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Structural basis for the inhibition of the eukaryotic ribosome

Nature volume 513, pages 517–522 (2014)Cite this article

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Abstract

The ribosome is a molecular machine responsible for protein synthesis and a major target for small-molecule inhibitors. Compared to the wealth of structural information available on ribosome-targeting antibiotics in bacteria, our understanding of the binding mode of ribosome inhibitors in eukaryotes is currently limited. Here we used X-ray crystallography to determine 16 high-resolution structures of 80S ribosomes from Saccharomyces cerevisiae in complexes with 12 eukaryote-specific and 4 broad-spectrum inhibitors. All inhibitors were found associated with messenger RNA and transfer RNA binding sites. In combination with kinetic experiments, the structures suggest a model for the action of cycloheximide and lactimidomycin, which explains why lactimidomycin, the larger compound, specifically targets the first elongation cycle. The study defines common principles of targeting and resistance, provides insights into translation inhibitor mode of action and reveals the structural determinants responsible for species selectivity which could guide future drug development.

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Figure 1: Chemical structures of the 16 small-molecule inhibitors.
Figure 2: Binding sites of inhibitors on the yeast ribosome.
Figure 3: Structure and function of 60S tRNA E-site inhibitors.
Figure 4: Structures of 60S peptidyl transferase centre inhibitors.
Figure 5: Structures of 40S mRNA and tRNA inhibitors.
Figure 6: Inhibitors action during protein synthesis in eukaryotes.

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Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported crystal structures have been deposited in the Protein Data Bank (http://www.pdb.org/pdb/home/home.do) under accession codes 4U3M (anisomycin), 4U56 (blasticidin S), 4U3N (CCA), 4U55 (cryptopleurine), 4U3U (cycloheximide), 4U53 (deoxynivalenol), 4U4N (edeine), 4U4O (geneticin G418), 4U4Q (homoharringtonine), 4U4R (lactimidomycin), 4U4U (lycorine), 4U52 (nagilactone C), 4U51 (narcilasine), 4U4Y (pactamycin), 4U4Z (phyllanthoside), 4U6F (T-2 toxin) and 4U50 (verrucarin).

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Acknowledgements

We thank J. Liu (Johns Hopkins Medical Institute), D. Wilson (Gene Center Munich), P. Hazendonk (Agriculture and Agri-Food Canada) and the NIH/NCI Developmental Therapeutics Program for providing materials. We acknowledge SOLEIL synchrotron (France), all staff members of PROXIMA1 beamline, especially A. Thompson and P. Legrand for their assistance during data collection. We thank A. Perez Lara, MPI Göttingen, for the help with the ITC experiments and S. Melnikov, IGBMC, for reading the manuscript. I.P. acknowledges support from AFM-Telethon post-doctoral fellowship. This work was supported by the SATT Conectus Technology Maturation grant I12-042 (to N.G.D.L.), the ERC Advanced grant 294312, the Human Frontier Science Program grant RGP0062/2012 and the Russian Government Program of Competitive Growth of Kazan Federal University (to M.Y.), the French National Research Agency grant ANR-11-BSV8-006 01 (to G.Y.) and the Deutsche Forschungsgemeinschaft grant (to M.V.R.).

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Authors and Affiliations

  1. Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U964, CNRS UMR7104, Université de Strasbourg, 67404, Illkirch, France ,

    Nicolas Garreau de Loubresse, Irina Prokhorova, Gulnara Yusupova & Marat Yusupov

  2. Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany,

    Wolf Holtkamp & Marina V. Rodnina

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  1. Nicolas Garreau de Loubresse
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Contributions

M.Y. supervised the study. N.G.D.L. designed the experiments. N.G.D.L. and I.P. conducted purification, crystallization and post-crystallization treatment experiments, collected X-ray diffraction data and carried out the structure determination. N.G.D.L., I.P., G.Y. and M.Y. analysed the crystal structures. M.V.R. and W.H. designed, performed and interpreted rapid kinetic experiments. N.G.D.L. wrote the initial manuscript to which M.V.R., G.Y. and M.Y. contributed specialist insights. All authors helped with refining the manuscript and approved the final version.

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Correspondence to Marat Yusupov.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Unbiased positive electron density of small-molecule inhibitors and CCA-trinucleotide.

Fo–Fc positive electron density maps of the 16 small-molecule inhibitors and the CCA tri-nucleotide. The maps were contoured at 3.0–3.5σ.

Extended Data Figure 2 Electron density of small-molecule inhibitors and CCA-trinucleotide.

2Fo–Fc electron density maps of the 16 small-molecule inhibitors and the CCA tri-nucleotide. The maps were contoured at 1.0–1.5σ.

Extended Data Figure 3 Structures of homoharringtonine, anisomycin, blasticidin S and pactamycin in eukaryotes, archaea and bacteria.

Complexes with bacterial and archaeal structures were aligned with the 25S rRNA or the 18S rRNA of the yeast ribosome. Differences in the binding pocket were found only in the case of anisomycin as described in Extended Data Fig. 8. Coordinates were taken from the PDB databank; PDB entries are indicated in parentheses.

Extended Data Figure 4 Structural differences in protein eL42 may preclude the binding of lactimidomycin and phyllanthoside to the archaeal ribosome.

In archaea, the protein eL42 (in red, PDB 1JJ2) is shorter than its eukaryotic counterpart (yellow) and adopts a markedly different conformation that clashes with lactimidomycin (pink) and phyllanthoside (cyan). Residues of protein eL42 from archaea involved in the steric clash with both inhibitors are depicted in red with sticks and van der Waals spheres. Although the 60S tRNA E-site is targeted by small-molecule inhibitors in archaea and eukaryotes, remarkably no antibiotics targeting this site in bacteria have been described.

Extended Data Figure 5 Close-up view of CCA tri-nucleotide binding site.

CCA tri-nucleotide (white) bound to the 60S tRNA E-site. The binding pocket is formed by 25S rRNA nucleotides (blue) and part of protein eL42 (yellow). In eukaryotes, the protein eL42 remodels the 60S E-site and participates actively in positioning the CCA-end. Although C75 is stabilized by stacking and hydrogen bonds interactions with eL42, the terminal residue A76 of deacylated tRNA enters the pocket and forms a non-canonical base pair with a conserved residue of the 25S rRNA.

Extended Data Figure 6 Kinetic study of lactimidomycin and cycloheximide.

a, Deacylated tRNA binding to the bacterial 70S and eukaryotic 80S ribosomes. Time courses of tRNAPhe (Prf) binding to the S. cerevisiae 80S (blue) and E. coli 70S (red) ribosomes measured by the stopped-flow technique. b, Competition binding assays. Dose response curves for lactimidomycin (closed circles) and cycloheximide (open circles). Inset, the binding of the tRNA to the 70S ribosome was not affected in the presence of lactimidomycin (blue) and cycloheximide (magenta). Control without inhibitors is shown in black. c, Measurement of cycloheximide affinity to the 80S ribosome by isothermal titration calorimetry. The curves present the thermodynamic parameters of cycloheximide binding to 80S ribosomes (black circles) and control buffer (red circles). N, number of binding sites. The affinity was determined in 4 independent experiments.

Extended Data Figure 7 Close-up view of blasticidin S binding site.

Blasticidin S (pink) bound to the 60S tRNA P-site. The binding pocket is formed exclusively by nucleotides of the 25S rRNA (yellow). Dashed lines indicate hydrogen contacts with G2619 that precludes the formation of the base pair with C75 of the tRNA in the P-site.

Extended Data Figure 8 Conformational changes in the peptidyl transferase centre and differences with the archaeal ribosome.

a, A-site inhibitors induce conformational changes upon binding to the peptidyl transferase centre of the yeast ribosome. Superimposition of the vacant 80S ribosome (PDB 3U5A–3U5D, blue) and the 80S ribosome in complexes with A-site inhibitors (25S rRNA in yellow). The structure of anisomycin (orange) was chosen as a reference to represent the peptidyl transferase centre A-site inhibitors. Residue U2875 (U2506) undergoes the most drastic change resulting in the breakdown of a canonical base pair formed by G2952 (U2583) and its subsequent flipping out. The reorientation of U2875 (U2506) participates in preventing the binding of aminoacyl-tRNA. b, U2875 adopts a different conformation upon binding of anisomycin (orange) to the peptidyl transferase centre A-site (yellow) in eukaryotes in contrast to its homologue (U2541) in archaea (magenta). Superimposition of the 50S large subunit from H. marismortui in complex with anisomycin (PDB 1K73) and the 80S ribosome in complex with anisomycin.

Extended Data Table 1 Summary of information for the 16 small-molecule inhibitors
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Extended Data Table 2 Resistant mutations in proteins of the yeast ribosome
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Garreau de Loubresse, N., Prokhorova, I., Holtkamp, W. et al. Structural basis for the inhibition of the eukaryotic ribosome. Nature 513, 517–522 (2014). https://doi.org/10.1038/nature13737

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