CIISB Research Highlights Archive

Cryo-EM structure of TEL bound to the yeast ribosome. a Transverse section of the cryo-EM map density (gray) of the large (60S) subunit of the yeast G2400A mutant ribosome with TEL (salmon) bound within the NPET. b Isolated cryo-EM density for TEL (gray mesh) with fitted molecular model for TEL. The similarly-oriented chemical structure of TEL is shown for reference. c TEL bound within the NPET with the surrounding nucleotides of the 25S rRNA (gray) and His133 residue of uL22 protein (purple). The N1 of A2400(A2058) forms a hydrogen bond interaction to the hydroxyl group of the desosamine sugar of TEL and a water molecule (W1) mediates interaction of the desosamine’s dimethylamine with the N6 of A2400(A2058), as was also observed in bacteria43. The alkyl–aryl sidechain of TEL stacks upon the base pair A884(A752)-U2978(U2609) and forms bridging interaction with water W2 (light blue) and O6 of G880(G748). d Superposition of TEL bound to the E. coli ribosome (green, PDB ID 4V7S15) with TEL (salmon) in complex with the S. cerevisiae 60S subunit (Sc60S) bearing the G2400A mutation. E. coli rRNA and ribosomal protein residues are light green, yeast rRNA and protein residues are gray.

Daniel N. Wilson and Alexander S. Mankin Research Group

Significance

Macrolide antibiotics bind in the nascent peptide exit tunnel of the bacterial ribosome and prevent polymerization of specific amino acid sequences, selectively inhibiting translation of a subset of proteins. Because preventing translation of individual proteins could be beneficial for the treatment of human diseases, we asked whether macrolides, if bound to the eukar- yotic ribosome, would retain their context- and protein-specific action. By introducing a single mutation in rRNA, we rendered yeast Saccharomyces cerevisiae cells sensitive to macrolides. Cryo-EM structural analysis showed that the macrolide telithromycin binds in the tunnel of the engineered eukaryotic ribosome. Genome-wide analysis of cellular translation and bio- chemical studies demonstrated that the drug inhibits eukaryotic translation by preferentially stalling ribosomes at distinct sequence motifs. Context-specific action markedly depends on the macrolide structure. Eliminating macrolide-arrest motifs from a protein renders its translation macrolide-tolerant. Our data illuminate the prospects of adapting macrolides for protein-selective translation inhibition in eukaryotic cells.

Svetlov, M. S.; Koller, T. O.; Meydan, S.; Shankar, V.; Klepacki, D.; Polacek, N.; Guydosh, N. R.; Vazquez-Laslop, N.; Wilson, D. N. & Mankin, A. S.: Context-specific action of macrolide antibiotics on the eukaryotic ribosome, Nature Commun. (2021)12:2803 https://doi.org/10.1038/s41467-021-23068-1

The essential fungal‐specific translation elongation factor 3 (eEF3) has been implicated in tRNA binding and release. Combined in vitro and in vivo analyses show that its critical is in release of E‐site‐tRNA from the ribosome during late steps of translocation.

Daniel N. Wilson Research Group

Significance

In addition to the conserved translation elongation factors eEF1A and eEF2, fungi require a third essential elongation factor, eEF3. While eEF3 has been implicated in tRNA binding and release at the ribosomal A and E sites, its exact mechanism of action is unclear. Here, we show that eEF3 acts at the mRNA–tRNA translocation step by promoting the dissociation of the tRNA from the E site, but independent of aminoacyl‐tRNA recruitment to the A site. Depletion of eEF3 in vivo leads to a general slowdown in translation elongation due to accumulation of ribosomes with an occupied A site. Cryo‐EM analysis of native eEF3‐ribosome complexes shows that eEF3 facilitates late steps of translocation by favoring non‐rotated ribosomal states, as well as by opening the L1 stalk to release the E‐site tRNA. Additionally, our analysis provides structural insights into novel translation elongation states, enabling presentation of a revised yeast translation elongation cycle.

Ranjan, N.; Pochopien, A.; Chih-Chien Wu, C.; Beckert, B.; Blanchet, S.; Green, R.; Rodnina, M. & Wilson, D. N.: Yeast translation elongation factor eEF3 promotes late stages of tRNA translocation during RF3-mediated recycling of RF1 EMBO J (2021) 40: e106449; https://doi.org/10.15252/embj.2020106449

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

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. b–g 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

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

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