CIISB Research Highlights Archive

  • J. Am. Chem. Soc. 2020

    J. Am. Chem. Soc. 2020

    Crystal structures of (A) PBD anthramycin covalently bound to DNA strands and (B) lincomycin targeting the peptidyl transferase center in the 50S ribosomal subunit of Staphylococcus aureus.

    Jiří Janata Research Group


    Antitumor pyrrolobenzodiazepines (PBDs), lincosamide antibiotics, quorumsensing molecule hormaomycin, and antimicrobial griselimycin are structurally and functionally diverse groups of actinobacterial metabolites. The common feature of these compounds is the incorporation of L -tyrosine- or L -leucine-derived 4-alkyl-L -proline derivatives (APDs) in their structures. In this study, the authors report that the last reaction in the biosynthetic pathway of APDs, catalyzed by F420 H2 -dependent Apd6 reductases, contributes to the structural diversity of APD precursors. Specifically, the heterologous overproduction of six Apd6 enzymes demonstrated that Apd6 from the biosynthesis of PBDs and hormaomycin can reduce only an endocyclic imine double bond, whereas Apd6 LmbY and partially GriH from the biosynthesis of lincomycin and griselimycin, respectively, also reduce the more inert exocyclic double bond of the same 4- substituted Δ 1-pyrroline-2-carboxylic acid substrate, making LmbY and GriH unusual, if not unique, among reductases. Furthermore, the differences in the reaction specificity of the Apd6 reductases determine the formation of the fully saturated APD moiety of lincomycin versus the unsaturated APD moiety of PBDs, providing molecules with optimal shapes to bind their distinct biological targets. Moreover, the Apd6 reductases establish the first F420 H2-dependent enzymes from the luciferase-like hydride transferase protein superfamily in the biosynthesis of bioactive molecules. Finally, bioinformatics analysis demonstrates that Apd6 and their homologues, widely distributed within several bacterial phyla, play a role in the formation of novel yet unknown natural products with incorporated L-proline-like precursors and likely in the microbial central metabolism.

    Steiningerova, L.; Kamenik, Z.*; Gazak, R.; Kadlcik, S.; Bashiri, G.; Man, P.; Kuzma, M.; Pavlikova, M. & Janata, J.: Different Reaction Specificities of F420H2Dependent Reductases Facilitate Pyrrolobenzodiazepines and Lincomycin To Fit Their Biological Targets, J. Am. Chem. Soc.  2020, 142, 3440-3448,

  • Nat. Commun. 2020

    Nat. Commun. 2020

    Cryo-EM structure of pT=4 quasi-icosahedral BDP and its penatameric and hexameric components. a Surface model of pT = 4 quasi-icosahedral BDP particle, displayed on the left side. A ribbon model of a cmcD pentamer and three cmcC′ hexamers is displayed on the right side. Pentameric cmcD protein is colored in yellow and hexameric cmcC′ is colored in green. Note that the fivefold symmetry axis is located at the center of cmcD pentamer and threefold axis is located in the middle between three cmcC′ hexamers. b Electrostatic surface potential of pentameric cmcD and hexameric cmcC′. Note the pores in the centers of pentamers and hexamers.

    Kaspas Tars Research Group


    Bacterial microcompartments (BMCs) are prokaryotic organelles consisting of a protein shell and an encapsulated enzymatic core. BMCs are involved in several biochemical processes, such as choline, glycerol and ethanolamine degradation and carbon fixation. Since non-native enzymes can also be encapsulated in BMCs, an improved understanding of BMC shell assembly and encapsulation processes could be useful for synthetic biology applications. Here we report the isolation and recombinant expression of BMC structural genes from the Klebsiella pneumoniae GRM2 locus, the investigation of mechanisms behind encapsulation of the core enzymes, and the characterization of shell particles by cryo-EM. We conclude that the enzymatic core is encapsulated in a hierarchical manner and that the CutC choline lyase may play a secondary role as an adaptor protein. We also present a cryo-EM structure of a pT = 4 quasi-symmetric icosahedral shell particle at 3.3 Å resolution, and demonstrate variability among the minor shell forms.

    Kalnins, G.; Cesle, E-E.; Jansons, J.; Liepins, J.; Filimonenko, A. & Tars, K.: Encapsulation mechanisms and structural studies of GRM2 bacterial microcompartment particles, Nature Comm. (2020) 11 (1), No. 388,

  • J. Am. Chem. Soc. 2019

    J. Am. Chem. Soc. 2019

    The unstructured C-terminal domain of delta subunit of bacterial RNA Polymerase is 90 aa long and highly charged. The charge distribution of this domain is distinct, with a conserved stretch of 9 residues (96−104) containing 7 positive charges followed by the rest of the domain with 51 acidic residues (K-D/E motif). A previous study demonstrated that the two parts of the motif transiently interact, and this affects the spatiotemporal properties of this domain. From the biological point of view, δ increases cell fitness and virulence of pathogens and was previously proposed to function as a nucleic acid mimic and affect RNAP− nucleic acid interactions.

    Lukáš Žídek Research Group


    Electrostatic interactions play important roles in the functional mechanisms exploited by intrinsically disordered proteins (IDPs). The atomic resolution description of long-range and local structural propensities that can both be crucial for the function of highly charged IDPs presents significant experimental challenges. Here, we investigate the conformational behavior of the δ subunit of RNA polymerase from Bacillus subtilis whose unfolded domain is highly charged, with 7 positively charged amino acids followed by 51 acidic amino acids. Using a specifically designed analytical strategy, we identify transient contacts between the two regions using a combination of NMR paramagnetic relaxation enhancements, residual dipolar couplings (RDCs), chemical shifts, and small-angle scattering. This strategy allows the resolution of long-range and local ensemble averaged structural contributions to the experimental RDCs, and reveals that the negatively charged segment folds back onto the positively charged strand, compacting the conformational sampling of the protein while remaining highly flexible in solution. Mutation of the positively charged region abrogates the long-range contact, leaving the disordered domain in an extended conformation, possibly due to local repulsion of like-charges along the chain. Remarkably, in-vitro studies show that this mutation also has a significant effect on transcription activity, and results in diminished cell fitness of the mutated bacteria in vivo. This study highlights the importance of accurately describing electrostatic interactions for understanding the functional mechanisms of IDPs.

    Kuban, V.; Srb, P.; Stegnerova, H.; Padrta, P.; Zachrdla, M.; Jasenakova, Z.; Sanderova, H.; Vitovska, D.; Krasny, L.; Koval, T.; Dohnalek, J.; Ziemska-Legiecka, J.; Grynberg, M.; Jarnot, P.; Gruca, A.; Jensen, M. R.; Blackledge, M. & Zidek, L.: Quantitative Conformational Analysis of Functionally Important Electrostatic Interactions in the Intrinsically Disordered Region of Delta Subunit of Bacterial RNA Polymerase,  J. Am. Chem. Soc.  2019, 141, 16817-16828, DOI:10.1021/jacs.9b07837

  • 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


    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


    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


    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.

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