EMBO REP- 2017
NMR structure shows how the CTD-interacting domain of Rtt103p recognizes threonine-4 phosphorylated CTD of RNA polymerase II (RNAPII).
Phosphorylation patterns of the C‐terminal domain (CTD) of largest subunit of RNApolymerase II (called the CTD code) orchestrate the recruitment of RNA processing and transcription factors. Recent studies showed that not only serines and tyrosines but also threonines of the CTD can be phosphorylated with a number of functional consequences, including the interaction with yeast transcription termination factor, Rtt103p. The solution structure of the Rtt103p CTD‐interacting domain (CID) bound to Thr4 phosphorylated CTD has been obtained by NMR. The structure reveals a direct recognition of the phospho‐Thr4 mark by Rtt103p CID and shows extensive interactions involving residues from three repeats of the CTD heptad. The structural data suggests that the recruitment of a CID‐containing CTD‐binding factor may be coded by more than one letter of the CTD code.
Jasnovidova, O., Krejcikova, M., Kubicek, K. & Stefl, R. Structural insight into recognition of phosphorylated threonine-4 of RNA polymerase II C-terminal domain by Rtt103p. Embo Reports 18, 906-913, doi:10.15252/embr.201643723 (2017).
Proc. Natl. Acad. Sci. U. S. A. 2017
The virus structure of deformed wing virus of honeybees determined by cryo-electron microscopy. (A) Surface of the virus is rainbow-colored according to its distance from the particle center. (B) Cartoon representation of structure of the P-domain that decorates deformed wing virus surface is rainbow-colored from residue 260 in blue to 416 in red. The background shows image of the deformed wing virus particles from electron microscope.
Honey bee populations in Europe and North America have been decreasing since the 1950s. Deformed wing virus (DWV), which is undergoing a worldwide epidemic, causes the deaths of individual honey bees and collapse of whole colonies. Three-dimensional structures of DWV determined at different conditions shows that the virus surface is decorated with protruding globular extensions of capsid proteins. The protruding domains contain a putative catalytic site that is probably required for the entry of the virus into the host cell. In addition, parts of the DWV RNA genome interact with the inside of the virus capsid. Identifying the RNA binding and catalytic sites within the DWV virion offers prospects for the development of antiviral treatments.
Skubnik, K. ; Novacek, J.; Fuezik, T.; Pridal, A.; Paxton, R. J. ; Plevka, P., Structure of deformed wing virus, a major honey bee pathogen, PNAS, 114, 3210-3215 (2017) DOI: 10.1073/pnas.1615695114
Nucleic Acids Res. 2017
Cryo-EM structure of the spinach chloroplast ribosome reveals the location of plastid-specific ribosomal proteins and extensions.
Ribosomes are the protein synthesizing machines of the cell. Recent advances in cryo-EM have led to the determination of structures from a variety of species, including bacterial 70S and eukaryotic 80S ribosomes as well as mitoribosomes from eukaryotic mitochondria, however, to date high resolution structures of plastid 70S ribosomes have been lacking. Here we present a cryo-EM structure of the spinach chloroplast 70S ribosome, with an average resolution of 5.4 Å for the small 30S subunit and 3.6 Å for the large 50S ribosomal subunit. The structure reveals the location of the plastid-specific ribosomal proteins (RPs) PSRP1, PSRP4, PSRP5 and PSRP6 as well as the numerous plastid-specific extensions of the RPs. We discover many features by which the plastid-specific extensions stabilize the ribosome via establishing additional interactions with surrounding ribosomal RNA and RPs. Moreover, we identify a large conglomerate of plastid-specific protein mass adjacent to the tunnel exit site that could facilitate interaction of the chloroplast ribosome with the thylakoid membrane and the protein-targeting machinery. Comparing the Escherichia coli 70S ribosome with that of the spinach chloroplast ribosome provides detailed insight into the co-evolution of RP and rRNA.
Graf, M., Arenz, S., Huter, P., Dönhöfer, A., Nováček, J., and Wilson D.N.: Cryo-EM structure of the spinach chloroplast ribosome reveals the location of plastid-specific ribosomal proteins and extensions. Nucleic Acids Research 45, 2887-2896, doi:10.1093/nar/gkw1272 (2017).
Proc. Natl. Acad. Sci. U. S. A. 2017
A model of RNA polymerase II bound to the transcription termination factor Rtt103. The structural model was created using integrative structural biology.
RNA polymerase II (RNAPII) not only transcribes protein coding genes and many noncoding RNA, but also coordinates transcription and RNA processing. This coordination is mediated by a long C-terminal domain (CTD) of the largest RNAPII subunit, which serves as a binding platform for many RNA/protein-binding factors involved in transcription regulation. In this work, we used a hybrid approach to visualize the architecture of the full-length CTD in complex with the transcription termination factor Rtt103. Specifically, we first solved the structures of the isolated subcomplexes at high resolution and then arranged them into the overall envelopes determined at low resolution by small-angle X-ray scattering. The reconstructed overall architecture of the Rtt103–CTD complex reveals how Rtt103 decorates the CTD platform.
Jasnovidova, O.; Klumpler, T.; Kubicek, K.; Kalynych, S.; Plevka, P.; Stefl, R. PNAS,Structure and dynamics of the RNAPII CTDsome with Rtt103, PNAS 2017 114 (42) 11133-11138; doi:10.1073/pnas.1712450114
J. Am. Chem. Soc. 2017
High-resolution structure of a stable G-hairpin calculated from NMR data (PDB ID: 5M1W). (A) Ten lowest-energy structures. Loop residues are colored orange and O4′ atoms are colored red. (B) Schematic representation of hairpin folding topology. Chain reversal arrangement of the backbone and the 3′-to-5′ stacking of the terminal residues are indicated by dark green and magenta arrows, respectively. Anti and syn guanines that form G:G base pairs are colored dark and light blue, respectively.
The first atomic resolution structure of a stable G-hairpin formed by a natively occurring DNA sequence is reported. An 11-nt long G-rich DNA oligonucleotide, 5′-d(GTGTGGGTGTG)-3′, corresponding to the most abundant sequence motif in irregular telomeric DNA from Saccharomyces cerevisiae adopts a novel type of mixed parallel/antiparallel fold-back DNA structure, which is stabilized by dynamic G:G base pairs that transit between N1-carbonyl symmetric and N1-carbonyl, N7-amino base-pairing arrangements. The structure reveals previously unknown principles of the folding of G-rich oligonucleotides that could be applied to the prediction of natural and/or the design of artificial recognition DNA elements. The structure also demonstrates that the folding landscapes of short DNA single strands is much more complex than previously assumed.
Gajarsky, M.; Zivkovic, M. L. ; Stadlbauer, P. ; Pagano, B.; Fiala, R.; Amato, J.; Tomaska, L.; Sponer, J.; Plavec, J.; Trantirek, L. Structure of a Stable G-Hairpin JACS 139, 3591-3594, doi:10.1021/jacs.6b10786 (2017)
J. Biol. Chem. 2017
Structural changes in crystal structure of of AfGcHK sensor protein induced by sodium dithionite soaking (PDB ID 5OHF) and conformational changes revealed by HDX-MS. (A) Two protein chains of the heme domain of the sensor protein observed in crystal structure including alternative B (yellow) occurring with dithionite soaking. (B) Conformational changes revealed by HDX-MS after 60 min of deuteration of the full-length AfGcHK proteins visualized on the protein structure. Differences between the Fe(III)-OH- form (active) and inactive Fe(II) form are color coded: grey - no difference, red - higher and blue - lower levels of deuteration.
The heme-based oxygen sensor histidine kinase AfGcHK is part of a two-component signal transduction system in bacteria. O2 binding to the Fe(II) heme complex of its N-terminal globin domain strongly stimulates autophosphorylation at His-183 in its C-terminal kinase domain. The 6-coordinate heme Fe(III)-OH- and -CN- complexes of AfGcHK are also active, but the 5-coordinate heme Fe(II) complex and the heme-free apo-form are inactive. The crystal structures of the isolated dimeric globin domains of the active Fe(III)-CN- and inactive 5-coordinate Fe(II) forms were determined, revealing striking structural differences on the heme-proximal side of the globin domain. Using hydrogen/deuterium exchange coupled with mass spectrometry (HDX–MS) the intramolecular signal transduction mechanisms was investigated in full length AfGcHK. The results suggest that structural changes at the heme proximal side, the globin domain-dimerization interface, and the ATP-binding site are important in the signal transduction mechanism of AfGcHK. For the first time, the conformational changes associated with signal transduction were studied in a full-length globin-coupled oxygen sensor protein and linked to directly observed structural changes in the globin domain.
Stranava, M.; Man, P; Skálová, T.; Kolenko, P; Blaha, J.; Fojtikova, V .; Martínek, V.; Dohnálek, J.; Lengalova, A.; Rosůlek, M.; Shimizu, T.; Martínková, M.: Coordination and redox state-dependent structural changes of the heme-based oxygen sensor AfGcHK associated with intraprotein signal transduction. J. Biol. Chem. first Published on November 1, 2017, doi: 10.1074/jbc.M117.817023jbc.M117.817023.
Mol. Cell 2017
Cryo-EM Structures of Polyproline-Stalled Ribosomes in the Presence of EF-P (A-C) Schematic representation (A) and cryo-EM reconstructions (B and C) of PPP-stalled ribosome complexes with (B) or without (C) of EF-P (salmon) bound in the E site. (D and E) Cryo-EM density (mesh) of the CCA end of the P-site tRNA (green) from cryo-EM maps in (C) without EF-P (D) and in (B) with EF-P (E), respectively, with aligned fMet (cyan, PDB: 1VY4) (Polikanov et al., 2014).
Ribosomes synthesizing proteins containing consecutive proline residues become stalled and require rescue via the action of uniquely modified translation elongation factors, EF-P in bacteria, or archaeal/eukaryotic a/eIF5A. To date, no structures exist of EF-P or eIF5A in complex with translating ribosomes stalled at polyproline stretches, and thus structural insight into how EF-P/eIF5A rescue these arrested ribosomes has been lacking. Here we present cryo-EM structures of ribosomes stalled on proline stretches, without and with modified EF-P. The structures suggest that the favored conformation of the polyproline-containing nascent chain is incompatible with the peptide exit tunnel of the ribosome and leads to destabilization of the peptidyl-tRNA. Binding of EF-P stabilizes the P-site tRNA, particularly via interactions between its modification and the CCA end, thereby enforcing an alternative conformation of the polyproline-containing nascent chain, which allows a favorable substrate geometry for peptide bond formation.
Huter, P., Arenz, S., Bock, L.V., Frister, J.O., Heuer, A., Peil, L., Starosta, A.L., Peske, F., Nováček, J., Berninghausen, O., Grubmüller, H., Tenson, T., Beckmann, R., Rodina, M.V., Vaiana, A.C., and Wilson D.N.: Structural Basis for Polyproline-Mediated Ribosome Stalling and Rescue by the Translation Elongation Factor EF-P, Moll Cell 68, No. 3., 515-527.e6 DOI: dx.doi.org/10.1016/j.molcel.2017.10.014
ANGEW. CHEM. INT. EDIT. 2018
Schematic of an intramolecular A) i-motif DNA structure and B) C.C+ base pair. C) Double-staining (PI/FAM) FCM analysis of transfected HeLa cells with the (FAM)-DAP construct (upper left corner). Percentages of viable DNA non-transfected cells, viable DNA-containing cells, non-transfected dead/compromised cells, and transfected dead/compromised cells with DNA are indicated in left-bottom, right-bottom, left-top, and right-top quadrants, respectively. Confocal microscope images of cells transfected with (FAM)-DAP (upper right corner). The green color indicates the localization of (FAM)-DAP. The blue color corresponds to a cell nucleus stained by Hoechst 33342. Imino region of 1D 1H NMR spectra of DAP in vitro in T-buffer (140 mm sodium phosphate, 5 mm KCl, 10 mm MgCl2, pH 7.0) (black) and in-cell (red). Imino region of 1D 1H NMR spectrum of extracellular fluid taken from the in-cell NMR samples after completion of the spectra acquisition (gray). The (in-cell) NMR spectra were acquired at 20oC.
C-rich DNA has the capacity to form a tetra-stranded structure known as an i-motif. The i-motifs within genomic DNA have been proposed to contribute to the regulation of DNA transcription. However, direct experimental evidence for the existence of these structures in vivo has been missing. Whether i-motif structures form in complex environment of living cells is not currently known. Using state-of- the-art in-cell NMR spectroscopy, Lukáš Trantírek and his colleagues from CEITEC Masaryk University in Brno has evaluated the stabilities of i-motif structures in the complex cellular environment. They showed that i-motifs formed from naturally occurring C-rich sequences in the human genome are stable and persist in the nuclei of living human cells. The obtained data show that i-motif stabilities in vivo are generally distinct from those in vitro. Results are the first to interlink the stability of DNA i-motifs in vitro with their stability in vivo and provide essential information for the design and development of i-motif-based DNA biosensors for intracellular applications.
Dzatko, S., Krafcikova, M., Hänsel-Hertsch, R., Fessl, T., Fiala, R., Loja, T., Krafcik, D., Mergny, J-L., Foldynova-Trantirkova, S., and Trantirek, L.: Evaluation of the Stability of DNA i-Motifs in the Nuclei of Living Mammalian Cells, Angew. Chem. Int. Edit. 2018, in press, DOI: 10.1002/anie.201712284
NAT. COMMUN. 2018
Automated structure determination using 4D-CHAINS/autoNOE-Rosetta. (a) Logo of 4D-CHAINS algorithm depicting its powerfulness. Chains squeeze the NMR spectrometer to unleash high-quality structures by using a minimal set of 4D spectra and fully automated data analysis. (b) 4D-CHAINS utilizes two complementary experimental datasets, a 4D-TOCSY and a 4D-NOESY, to yield correct assignments for at least 95% of residues and an error rate of less than 1.5% (middle bar; TOCSY-NOESY). (c-d) Performance of different 4D-CHAINS assignment scenarios for a 20 kDa protein structure, α-lytic protease, calculated using autoNOE-Rosetta. (c) Goodness of structure ensembles is measured using the Rosetta all-atom energy function, backbone heavy atom RMSD to X-ray structure and degree of structural convergence. (d) Lowest-energy structures in each ensemble colored as the points in (c) superimposed on the X-ray reference structure (gray).
The automation of NMR structure determination remains a significant bottleneck towards increasing the throughput and accessibility of NMR as a structural biology tool to study proteins. The chief barrier currently is that obtaining NMR assignments at sufficient levels of completeness to accurately define the structures by conventional methods requires a significant amount of spectrometer time (several weeks), and effort by a trained expert (up to several months). Here, we describe 4D-CHAINS/autoNOE-Rosetta, a complete pipeline for NOE-driven structure determination of medium- to larger-sized proteins. The 4D-CHAINS algorithm analyzes two 4D spectra in an iterative ansatz where common NOEs between different spin systems supplement conventional through-bond connectivities to establish assignments of sidechain and backbone resonances at high levels of completeness and with a minimum error rate. The 4D-CHAINS assignments are then used to guide automated assignment of long-range NOEs and structure refinement in autoNOE-Rosetta. Our results on four targets ranging in size from 15.5 to 27.3 kDa illustrate that the NMR structures of proteins can be determined accurately and in an unsupervised manner in a matter of days.
4D-CHAINS software is free for non-commercial usage and can be downloaded from https://github.com/tevang/4D-CHAINS
Evangelidis, T. et al. Automated NMR resonance assignments and structure determination using a minimal set of 4D spectra. Nature Communications 9, 13, doi:10.1038/s41467-017-02592-z (2018).
CHEMISTRY – A EUROPIAN JOURNAL 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.
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
NAT. COMMUN. 2018
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.
Fuzik, 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). doi:10.1038/s41467-018-02882-0
Proc. Natl. Acad. Sci. U. S. A. 2018
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.
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
Proc. Natl. Acad. Sci. U. S. A. 2018
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
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.
NAT. COMMUN. 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.
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., and 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
THE FEBS JOURNAL 2018
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.
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., and 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).
PLOS BIOLOGY 2019
(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.
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., and 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
NAT. COMMUN. 2019
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
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., and 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
NAT. COMMUN. 2019
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
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., and 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
Nature Index Journal
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.
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 bambusuril macrocycles functionalized with fluorinated benzyl groups, which are ableto exchange Cl-and HCO3-efficiently. Remarkably, rates for Cl-/NO3- exchangeare 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. and 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
SCIENCE ADVANCES 2019
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.
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., and 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
Anal. Chem. 2019
Utility of phenylhydrazine (PHN) labeling for structural studies of fucosylated N-glycans by tandem MALDI mass spectrometry (MS) in the positive ion mode is proposed. PHN-tag influences the production of specific ion types, and the MS/MS fragmentation pattern provides useful structural information.
Fucosylation is a common modification, and its site in glycans refers to different normal and pathological processes. Despite intensive research, there is still a lack of methods to discriminate unambiguously the fucose position in one-step. In this work, we propose utility of phenylhydrazine (PHN) labeling for structural studies of fucosylated N-glycans by tandem MALDI mass spectrometry (MS) in the positive ion mode. PHN-tag influences the production of specific ion types, and the MS/MS fragmentation pattern provides useful structural information. All types of core fucosylated N-glycans have produced two abundant ions consistent with B- and C-glycosidic cleavages corresponding to the loss of the FucGlcNAcPHN residue with a mass 457 and 441 Da from the parent ions. These types of fragment ions in N-glycans without a core fucose were associated with the loss of the GlcNAcPHN unit (311 and 295 Da), and fucose cleavage followed the loss of the chitobiose residue. Since diagnostic useful cleavages produce peaks with significant intensities, this approach is also beneficial for rapid recognition of antenna from core fucosylation in glycans detected with low abundances. The practical applicability of the approach is demonstrated on the analysis of multifucosylated N-glycans detected with lower abundances in lung cancer samples
Lattová, E., Skřičková, J., and Zdráhal, Z. Applicability of Phenylhydrazine Labeling for Structural Studies of Fucosylated N-Glycans, Anal. Chem. 2019 91, 13,7985-7990. https://doi.org/10.1021/acs.analchem.9b01321
Proc. Natl. Acad. Sci. U.S.A. 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.
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., and 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.https://doi.org/10.1073/pnas.1904732116
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.
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 DNA−ligand 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., and 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
Science Advances 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).
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., and 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
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.
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., and 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
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.
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., and Tars, K.: Encapsulation mechanisms and structural studies of GRM2 bacterial microcompartment particles, Nature Comm. (2020) 11 (1), No. 388, doi.org/10.1038/s41467-019-14205-y
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.
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., and Janata, J.: Different Reaction Specificities of F420H2‑Dependent Reductases Facilitate Pyrrolobenzodiazepines and Lincomycin To Fit Their Biological Targets, J. Am. Chem. Soc. 2020, 142, 3440-3448, https://dx.doi.org/10.1021/jacs.9b11234
Nucleic Acids Res. 2020
Structure of STAU1 dsRBD4 and dsRBD3/4 in complex with sARF1 SBS dsRNA. (A) Structural ensemble of the STAU1 dsRBD4–sARF1 SBS dsRNA complex. Heavy-atom superposition of the ensemble of the 20 lowest-energy structures. The protein backbone is shown in dark green and the RNA heavy atoms of the bases in orange and those of the ribose-phosphodiester backbone are shown in gold (omitting phosphate and 2’-OH oxygens). (B) Schematic representation of sARF1 SBS dsRNA showing interactions of dsRBD3 as well as dsRBD4 as dotted lines. Interactions with the ribose-phosphodiester backbone are circled in dark green for dsRBD4 and light green for dsRBD3 while base interactions are shown as filled circles.
Staufen1 (STAU1) is a dsRNA binding protein mediating mRNA transport and localization, translational control and STAU1-mediated mRNA decay (SMD). The STAU1 binding site (SBS) within human ADP-ribosylation factor1 (ARF1) 3’ UTR binds STAU1 and this downregulates ARF1 cytoplasmic mRNA levels by SMD. However, how STAU1 recognizes specific mRNA targets is still under debate. The structure of the ARF1 SBS–STAU1 complex, presented in this study, uncovers target recognition by STAU1. STAU1 dsRNA binding domain (dsRBD) 4 interacts with two pyrimidines and one purine from the minor groove side via helix a1, the b1–b 2 loop anchors the dsRBD at the end of the dsRNA and lysines in helix a2 bind to the phosphodiester backbone from the major groove side. STAU1 dsRBD3 displays the same binding mode with specific recognition of one guanine base. Mutants disrupting minor groove recognition of ARF1 SBS affect in vitrobinding and reduce SMD in vivo. Our data thus reveal how STAU1 recognizes minor groove features in dsRNA relevant for target selection.
Yadav, DP, Zigáčková, D., Zlobina, M., Klumpler, Beaumont, TC., Kubíčková, M., Vaňáčová, Š, and Lukavsky, PJ.: Staufen1 reads out structure and sequence features in ARF1 dsRNA for target recognition, Nucleic Acids Res. 2020, 48, 2091-2106, doi:10.1093/nar/gkz1163
J. Am. Chem. Soc. 2020
(−)-Bactobolin A and selected related natural products.
(−)-Bactobolin A (1) is a polyketide–peptide natural product first isolated in the late 1970s as a secondary metabolite of Pseudomonas sp. Early biological evaluations revealed that 1 exhibits broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens and in vivo antiproliferative effects on certain cancer cell lines. (−)-Bactobolin A (1) was subsequently identified as a strong inhibitor of protein synthesis in intact prokaryotic and eukaryotic cells, and mammalian cell-free systems.
A stereoselective synthesis of the ribosome-binding antitumor antibiotic (−)-bactobolin A is reported. The presented approach makes effective use of (−)-quinic acid as a chiral pool starting material and substrate stereocontrol to establish the five contiguous stereocenters of (−)-bactobolin A. The key steps of the synthesis include a stereoselective vinylogous aldol reaction to introduce the unusual dichloromethyl substituent, a completely diastereoselective rhodium(II)-catalyzed C–H amination reaction to set the configuration of the axial amine, and an intramolecular alkoxycarbonylation to build the bicyclic lactone framework. The developed synthetic route was used to prepare 90 mg of (−)-bactobolin A trifluoroacetate in 10% overall yield.
Vojáčková, P., Michalska, L., Nečas, M., Shcherbakov, D., Böttger, E. C., Šponer, J., Šponer, J.E., and Švenda, J Stereocontrolled Synthesis of (−)-Bactobolin A, J. Am. Chem. Soc. 2020, 142, 7306-7311, doi.org/10.1021/jacs.0c01554
The NMR structure of the self-processing module (SPM) of the Neisseria meningitidis FrpC protein. (A) Schematic representation of the Ca2+-dependent clip-and-link of FrpC. The Ca2+-induced folding of FrpC is associated with a Ca2+-dependent conformational switch of SPM (residues 415 to 591 of FrpC, in orange), which promotes autocatalytic processing of the D414-P415 peptide bond and covalent linkage of the released D414 residue to an e-amino group of a neighboring lysine residue through an Asp-Lys isopeptide bond. The residues of the putative EF-hand-like Ca2+-binding motifs are underlined. (B) Overlay of the 1H-15N HSQC spectra of 15N-labeled SPM in the absence (-) and in the presence (+) of 10 mM CaCl2. (C) Overlay of backbone traces of the 20 lowest energy structures of Ca-SPM solved by NMR, shown in a rainbow representation from blue (N terminus) to red (C terminus). (D) Topology of secondary structure elements of Ca-SPM generated by Pro-origami.
The posttranslational Ca2+-dependent “clip-and-link” activity of large repeat-in-toxin (RTX) proteins starts by Ca2+-dependent structural rearrangement of a highly conserved self-processing module (SPM). Subsequently, an internal aspartate-proline (Asp-Pro) peptide bond at the N-terminal end of SPM breaks, and the liberated C-terminal aspartyl residue can react with a free e-amino group of an adjacent lysine residue to form a new isopeptide bond. Here, we report a solution structure of the calcium-loaded SPM (Ca-SPM) derived from the FrpC protein of Neiseria meningitidis. The Ca-SPM structure defines a unique protein architecture and provides structural insight into the autocatalytic cleavage of the Asp-Pro peptide bond through a “twisted-amide” activation. Furthermore, in-frame deletion of the SPM domain from the ApxIVA protein of Actinobacillus pleuropneumoniae attenuated the virulence of this porcine pathogen in a pig respiratory challenge model. We hypothesize that the Ca2+-dependent clip-and-link activity represents an unconventional strategy for Gram-negative pathogens to adhere to the host target cell surface.
Kuban, V., Macek, P., Hritz, J., Nechvatalova, K., Nedbalcova, K., Faldyna, M., Zidek, L, and Bumba, L.: Structural Basis of Ca2-Dependent Self-Processing Activity of Repeat-in-Toxin Proteins, mBio. 2020, 11:e00226-20, https://doi.org/10.1128/mBio.00226-20.
Int. J. Biol. Macromol. 2020
Crystal structure of BP39L and CV39L. (A) Crystal structure of BP39L shown in secondary structure representation (β-strands in yellow and loops in green) (B) Surface representation (yellow) of BP39L color-coded according to putative binding sites (blue). (C) Crystal structure of CV39L shown in secondary structure representation (β-strands in violet, α-helices in cyan and loops in pink). (D) Surface representation (violet) of CV39L color-coded according to putative binding sites (blue), α-helices shown in cyan.
Burkholderia pseudomallei and Chromobacterium violaceum are bacteria of tropical and subtropical soil and water that occasionally cause fatal infections in humans and animals. Microbial lectins mediate the adhesion of organisms to host cells, which is the first phase in the development of infection. Here we report the discovery of two novel lectins from the above-mentioned bacteria – BP39L and CV39L. The crystal structures revealed that the lectins possess a seven-bladed β-propeller fold. Functional studies conducted on a series of oligo- and polysaccharides confirmed the preference of BP39L for mannosylated saccharides and CV39L for rather more complex polysaccharides with a monosaccharide preference for β-l-fucose. The presented data indicate that the proteins belong to a currently unknown family of lectins.
Sykorova, P., Novotna, J. Demo, G., Pompidor, G., Dubska, E., Komarek, J., Fujdiarova, E., Houser, J., Haronikova, L., Varrot, A., Shilova, N., Imberty, A., Bovin, N., Pokorna, M., and Wimmerova, M.: Characterization of novel lectins from Burkholderia pseudomallei and Chromobacterium violaceum with seven-bladed beta-propeller fold, Int. J. Biol. Macromol. 2019, 152, 1113-1124, doi.org/10.1016/j.ijbiomac.2019.10.200
Structure of the RcGTA particle and organization of segments of the R. capsulatus genome encoding protein components of RcGTA particles. a Cryo-EM reconstruction of a native particle of RcGTA from R. capsulatus strain DE442 calculated from 42,242 particle images. The left part of the panel shows the complete particle, whereas on the right the front half of the particle has been removed to show DNA and internal proteins. Individual proteins in the density map are colored according to the gene map in panel b. Yellow mesh highlights the structural organization of capsid proteins within the RcGTA head. The inset shows an example of a two-dimensional class average and an electron micrograph of an RcGTA particle. The scale bar within the inset represents 20 nm. b Gene map of three genome segments encoding fourteen structural proteins of RcGTA particles. c Cryo-EM reconstruction of an RcGTA particle from R. capsulatus strain DE442 with T = 3 quasi-icosahedral head. The reconstruction is based on 1076 particle images. The structure is at the scale of those shown in panel a. The inset shows an example of a two-dimensional class average and an electron micrograph of RcGTA particle with an icosahedral head. Scale bar represents 20 nm. d Organization of capsomers in the oblate capsid of RcGTA. Capsomers forming one fifth of the capsid are highlighted in different colors and marked with P for pentamer and H for hexamer.
Alphaproteobacteria, which are the most abundant microorganisms of temperate oceans, produce phage-like particles called gene transfer agents (GTAs) that mediate lateral gene exchange. However, the mechanism by which GTAs deliver DNA into cells is unknown. Here we present the structure of the GTA of Rhodobacter capsulatus (RcGTA) and describe the conformational changes required for its DNA ejection. The structure of RcGTA resembles that of a tailed phage, but it has an oblate head shortened in the direction of the tail axis, which limits its packaging capacity to less than 4,500 base pairs of linear double-stranded DNA. The tail channel of RcGTA contains a trimer of proteins that possess features of both tape measure proteins of long-tailed phages from the family Siphoviridae and tail needle proteins of short-tailed phages from the family Podoviridae. The opening of a constriction within the RcGTA baseplate enables the ejection of DNA into bacterial periplasm.
Bárdy,P., Füzik, T., Hrebík, D., Pantůček, R., Beatty, T.J. and Plevka, P: Structure and mechanism of DNA delivery of a gene transfer agent, Nat. Commun. (2020) 11, 3034.doi.org/10.1038/s41467-020-16669-9
Schematic illustration of the TRAK1-mediated anchoring of KIF5B. a Top: in absence of TRAK1, KIF5B (green) can either continue its walk by rebinding the disengaged motor domain to the microtubule or dissociate from the microtubule when the engaged motor domain unbinds from the microtubule. Bottom: in presence of microtubule-bound TRAK1 (magenta), when both motor domains of KIF5B disengage from the microtubule, KIF5B remains tethered to the microtubule through a diffusive interaction of TRAK1 with the microtubule and thereby enables the rebinding of a motor domain of KIF5B to the microtubule. In this state, TRAK1 might facilitate navigation around obstacles by diffusion along the microtubule surface. b Overview of the functions of TRAK1. Top: TRAK1 activates auto-inhibited KIF5B, enabling its processive movement along microtubules. Middle: TRAK1 increases the processivity of KIF5B in crowded environments. Bottom: TRAK1 enables KIF5B-based transport of isolated mitochondria along microtubules in vitro.
Intracellular trafficking of organelles, driven by kinesin-1 stepping along microtubules, underpins essential cellular processes. In absence of other proteins on the microtubule surface, kinesin-1 performs micron-long runs. Under crowding conditions, however, kinesin-1 motility is drastically impeded. It is thus unclear how kinesin-1 acts as an efficient transporter in intracellular environments. Here, we demonstrate that TRAK1 (Milton), an adaptor protein essential for mitochondrial trafficking, activates kinesin-1 and increases robustness of kinesin- 1 stepping on crowded microtubule surfaces. Interaction with TRAK1 i) facilitates kinesin-1 navigation around obstacles, ii) increases the probability of kinesin-1 passing through cohesive islands of tau and iii) increases the run length of kinesin-1 in cell lysate. We explain the enhanced motility by the observed direct interaction of TRAK1 with microtubules, pro- viding an additional anchor for the kinesin-1-TRAK1 complex. Furthermore, TRAK1 enables mitochondrial transport in vitro. We propose adaptor-mediated tethering as a mechanism regulating kinesin-1 motility in various cellular environments.
Henrichs, V., Grycova, L., Bařinka, C., Nahačka, Z., Neužil, J., Diez, S., Rohlena, J., Braun, M., and Lánský, Z.:Mitochondria-adaptor TRAK1 promotes kinesin-1 driven transport in crowded environments, Nat. Commun. (2020) 11, 3123, https://doi.org/10.1038/s41467-020-16972-5
Sinefungin recognition by the nsp16 MTase. A) SARS CoV-2 nsp10-nsp16 protein complex bound to sinefungin (white sticks), nsp16 in surface representation (cyan), nsp10 in cartoon representation (orange) and zinc ions as gray spheres. B) Detailed view of sinefungin recognition, important amino acid residues are shown in stick representation, water as red spheres and hydrogen bonds are shown as dashed lines.
COVID-19 pandemic is caused by the SARS-CoV-2 virus that has several enzymes that could be targeted by antivirals including a 2'-O RNA methyltransferase (MTase) that is involved in the viral RNA cap formation; an essential process for RNA stability. This MTase is composed of two nonstructural proteins, the nsp16 catalytic subunit and the activating nsp10 protein. We have solved the crystal structure of the nsp10-nsp16 complex bound to the pan-MTase inhibitor sinefungin in the active site. Based on the structural data we built a model of the MTase in complex with RNA that illustrates the catalytic reaction. A structural comparison to the Zika MTase revealed low conservation of the catalytic site between these two RNA viruses suggesting preparation of inhibitors targeting both these viruses will be very difficult. Together, our data will provide the information needed for structure-based drug design.
Krafčíková, P., Šilhan, J., Nencka, R., and Bouřa, E.: Structural analysis of the SARS-CoV-2 methyltransferase complex involved in coronaviral RNA cap creation, Nat. Commun. (2020) 11, 3717, https://doi.org/10.1038/s41467-020-17495-9