CIISB Research Highlights Archive

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

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 Cl^- and 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 

Summary showing CK1ε role in DVL3 conformational dynamics. A summarizing model which proposes at least three DVL conformations in vivo: (i) a closed (CK1ε present and inactive), (ii) open (CK1ε active), and (iii) non-physiological open, which occurs when CK1ε is absent or the DVL-CK1ε interaction is disrupted. Position of insertion of FlAsH III binding tag is indicated. The CK1-induced phosphorylation events are depicted as P in red circle and the C-terminus of DVL as red thick line. The molecular distance analysed in the FRET FlAsH sensor III is shown as a dashed red line; ECFP, enhanced cyan fluorescent protein

Vítězslav Bryja Research Group

Significance

Dishevelled (DVL) is the key component of the Wnt signalling pathway. Currently, DVL conformational dynamics under native conditions is unknown. To overcome this limitation, we develop the Fluorescein Arsenical Hairpin Binder- (FlAsH-) based FRET in vivo approach to study DVL conformation in living cells. Using this single-cell FRET approach, we demonstrate that (i) Wnt ligands induce open DVL conformation, (ii) DVL variants that are predominantly open, show more even subcellular localization and more efficient membrane recruitment by Frizzled (FZD) and (iii) Casein kinase 1 ɛ (CK1ɛ) has a key regulatory function in DVL conformational dynamics. In silico modelling and in vitro biophysical methods explain how CK1ɛ-specific phosphorylation events control DVL conformations via modulation of the PDZ domain and its interaction with DVL C-terminus. In summary, our study describes an experimental tool for DVL conformational sampling in living cells and elucidates the essential regulatory role of CK1ɛ in DVL conformational dynamics. 

Harnoš, J.; Cañizal, M. C. A.; Jurásek, M.; Kumar, J.; Holler, C.; Schambony, A.; Hanáková, K.; Bernatík, O.; Zdráhal, Z.; Gömöryová, K.; Gybel’, T.; Radaszkiewicz, T. W.; Kravec, M.; Trantírek, L.; Ryneš, J.; Dave, Z.; Fernández-Llamazares, A. I.; Vácha, R.; Tripsianes, K.; Hoffmann, C. & Bryja, V.: Dishevelled-3 conformation dynamics analyzed by FRET-based biosensors reveals a key role of casein kinase 1, Nat. Commun. (2019) 10, 1804, 1-18.  doi.org/10.1038/s41467-019-09651-7

Scheme of enterovirus genome release. Binding to receptors or exposure to acidic pH in endosomes induces conformational transition of virions to activated particles. The structural changes within the capsid and virus RNA enable the expulsion of pentamers from the capsid, resulting in the formation of open particles. The RNA genomes are released from the open particles. After the genome release, the pentamers may re-associate with the open capsids. Scale bar represents 10 nm

Pavel Plevka Research Group

Significance

Viruses from the genus Enterovirusare important human pathogens. Receptor binding or exposure to acidic pH in endosomes converts enterovirus particles to an activated state that is required for genome release. However, the mechanism of enterovirus uncoating is not well understood. Here, we use cryo-electron microscopy to visualize virions of human echovirus 18 in the process of genome release. We discover that the exit of the RNA from the particle of echovirus 18 results in a loss of one, two, or three adjacent capsid-protein pentamers. The opening in the capsid, which is more than 120 Å in diameter, enables the release of the genome without the need to unwind its putative double-stranded RNA segments. We also detect capsids lacking pentamers during genome release from echovirus 30. Thus, our findings uncover a mechanism of enterovirus genome release that could become target for antiviral drugs.

Buchta, D.; Füzik, T.; Hrebík, D.; Levdansky, Y.; Sukeník, L.; Mukhamedova, L.; Moravcová, J.; Vácha, R. & Plevka, P: Enterovirus particles expel capsid pentamers to enable genome release, Nature Commun. (2019)10, 1138, 1-9 doi.org/10.1038/s41467-019-09132-x

(A) Model of TvTom40-2 was built using the N. crassa Tom40 structure (PDB ID 5o8o) as a template. The asterisk shows the extra loop between β-strands five and six, and the arrow shows the loop between β-strands four and five. (B) Comparison of 3D structures of N. crassa Tom40 (5o8o), TvTom40-2, and Mus musculus VDAC (3emn). Mouse VDAC is almost uniformly positively charged inside the barrel to bind negatively charged small molecules (ATP), while TvTom40-2 and N. crassa Tom40 share both positively and negatively charged patches inside the barrel. The scale of the electrostatic potential ranges from −5 to +5 kT/e.

Jan Tachezy Research Group

Significance

Mitochondria carry out many vital functions in the eukaryotic cells, from energy metabolism to programmed cell death. These organelles descended from bacterial endosymbionts, and during their evolution, the cell established a mechanism to transport nuclear-encoded proteins into mitochondria. Embedded in the mitochondrial outer membrane is a molecular machine, known as the translocase of the outer membrane (TOM) complex, that plays a key role in protein import and biogenesis of the organelle. Here, we provide evidence that the TOM complex of hydrogenosomes, a metabolically specialised anaerobic form of mitochondria in Trichomonas vaginalis, is composed of highly divergent core subunits and lineage-specific peripheral subunits. Despite the evolutionary distance, the T. vaginalis TOM (TvTOM) complex has a conserved triplet-pore structure but with a unique skull-like shape suggesting that the TOM in the early mitochondrion could have formed three pores. Our results contribute to a better understanding of the evolution and adaptation of protein import machinery in anaerobic forms of mitochondria.

Makki, A.; Rada, P.; Žárský, V.; Kereïche, S.; Kováčik, L.; Novotný, M.; Jores, T.; Rapaport, D. & Tachezy, J.: Triplet-pore structure of a highly divergent TOM complex of hydrogenosomes in Trichomonas vaginalis, Plos Biol. (2019)17, No. 1, doi.org/10.1371/journal.pbio.3000098

SAXS‐based structural modeling of the proC2:14‐3‐3ζ complex. (A) Best‐scoring AllosMod‐FoXS model of the proC2:14‐3‐3ζ complex shown in two perpendicular views. The N‐terminal linker, the p19 and the p12 domains and phosphorylation sites are indicated in brown, salmon, yellow, and red spheres, respectively. The protomers of 14‐3‐3ζ are shown in blue. 14‐3‐3 helices are identified with capital letters, whereas proC2 helices and β‐strands are identified with Greek letters. (B) Intermolecular cross‐links connecting the NLS region of proC2 (Lys¹⁵³) to helices H1 and H3 of 14‐3‐3 (Lys¹¹ and Lys⁶⁸); and the proC2 domain p12 (Lys³⁷², Lys³⁸¹) to the 14‐3‐3ζ helix H3 (Lys⁶⁸). Lysine residues of proC2 are shown in brown. (C) Cross‐links between the N terminus of proC2 (Ser¹²³) and the 14‐3‐3ζ helix H4 and H5/H6 loop (Lys⁷⁵, Lys¹³⁸). Lysine residues of proC2 are shown in brown.

Tomáš Obšil Research Group

Significance

The main goal of this work is to provide the structural basis for the role of 14‐3‐3 protein binding in regulating caspase‐2 activation. Because all our previous attempts to crystallize the complex between Ser139‐ and Ser164‐ phosphorylated caspase‐2 (residues 123–452 without the CARD domain, hereafter referred to as proC2) and 14‐3‐3ζ had been unsuccessful, we decided to use small angle X‐ray scattering (SAXS) combined with NMR, with chemical cross‐linking coupled to MS and with fluorescence spectroscopy to characterize the solution structure and conformational behavior of this complex.

The structural analysis of the 14‐3‐3:caspase‐2 complex reported in this study suggested that 14‐3‐3 protein binding may inhibit caspase‐2 activation through interference with caspase‐2 oligomerization and/or its nuclear localization by sterically occluding caspase‐2 p12 domain as well as NLS, which is bordered by the two phosphorylated 14‐3‐3‐binding motifs of caspase‐2. Thus, these results corroborate the hypothesis that 14‐3‐3 binding is an important regulatory element of caspase‐2 activation. Further research should be directed to study the effect of 14‐3‐3 on the caspase‐2 dimerization and cellular localization in vivo.

Smidova, A; Alblova, M.; Kalabova, D.; Psenakova, K.; Rosulek, M; Herman, P.; Obsil. T. & Obsilova, V.: 14-3-3 protein masks the nuclear localization sequence of caspase-2. Febs Journal 285, 4196-4213, doi:10.1111/febs.14670 (2018).