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Structural basis for tRNA modification by Elp3 from Dehalococcoides mccartyi

Nature Structural & Molecular Biology volume 23, pages 794–802 (2016)Cite this article

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Abstract

During translation elongation, decoding is based on the recognition of codons by corresponding tRNA anticodon triplets. Molecular mechanisms that regulate global protein synthesis via specific base modifications in tRNA anticodons are receiving increasing attention. The conserved eukaryotic Elongator complex specifically modifies uridines located in the wobble base position of tRNAs. Mutations in Elongator subunits are associated with certain neurodegenerative diseases and cancer. Here we present the crystal structure of D. mccartyi Elp3 (DmcElp3) at 2.15-Å resolution. Our results reveal an unexpected arrangement of Elp3 lysine acetyltransferase (KAT) and radical S-adenosyl methionine (SAM) domains, which share a large interface and form a composite active site and tRNA-binding pocket, with an iron–sulfur cluster located in the dimerization interface of two DmcElp3 molecules. Structure-guided mutagenesis studies of yeast Elp3 confirmed the relevance of our findings for eukaryotic Elp3s and should aid in understanding the cellular functions and pathophysiological roles of Elongator.

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Figure 1: Crystal structure of DmcElp3.
Figure 2: Dimerization of DmcElp3.
Figure 3: DmcElp3 tRNA interaction.
Figure 4: RNase protection assays and tRNA–DmcElp3 model.
Figure 5: Model of the individual steps of the Elp3 modification reaction.

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Acknowledgements

We thank H. Lin (Cornell University) for providing the Azotobacter vinelandii isc operon–containing plasmid, G. Sawers (Martin-Luther-University Halle) for providing genomic DNA from D. mccartyi, G. Fritz (University of Freiburg) for discussion and H. Grötsch for support. We acknowledge support from the EMBL Heidelberg Crystallization Platform. We also acknowledge access and support provided by the EMBL/ESRF Joint Structural Biology Group at ESRF beamlines. R.Z. and K.D.B. acknowledge funding from the German Science Foundation (SFB 648). EPR work was supported by TGE RPE FR3443 (F. Baymann). This work was also supported by the Ligue contre le Cancer (Equipe labellisée 2014) (B.S.), the Centre National pour la Recherche Scientifique (B.S.), the CERBM–IGBMC, the project Elongator from the Agence Nationale pour la Recherche (grant ANR-13-BSV8-0005-01) (B.S.) and grant ANR-10-LABX-0030-INRT managed under the program Investissements d'Avenir ANR-10-IDEX-0002-02 (B.S.).

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Author notes

  1. Rene Zabel and Olga Kolaj-Robin: These authors contributed equally to this work.

Authors and Affiliations

  1. European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany

    Sebastian Glatt, Florence Baudin, Andrea Graziadei & Christoph W Müller

  2. Max Planck Research Group at the Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland

    Sebastian Glatt & Ting-Yu Lin

  3. Institute of Biology, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany

    Rene Zabel, Osita F Onuma & Karin D Breunig

  4. Université de Strasbourg, IGBMC UMR 7104, Illkirch, France

    Olga Kolaj-Robin, Valerio Taverniti & Bertrand Séraphin

  5. CNRS, IGBMC UMR 7104, Illkirch, France

    Olga Kolaj-Robin, Valerio Taverniti & Bertrand Séraphin

  6. Inserm, IGBMC U964, Illkirch, France

    Olga Kolaj-Robin, Valerio Taverniti & Bertrand Séraphin

  7. Unit of Virus Host-Cell Interactions (UMI 3265), University Grenoble Alpes–CNRS–EMBL, Grenoble, France

    Florence Baudin

  8. Laboratoire de Bioénergétique et Ingénierie des Protéines, BIP/CNRS, UMR7281, AMU, Marseille, France

    Frauke Baymann

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Contributions

S.G. performed the biochemical, biophysical and crystallographic analyses of DmcElp3 with the help of T.-Y.L. R.Z. and O.F.O. characterized mutant phenotypes in yeast. O.K.-R. and V.T. carried out anaerobic purifications, acetylation assays and spectroscopic analyses. F. Baymann. performed EPR measurements. F. Baudin performed RNase protection assays. A.G. collected and analyzed SAXS data. S.G., O.K.-R., B.S., K.D.B. and C.W.M. designed experiments and analyzed the data. S.G. and C.W.M. wrote the manuscript with input from all other authors.

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Correspondence to Christoph W Müller.

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Integrated supplementary information

Supplementary Figure 1 Topology and structural features of DmcElp3.

(a) Anomalous difference Fourier map of DmcElp3 collected at the selenium absorption edge shown at 5 I/σ. Selenomethionine residues are shown in ball and sticks representation. (b) Topology of DmcElp3 indicating secondary structure elements. (c) Secondary structure elements are shown above the amino acid sequence showing only residues present in the structural model and highlighting cysteine residues involved in iron-sulfur cluster or zinc coordination. (d) Stereo figure showing the different indicated substrate peptides bound to Gcn5 resulting from a superimposition of Gcn5 with the KAT domain of DmcElp3. Used structures were 1Q2C (H4), 1QSN (H3), 1PUA (pH3) and 1Q2D (p53) (Clements, A. et al., Mol Cell 12, 461–473, 2003; Rojas, J.R. et al., Nature 401, 93-98; Poux, A.N. and Marmorstein, R., Biochemistry 42, 14366-14374).

Supplementary Figure 2 Analyses of purified DmcElp3.

(a) UV/Vis absorption spectra of anaerobically purified DmcElp3 showing the broad absorption band characteristic of [4Fe-4S]2+ cluster at 410 nm. (b) EPR spectra of anaerobically (upper traces, protein concentration 7.5 mg/ml) and aerobically (lower traces, protein concentration 10.6 mg/ml) purified DmcElp3 protein recorded at 6 K with a microwave power of 128 mW and a modulation amplitude of 0.00303 T (left panel) and at 50 K with a microwave power of 6.4 mW and a modulation amplitude of 0.001 T (right panel) on a Bruker Elexsys E500 X-band spectrometer fitted with an Oxford Instrument He-cryostat ESR900 and temperature control system. Microwave frequency was 9.48 GHz. (c) Gelfiltration profiles of indicated purified DmcElp3 mutants. (d) Thermofluor analyses of indicated monomeric and dimeric DmcElp3 mutants. (e) Fitting of experimental scattering data (dots) for Elp3 monomer (red, 0.58 mg/ml) and dimer (black, 0.54 mg/ml) to theoretical scattering curves calculated from the structure (lines) show that the model predicts the low-resolution features of both the monomer and the dimer in solution. Furthermore, the Guinier analysis in the inset shows a good agreement in terms of radius of gyration (Rg) between theoretical and experimental data. Fitting calculated using CRYSOL (ATSAS suite). Theoretical Rg Dimer =3.33 nm. Theoretical Rg Monomer=2.22 nm. (f) Titration of acetyl-CoA and SAM onto Elp3 monomer and dimer. No large conformational change is observed upon addition of acetyl-CoA or SAM, with the protein maintaining a conformation compatible with that of the crystal structure. The curves displayed are extrapolations to infinite dilution calculated using PRIMUS (ATSAS suite) from concentration series, in order to minimize inter-particle effects (see methods).

Supplementary Figure 3 tRNA binding of DmcElp3 and structural surface analyses.

(a) Unspecific acetylation of DmcElp3 and unrelated proteins (human BTG2, yeast Kti13 and E.coli NorW) in the presence of Na2O4S2 detected by using radioactive labelled (C14) acetyl-CoA. (b) EMSA analysis of monomeric and dimeric DmcElp3 using tRNAGlu from D. mccartyi (left) and S. cerevisiae (right). (c) Structure of DmcElp3 shown in cartoon representation (top), surface representation showing conservation scores of 394 unique sequences from ConSurf analyses (www.consurf.tau.ac.il/) (middle) and surface representation showing surface charge distribution from APBS analysis (bottom). The conserved basic tRNA binding cavity is indicated by a dotted circle.

Supplementary Figure 4 tRNA binding and phenotypes of additional DmcElp3 mutants.

(a,b) EMSA analysis of indicated point mutants using radioactively labelled tRNA and increasing amounts of protein (1.6, 7.5, 15 μM) on a 6% native gel. Samples were also analyzed using denaturing SDS-PAGE as loading control. (c,d) Phenotypes of yeast strains carrying the indicated Scelp3 mutant alleles using γ-toxin and tRNA suppression assays. γ-toxin and SUP4 suppression assays are described in the Online Methods.

Supplementary Figure 5 Comparing crystal structures of full-length and 390–407(GSGSG) DmcElp3.

(a) Comparison between binding affinities of full length DmcElp3 and 390-407GSGSG DmcElp3 using EMSA analysis of indicated point mutants using radioactively labelled tRNA and increasing amounts of protein (0.9, 1.8, 3.7, 7.5, 15 μM) on a 6% native gel. Samples were also analyzed using denaturing SDS-PAGE as loading control. (b) 2mFo-Fc density of full length DmcElp3 (left) and 390-407GSGSG DmcElp3 (right) shown at 1 I/σ. (c) Close up of the acetyl-CoA binding site showing 2mFo-Fc density for the acetyl-CoA blocking loop and the 5GS linker region (left, middle). Model of fitted CoA (green) bound to the acetyl-CoA binding site of 390-407GSGSG using CoA from 1QSR (right). 2mFo-Fc density with omitted acetyl-CoA is shown in green.

Supplementary Figure 6 Localization of known Elp3 mutations in the DmcElp3 structure.

(a) Summary of previously tested Elp3 mutations. Tested organisms (D. melanogaster (Dm), S. cerevisiae (Sc), A. thaliana (At), H. sapiens (Hs), M. infernus (Min), M. jannaschii (Mj), M. musculus (Mm)), original mutations and equivalent positions are listed. Identical residues are in bold and cysteine residues involved in zinc coordination are underlined. All mutants are mapped on the DmcElp3 structure and are highlighted (blue/green) using balls and sticks representation. (b) Summary of identified alterations of human Elp3 from ICGC (www.icgc.com) and TCGA (www.cancergenome.nih.org). Equivalent positions are highlighted red and using balls and sticks representation. (c) Complementation phenotypes from the indicated genomic integrated Scelp3 mutants using γ-toxin and tRNA suppression assays. Tested mutations related to neurodegenerative diseases in (a) are labelled with an asterisk (*) and mutations identified in cancer patients in (b) are labelled by two asterisks (**).

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Glatt, S., Zabel, R., Kolaj-Robin, O. et al. Structural basis for tRNA modification by Elp3 from Dehalococcoides mccartyi. Nat Struct Mol Biol 23, 794–802 (2016). https://doi.org/10.1038/nsmb.3265

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  • DOI: https://doi.org/10.1038/nsmb.3265

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