumulation of viral RNA and systemic movement of selected PVY mutants in plants.

    Kežar, A.; Kavčič, L.; Polák, M.; Nováček, J.; Gutiérrez-Aguirre, I.; Tušek Žnidarič, M.; Coll, A.; Stare, K.; Gruden, K.; Ravnikar, M.; Pahovnik, D.; Žagar, E.; Merzel, F.; Anderluh, G. & Podobnik, M.: Structural basis for the multitasking nature of the potato virus Y coat protein, Sci. Adv. (2019) 5(7), eaaw3808, DOI: 10.1126/sciadv.aaw3808

  • Chem 2019

    Chem 2019

    In biology, the transport of ions across lipid membranes is crucial and is generally performed by membrane proteins. Deficiencies in transport are at the origin of various diseases, such as cystic fibrosis. In this context, synthetic anion carriers incorporated within the lipid bilayer could play a remedial role. They extract ions from one side of the membrane, move across, and release the ions on the other side.

    Vladimír Šindelář Research Group

    Significance

    The exchange of chloride and bicarbonate across lipid bilayers is an important biological process. Synthetic molecules can act as mobile carriers for these anions, although most show little selectivity. Here we report on three bambus[6]uril macrocycles functionalized with fluorinated benzyl groups, which are ableto exchange Cl- and HCO3-efficiently. Remarkably, rates for Cl-/NO3- exchange are two orders of magnitude lower. The higher rates of Cl-/HCO3- transport can be explained by the ability of the bambusurils to complex Cland HCO3- simultaneously, facilitating their exchange at the bilayer interface.

    Furthermore, the exceptionally high affinity and selectivity of these systemsfor NO3- appear to contribute to the poor Cl-/NO3- exchange. This worknot only demonstrates the importance of anion binding characteristics onanion transport but also the potential relevance of bambusurils for aniontransport applications considering the high rate observed for Cl-/HCO3- exchange.

    Valkenier, H.; Akrawi, O.; Jurcek, P.; Sleziakova, K.; Lizal, T.; Bartik, K. & Sindelar, V.: Fluorinated Bambusurils as Highly Effective and Selective Transmembrane Cl-/HCO3- Antiporters, Chem (2019) 5, 429-444. doi:10.1016/j.chempr.2018.11.008 

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