CIISB Research Highlights Archive

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).

RF3-induced subunit rotation destabilizes RF1 binding. a Cryo-EM map of SSU (light blue) and RF1 (orange) from state III compared with SSU (dark blue) and RF1 (red) from state IV. b Isolated cryo-EM electron densities (grey mesh) with molecular models for RF1 from state III (orange) and state IV (red) shown at the same contour level based on comparison with the SSU density. c Domain 2/4 of RF1 from state III (sIII:RF1, orange) is rotated by 6° and shifted by 4 Å compared to RF1 from state IV (sIV:RF1, red). d, e Contacts (arrowed) between RF1 (orange) and P/P-tRNA (green) are lost upon formation of the hybrid P/E-tRNA (light blue). Amino acids of RF1 that contact P/P-tRNA are shown as spheres. e Zoom of d showing the presence or absence of RF1 contacts with the ASL of P/P- or P/E-tRNA, respectively.

Daniel N. Wilson Research Group

Significance

During translation termination in bacteria, the release factors RF1 and RF2 are recycled from the ribosome by RF3. While high-resolution structures of the individual termination factors on the ribosome exist, direct structural insight into how RF3 mediates dissociation of the decoding RFs has been lacking. Here we have used the Apidaecin 137 peptide to trap RF1 together with RF3 on the ribosome and visualize an ensemble of termination intermediates using cryo-electron microscopy. Binding of RF3 to the ribosome induces small subunit (SSU) rotation and swivelling of the head, yielding intermediate states with shifted P-site tRNAs and RF1 conformations. RF3 does not directly eject RF1 from the ribosome, but rather induces full rotation of the SSU that indirectly dislodges RF1 from its binding site. SSU rotation is coupled to the accommodation of the GTPase domain of RF3 on the large subunit (LSU), thereby promoting GTP hydrolysis and dissociation of RF3 from the ribosome.

Graf, M.; Huter, P.; Maracci, C.; Peterek, M.; Rodnina, M. V. & Wilson D. N.: Visualization of translation termination intermediates trapped by the Apidaecin 137 peptide during RF3-mediated recycling of RF1 Nat. Commun. (2018)9, 3053 doi:10.1038/s41467-018-05465-1

Structure of the MiCP and its interaction with other capsid proteins. (A) Structure of the MiCP shown in stick representation with carbon atoms in magenta. The first and last structured residues of MiCP are labeled. The electron density map of the MiCP contoured at 2σ is shown as a blue mesh. Major capsid proteins are shown in cartoon representation with VP1 in blue, VP2 in green, and VP3 in red. (B and C) Comparison of capsid proteins VP2 of SBV (B) and poliovirus 1 (C). The MiCP of SBV, which is highlighted in magenta (B), occupies the volume of the puff loop in VP2 of poliovirus 1, highlighted in orange (C). (D and E) The VP3 subunit of SBV (D) lacks the knob loop present in poliovirus 1 (E), highlighted in green

Pavel Plevka Research Group

Significance

Infection by sacbrood virus (SBV) from the family Iflaviridae is lethal to honey bee larvae but only rarely causes the collapse of honey bee colonies. Despite the negative effect of SBV on honey bees, the structure of its particles and mechanism of its genome delivery are unknown. Here we present the crystal structure of SBV virion and show that it contains 60 copies of a minor capsid protein (MiCP) attached to the virion surface. No similar MiCPs have been previously reported in any of the related viruses from the order Picornavirales. The location of the MiCP coding sequence within the SBV genome indicates that the MiCP evolved from a C-terminal extension of a major capsid protein by the introduction of a cleavage site for a virus protease. The exposure of SBV to acidic pH, which the virus likely encounters during cell entry, induces the formation of pores at threefold and fivefold axes of the capsid that are 7 Å and 12 Å in diameter, respectively. This is in contrast to vertebrate picornaviruses, in which the pores along twofold icosahedral symmetry axes are currently considered the most likely sites for genome release. SBV virions lack VP4 subunits that facilitate the genome delivery of many related dicistroviruses and picornaviruses. MiCP subunits induce liposome disruption in vitro, indicating that they are functional analogs of VP4 subunits and enable the virus genome to escape across the endosome membrane into the cell cytoplasm.

Procházková, M. et al.: Virion structure and genome delivery mechanism of sacbrood honeybee virus. PNAS 2018, 115 (30) 7759-7764. doi.org/10.1073/pnas.1722018115.

LEDGF/p75 IBD binding partners interact in a structurally conserved manner. Solution structures of the IBD in complex with the binding motifs from POGZ, JPO2 motif 1 and 2 (M1, M2), IWS1, and MLL1 determined by NMR spectroscopy.

Zeger Debyser and Václav Veverka Research Groups

Significance

The transcription coactivator LEDGF/p75 contributes to regulation of gene expression by tethering other factors to actively transcribed genes on chromatin. Its chromatin-tethering activity is hijacked in two important disease settings, HIV and mixed-lineage leukemia; however, the basis for the biological regulation of LEDGF/p75’s interaction to binding partners has remained unknown. This has represented a gap in our understanding of LEDGF/p75’s fundamental biological function and a major limitation for development of therapeutic targeting of LEDGF/p75 in human disease. Our work provides a mechanistic understanding of how the lens epithelium-derived growth factor interaction network is regulated at the molecular level. We reveal that structurally conserved IBD-binding motifs (IBMs) on known LEDGF/p75 binding partners can be regulated by phosphorylation, permitting switching between low- and high-affinity states. Finally, we show that elimination of IBM phosphorylation sites on MLL1 disrupts the oncogenic potential of primary MLL1-rearranged leukemic cells. Our results demonstrate that kinase-dependent phosphorylation of MLL1 represents a previously unknown oncogenic dependency that may be harnessed in the treatment of MLL-rearranged leukemia.

Sharma, S. et. al.: Affinity switching of the LEDGF/p75 IBD interactome is governed by kinase-dependent phosphorylation, PNAS 2018, 115 (30) E7053-E7062.doi.org/10.1073/pnas.1803909115

Interaction of TBEV virions with Fab fragments of neutralizing antibody 19/1786. a Cryo-EM micrograph of TBEV virions incubated with Fab fragments of 19/1786. Scale bar represents 100 nm. b Electron-density map of Fab-covered TBEV virion. c Molecular surface of TBEV virion covered with Fab 19/1786 fragments low-pass filtered to 7 Å resolution. E-proteins are shown in red, green, and blue. Fab fragments are shown in magenta (heavy chain) and pink (light chain). Scale bars in band c represent 10 nm. d Footprints of Fab 19/1786 on TBEV surface. e The Fab 19/1786 binds to the domain III at an angle of 135° relative to the axis of the E-protein ectodomain.

Pavel Plevka Research group

Significance

Tick-borne encephalitis virus (TBEV) causes 13,000 cases of human meningitis and encephalitis annually. However, the structure of the TBEV virion and its interactions with antibodies are unknown. Here, Pavel Plevka and his coworkers present cryo-EM structures of the native TBEV virion and its complex with Fab fragments of neutralizing antibody 19/1786. Flavivirus genome delivery depends on membrane fusion that is triggered at low pH. The virion structure indicates that the repulsive interactions of histidine side chains, which become protonated at low pH, may contribute to the disruption of heterotetramers of the TBEV envelope and membrane proteins and induce detachment of the envelope protein ectodomains from the virus membrane. The Fab fragments bind to 120 out of the 180 envelope glycoproteins of the TBEV virion. Unlike most of the previously studied flavivirus-neutralizing antibodies, the Fab fragments do not lock the E-proteins in the native-like arrangement, but interfere with the process of virus-induced membrane fusion. 

Füzik, T. et al. Structure of tick-borne encephalitis virus and its neutralization by a monoclonal antibody. Nature Communications 9, 11, doi:10.1038/s41467-018-02882-0 (2018)

Structure of PHL complex with propargyl a-l-fucoside. (A) PHL monomer (chain A) overall architecture with propargyl a-l-fucoside shown as magenta sticks. Individual binding sites are labelled in black (front plane) or grey (back plane) (B) Side view of PHL dimer with chain B shown in grey and without ligands. (C) Individual PHL fucose‐type binding sites with Compound 5 (magenta) bound. Amino acids responsible in ligand binding are highlighted and labelled. (D) PHL galactose‐type binding site with propargyl a-l-fucoside (magenta) bound and PHL galactose‐type binding site with d‐Gal (yellow) bound (PDB ID 5MXH). Colour code for panels C/D: amino acids involved in ligand‐binding through H‐bond=cyan, CH–π interaction=orange, or water bridge=grey.

Michaela Wimmerova Research Group

Significance

Photorhabdus asymbiotica is a gram‐negative bacterium that is not only as effective an insect pathogen as other members of the genus, but it also causes serious diseases in humans. The recently identified lectin PHL from P. asymbiotica verifiably modulates an immune response of humans and insects, which supports the idea that the lectin might play an important role in the host–pathogen interaction. Dimeric PHL contains up to seven l‐fucose‐specific binding sites per monomer, and in order to target multiple binding sites of PHL, α‐l‐fucoside‐containing di‐, tri‐ and tetravalent glycoclusters were synthesized. The interaction between fucoside derivates and PHL was investigated by several biophysical and biological methods, ITC and SPR measurements, hemagglutination inhibition assay, and an investigation of bacterial aggregation properties were carried out. Details of the interaction between PHL and propargyl α‐l‐fucoside as a monomer unit were revealed using X‐ray crystallography. Besides this, the interaction with multivalent compounds was studied by NMR techniques. The newly synthesized multivalent fucoclusters proved to be up to several orders of magnitude better ligands than the natural ligand, l‐fucose.

Jancarikova, G. et al. Synthesis of a-l-Fucopyranoside-Presenting Glycoclusters and Investigation of Their Interaction with Photorhabdus asymbiotica Lectin (PHL). Chemistry – A European Journal, 24, 4055-4068, doi.org/10.1002/chem.201705853