CIISB Research Highlights Archive

  • Sci. Adv. 2021

    Sci. Adv. 2021

    Organization of protein IX.

    (A) External remnant density (green and yellow, unsharpened, 0.2σ). Modelled triskelions are in cyan (I3 axis, I3 NT) and magenta (L3 axes, L3 NT). Dotted rectangles: location of helix bundles in HAdV-C5. (B) Interpretation of the remnant map. Apart from elements shown in (A), density protruding at the L3 axes is in blue, and the proposed path for the rope domains corresponding to each triskelion in dashed lines. (C) Cartoon representing the hypothesis that the C-terminal domains associate forming a mobile, spike-like structure whose density is averaged away. Hexon HVR2 loops would constrain the flexibility of IX, producing the most evident blurry density (dark blue splotch). Less intense dark blue represents weaker density observed in some localized reconstruction classes (fig. S5B). (D) The rope domain of the IX molecules forming the I3 triskelion follows different paths in HAdV-C5/D26 and HAdV-F41. One of the IX monomers forming the HAdV-D26 I3 triskelion is in green; the equivalent monomer in HAdV-F41 in cyan; and the HAdV-F41 L3 triskelion in magenta. Grey surface: HAdV-F41 remnant map. Hexons (H3, H3′, H4) in semi-transparent surface. Top: view from outside the capsid as in (A). Bottom: side view after rotating as indicated. Notice that the blurry density at L3 protrudes above the hexon towers. (E) Zoom in on the region where the triskelion ends and the rope domain turns. Green, overlapped structures of IX in HAdV-C5 and HAdV-D26. HAdV-D26 residue labels are underlined. Dashed lines: untraced rope domains in HAdV-C5 and HAdV-F41. Grey mesh: HAdV-F41 remnant density corresponding to the rope domain. Hexon amino acids with RMSD above 2 Å are in red.

    Carmen San Martin Research Group

    Significance

    Enteric adenoviruses, one of the main causes of viral gastroenteritis in the world, must withstand the harsh conditions found in the gut. This requirement suggests that capsid stability must be different from that of other adenoviruses. Carmen San Martin et. al. report the 4-Å-resolution structure of a human enteric adenovirus, HAdV-F41, and compare it with that of other adenoviruses with respiratory (HAdV-C5) and ocular (HAdV-D26) tropisms. While the overall structures of hexon, penton base, and internal minor coat proteins IIIa and VIII are conserved, they observe partially ordered elements reinforcing the vertex region, which suggests their role in enhancing the physicochemical capsid stability of HAdV-F41. Unexpectedly, theyfind an organization of the external minor coat protein IX different from all previously characterized human and nonhuman mastadenoviruses. Knowledge of the structure of enteric adenoviruses provides a starting point for the design of vectors suitable for oral delivery or intestinal targeting.

    Peréz-Illana, M.; Martinéz, M.; Condezo, G. N.; Hernando-Peréz, M.; Mangroo, C.; Marabini, R. & San Martin, C.: Cryo-EM structure of enteric adenovirus HAdV-F41 highlights structural variations among human adenoviruses, Sci. Adv. 2021, 7, eabd9421, https://doi.org/10.1126/sciadv.abd9421

  • Nat. Commun. 2021

    Nat. Commun. 2021

    EH domains of AtEH1/Pan1 differ in their Ca2+-binding capacities. a Domain organization of AtEH1/Pan1 and AtEH2/Pan1. Both proteins contain two Eps15 homology domains (EH), a coiled-coil domain (CC), and an acidic (A)-motif. A schematic representation of a multiple sequence alignment (MSA) - shows strong conservation of the EH domains (blue lines) across the plant kingdom. Percentages indicate the relative number of identical amino acids. bg Cartoon representation of the X-ray structure of EH1.1 and NMR/all-atom molecular dynamics structure of EH1.2. Ions are shown as orange (Ca2+) or grey (Na+) spheres. Insets show the ion coordination in each EF-hand loop. Ca2+ coordinating residues and water molecules (W) are indicated in (c, d) and (f, g).

    Savvas N. Savvides, Kostas Tripsianes, Roman Pleskot, and Daniel Van Damme Research Groups

    Significance

    Clathrin-mediated endocytosis (CME) is the gatekeeper of the plasma membrane. In contrast to animals and yeasts, CME in plants depends on the TPLATE complex (TPC), an evolutionary ancient adaptor complex. The mechanistic contribution of the individual TPC subunits to plant CME remains however elusive. In this study, we used a multidisciplinary approach to elucidate the structural and functional roles of the evolutionary conserved N-terminal Eps15 homology (EH) domains of the TPC subunit AtEH1/Pan1. By integrating high-resolution structural information obtained by X-ray crystallography and NMR spectroscopy with all-atom molecular dynamics simulations, we provide structural insight into the function of both EH domains. Both domains bind phosphatidic acid with a different strength, and only the second domain binds phosphatidylinositol 4,5-bisphosphate. Unbiased peptidome profiling by mass-spectrometry revealed that the first EH domain preferentially interacts with the double N-terminal NPF motif of a previously unidentified TPC interactor, the integral membrane protein Secretory Carrier Membrane Protein 5 (SCAMP5). Furthermore, we show that AtEH/Pan1 proteins control the internalization of SCAMP5 via this double NPF peptide interaction motif. Collectively, our structural and functional studies reveal distinct but complementary roles of the EH domains of AtEH/Pan1 in plant CME and connect the internalization of SCAMP5 to the TPLATE complex.

    Yperman, K.; Papageorgiou, A. C.; Merceron, R.; De Munck, S.; Bloch, Y.; Eeckhout, D.; Jiang, Q.; Tack, P.; Grigoryan, R.; Evangelidis, T.; Van Leene, J.; Vincze, L.; Vandenabeele, P.; Vanhaecke, F.; Potocký, M.; De Jaeger, G.; Savvides, S. N.; Tripsianes, K.; Pleskot, R. & Van Damme D.: Distinct EH domains of the endocytic TPLATE complex confer lipid and protein binding, Nature Comm. (2021) **, **  https://doi.org/10.1038/s41467-021-23314-6

  • Sci. Adv. 2021

    Sci. Adv. 2021

    Structural changes in iflavirus particles that enable genome release of SBV, SBPV, and DWV. Native virions (A, F, and K), genome-containing particles at acidic pH (B, G, and L), open particles containing genomes (C, H, and M), open particles without genomes (D, I, and N), and empty capsids resulting from genome release (E, J, and O). Individual panels show cryo-EM reconstructions of particles rainbow colored on the basis of the distance of the particle surface from its center. (C), (H), and (N) show projection images of representative particles, since 3D reconstructions could not be calculated because of structural heterogeneity of the particles. Scale bar, 10 nm.

    Pavel Plevka Research Group

    Significance

    The family Iflaviridae includes economically important viruses of the western honeybee such as deformed wing virus, slow bee paralysis virus, and sacbrood virus. Iflaviruses have nonenveloped virions and capsids organized with icosahedral symmetry. The genome release of iflaviruses can be induced in vitro by exposure to acidic pH, implying that they enter cells by endocytosis. Genome release intermediates of iflaviruses have not been structurally characterized. Here, P. Plevka et.al. show that conformational changes and expansion of iflavirus RNA genomes, which are induced by acidic pH, trigger the opening of iflavirus particles. Capsids of slow bee paralysis virus and sacbrood virus crack into pieces. In contrast, capsids of deformed wing virus are more flexible and open like flowers to re- lease their genomes. The large openings in iflavirus particles enable the fast exit of genomes from capsids, which decreases the probability of genome degradation by the RNases present in endosomes.

    Škubník, K.; Sukeník, L.; Buchta, D.; Füzik, T.; Procházková, M.; Moravcová, J.; Šmerdová, L.; Přidal, A.; Vácha, R. & Plevka, P.: Capsid opening enables genome release of iflaviruses, Sci. Adv. 2021, 7, eabd7130, DOI: 10.1126/sciadv.abd7130

  • Nat. Commun. 2020

    Nat. Commun. 2020

    Three states of HelD color-coded according to the domain structure

    Libor Krásný and Jan Dohnálek Research Groups

    Significance

    RNA synthesis is central to life, and RNA polymerase (RNAP) depends on accessory factors for recovery from stalled states and adaptation to environmental changes. Here, T. Kouba, J. Dohnálek, L. Krásný et.al.investigated the mechanism by which a helicase-like factor HelD recycles RNAP. They report a cryo-EM structure of a complex between the Mycobacterium smegmatis RNAP and HelD. The crescent-shaped HelD simultaneously penetrates deep into two RNAP channels that are responsible for nucleic acids binding and substrate delivery to the active site, thereby locking RNAP in an inactive state. They show that HelD prevents non-specific interactions between RNAP and DNA and dissociates stalled transcription elongation complexes. The liberated RNAP can either stay dormant, sequestered by HelD, or upon HelD release, restart transcription. Their results provide insights into the architecture and regulation of the highly medically-relevant mycobacterial transcription machinery and define HelD as a clearing factor that releases RNAP from nonfunctional complexes with nucleic acids.

    Kouba, T.; Koval’, T.; Sudzinová, P.; Pospíšil, J.; Brezovská, B.; Hnilicová, J.; Šanderová, H.; Janoušková, M.; Šiková, M.; Halada, P.; Sýkora, M.; Barvík, I.; Nováček, J.; Trundová, M.; Dušková, J.; Skálová, T.; URee Chon; Murakami, K. S.; Dohnálek, J. & Krásný, L.: Mycobacterial HelD is a nucleic acids-clearing factor for RNA polymerase, Nature Comm. (2020) 11, 6419, https://doi.org/10.1038/s41467-020-20158-4

  • Angew. Chem. Int. Edit. 2020

    Angew. Chem. Int. Edit. 2020

    A) Confocal microscopy images of cells transfected with aptamer–ligand complex. The green color indicates the localization of (FAM)-aptamer/(FAM)-aptamer–ligand complex. The blue color corre- sponds to a cell nucleus stained by Hoechst 33342. B) Double-staining (PI/FAM) FCM analysis of transfected HeLa cells with the aptamer– ligand complex. Percentages of a viable non-transfected cells, viable aptamer–ligand complex containing cells, non-transfected dead/com- promised cells, and transfected dead/compromised cells with apta- mer–ligand complex are indicated in left-bottom, right-bottom (red), left-top, and right-top quadrants, respectively. C) Imino region of 1D 1H NMR spectra of aptamer–ligand complex in vitro (TOP) and corresponding spectrum of HeLa cells transfected with aptamer– ligand complex (MIDDLE). Imino region of 1D 1H NMR spectrum of extracellular fluid (supernatant) taken from the in-cell NMR samples after completion of the spectra acquisition (BOTTOM). D) 1D 13C- edited NMR spectra of the aptamer–ligand complex in vitro in EB- buffer (TOP) and corresponding spectra of HeLa cells transfected with aptamer–ligand complex.

    Lukáš Trantírek and Harald Schwalbe Research Groups

    Significance

    L. Trantírek and H. Schwalbe research groups report here the in-cell NMR-spectroscopic observation of the binding of the cognate ligand 2’-deoxyguanosine to the aptamer domain of the bacterial 2’-deoxyguanosine-sensing riboswitch in eukaryotic cells, namely Xenopus laevis oocytes and in human HeLa cells. The riboswitch is sufficiently stable in both cell types to allow for detection of binding of the ligand to the riboswitch. Most importantly, they show that the binding mode established by in vitro characterization of this prokaryotic riboswitch is maintained in eukaryotic cellular environment. Data also bring important methodological insights: Thus far, in-cell NMR studies on RNA in mammalian cells have been limited to investigations of short (< 15 nt) RNA fragments that were extensively modified by protecting groups to limit their degradation in the intra-cellular space. Here, they show that the in-cell NMR setup can be adjusted for characterization of much larger (~ 70 nt) functional and chemically non-modified RNA.

    Broft, P., S.; Dzatko, S.; Krafcikova, M.; Wacker, A.; Hänsel-Hertsch, R.; Dötsch, V.; Trantirek, L. & Schwalbe, H.: In-Cell NMR Spectroscopy of Functional Riboswitch Aptamers in Eukaryotic Cells, Angew. Chem. Int. Edit. 2020, 59, 2-11 doi.org/10.1002/anie.202007184

  • Science 2020

    Science 2020

    (−)-Bactobolin A and selected related natural products.

    (A) Overall structure of ovine complex I. Core subunits necessary for the reaction of complex I are labeled with corresponding colors, and mammalian supernumerary subunits are shown in gray. NADH and quinone binding sites are indicated. The membrane arm contains four separate proton-pumping channels: three in the antiporter-like subunits ND2, ND4, and ND5 and one in the E-channel, composed of subunits ND1, ND6, and ND4L. Q, quinone. 

    Leonid A. Sazanov Research Group

    Significance

    Complex I is the first and, with 45 subunits and a total mass of ~1 MDa, the most elaborate of the mitochondrial electron transfer chain enzymes. Complex I converts energy stored in chemical bonds into a proton gradient across the membrane that drives the synthesis of adenosine triphosphate (ATP), the universal energy currency of the cell. In each catalytic cycle, the transfer of two electrons from nicotinamide adenine dinucleotide (NADH) to a hydrophobic electron carrier quinone, which happens in the peripheral arm of the enzyme, is coupled to the translocation of four protons across the inner mitochondrial membrane in the membrane arm. The exact mechanism of this energy conversion currently presents an enigma because of complex I’s size and the spatial separation between the two reactions.

    To understand the coupling mechanism of complex I, we solved its cryo–electron microscopy (cryo-EM) structures in five different conditions, including the substrate- and inhibitor-bound states and during active turnover, unlocking the various conformations that the enzyme goes through during the catalytic cycle. We also improved the resolution to up to 2.3 to 2.5 Å, allowing us to directly observe water molecules critical for proton pumping.

    Kampjut, D. & Sazanov, L. A. The coupling mechanism of mammalian respiratory complex I, Science, 2020, 370, eabc4209, https://doi.org/10.1126/science.abc4209

Previous 1 2 3 4 5 6 7 11 Next

You are running an old browser version. We recommend updating your browser to its latest version.