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Structural Basis for the Inhibition of the Eukaryotic Ribosome

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Structural Basis for the Inhibition of the Eukaryotic Ribosome ARTICLE doi:10.1038/nature13737 Structural basis for the inhibition of the eukaryotic ribosome Nicolas Garreau de Loubresse1, Irina Prokhorova1, Wolf Holtkamp2, Marina V. Rodnina2, Gulnara Yusupova1 & Marat Yusupov1 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 under- standing of the binding mode of ribosome inhibitors in eukaryotes is currently limited. Here we used X-ray crystallo- graphy 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. In all living cells, ribosomes are large ribonucleoprotein assemblies re- decoding centre (geneticin G418) and the mRNA–tRNA binding site sponsible for the accurate conversion of the genetic information encoded on the small subunit (pactamycin, edeine). Eukaryotic-specific inhibi- within mRNA into a corresponding protein. Although core functions tors were chosen on the basis of their capacity to alter cell proliferation of the ribosome are conserved in all kingdoms of life, eukaryotic ribo- and/or protein synthesis and their selectivity restricted to eukaryotes. somes are at least 40% larger than their bacterial counterparts with a The list comprises cycloheximide, lactimidomycin, phyllanthoside, total mass ranging from ,3.3 MDa (yeasts and plants) up to ,4.5 MDa T-2 toxin, deoxynivalenol, verrucarin A, narciclasine, lycorine, nagi- (mammals)1–3. The additional complexity of the eukaryotic ribosome lactone C, anisomycin, homoharringtonine and cryptopleurine. structure is reflected in differences in terms of functions and aspects of The present study illustrates the chemical diversity of the small- translation and its regulation4. molecule inhibitors targeting the eukaryotic ribosome. All of them were Given the central role of the ribosome in the cell, living organisms systematically found in a clash with or in a close proximity to mRNA or have elaborated defence strategies using small-molecule inhibitors to transfer RNA (tRNA) binding sites on both subunits (Fig. 2a, b). In impair ribosomal functions. Decades of studies have revealed the great contrast to bacterial antibiotics, none were located in the peptide exit diversity of molecular mechanisms used by a multitude of antibacterial tunnel, which correlates with the increased number of rRNA modifica- agents (antibiotics)5,6. Atomic structures of prokaryotic ribosomes pro- tions in this region of the eukaryotic ribosomes16. Although the 80S vided the basis for the development of novel antibiotics and in turn ribosome contains 1.4 MDa of additional features absent in bacteria, itis ribosome inhibitors served as tools to study protein synthesis in bacteria7. noteworthy that all eukaryote-specific inhibitors target primary func- Similarly, the eukaryotic ribosome is a major target for broad-spectrum tional sites. This observation highlights the role of nucleotide substitu- and eukaryote-specific small-molecule inhibitors isolated from natural tions in the formation of eukaryotic-specific pockets in the core of the sources. Despite limited understanding of their molecular mechanism, machinery. Remarkably, the structures also explain the predominance eukaryote-specific ribosomal inhibitors are increasingly used in research of resistance mutations found in ribosomal proteins in or near the and hold potential for new therapeutics against a wide range of infectious inhibitor binding sites (Extended Data Table 2). In contrast to bacteria, diseases, cancers and genetic disorders8–12. the high-copy number of rDNA in eukaryotic genomes limits the To date, no structural data are available for small-molecule inhibitors appearance of point mutations in rRNA. Instead, resistance mutations in complexes with either the complete eukaryotic ribosome or its sub- emerge exclusively within ribosomal proteins, encoded by one or two units. Some eukaryote-specific inhibitors were investigated using crys- genes, sometimes located further away and separated by a layer of tals of the 50S subunit of the archaea Haloarcula marismortrui given its rRNA nucleotides like in the case of protein uL3. Consequently, deve- similarity with some parts of the eukaryotic ribosome13,14. A previous lopment of drugs targeting the eukaryotic ribosome can be facilitated X-ray study of the large subunit of Tetrahymena thermophila did not to prevent drug resistance by focusing on regions with the smallest succeed to unambiguously place cycloheximide in its density due to number of proteins. limited resolution15. To gain insight into the mode of action of ribosome inhibitors in eukaryotes and to identify principles for drug development, The 60S tRNA E-site we determined 16 crystal structures at high resolution, up to 2.9 A˚ ,of Among all three tRNA binding sites on the large subunit, the E-site is the the S. cerevisiae 80S ribosome in complexes with 12 eukaryote-specific most diverse across species, showing different nucleotide and protein and 4 broad-spectrum inhibitors (Fig. 1, Extended Data Figs 1–3 and content in bacteria, archaea and eukaryotes. Following peptide bond Extended Data Table 1). The broad-spectrum inhibitors target the formation, this site accommodates deacylated tRNAs before their re- peptidyl transferase centre on the large subunit (blasticidin S), the lease in the cytoplasm. 1Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire (IGBMC), INSERM U964, CNRS UMR7104, Universite´ de Strasbourg, 67404, Illkirch, France. 2Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Go¨ttingen, Germany. 25 SEPTEMBER 2014 | VOL 513 | NATURE | 517 ©2014 Macmillan Publishers Limited. All rights reserved RESEARCH ARTICLE Eukaryote-specific Phyllanthoside Anisomycin Cryptopleurine Nagilactone C CH3 H C H C Cycloheximide Lactimidomycin 3 O 3 CH HO O O 3 O O OH O O CH3 H3C O O O OH HN CH3 O N O O O OH O O CH3 O CH3 HO O O CH3 HN OH O O OH CH3 CH3 HO CH3 O O O O O O O O HN H3C CH3 O CH CH 3 3 O O CH 3 O H3C Lycorine Narciclasine Homoharringtonine Broad-spectrum O N O Geneticin G418 Pactamycin Blasticidin S N H2N NH HO H C CH3 NH 3 O H C OH N OH 3 N OH O OH H3C OH O HO H N NH HN O 2 2 NH2 OH H3C NH OH O O 2 O O HO CH3 H3C O O H3C H3C O OH HO O O NH HO H C NH OH OH 3 2 OH HN O O O CH3 O O O NH OH O N O HO CH 3 CH3 OH H3C O N NH T-2 toxin Deoxynivalenol Verrucarin A 2 H H H H H3C O O H O O Edeine OH OH O O HO HO O O O O O CH3 O OH O NH2 O NH2 O O H H H O O HO N N N HO CH3 N N N NH HO H H H O O NH O O OH O O 2 NH2 O Figure 1 | Chemical structures of the 16 small-molecule inhibitors. alkaloids known for their medicinal and toxic properties38. T-2 toxin, a, Chemical structures of small-molecule inhibitors analysed in the study. deoxynivalenol and verrucarin A are widespread mycotoxins representatives of Cycloheximide and lactimidomycin have an instrumental role in ribosome the three major trichothecenes subclasses39. The aminoglycoside geneticin profiling experiments18,20. Homoharringtonine is a marketed drug for the (G418) promotes the read-through of premature termination codons11. treatment of chronic myeloid leukaemia37. Lycorine and narciclasine are The glutarimide inhibitors, cycloheximide and lactimidomycin, bears an additional lactone ring that is positioned on top of eL42 and were located in the E-site on the large subunit in a pocket formed directed towards the subunit interface. Although chemically unrelated by universally conserved nucleotides of the 25S rRNA and a stretch to glutarimides, phyllanthoside makes contact with the same rRNA of the eukaryote-specific protein eL42 (Fig. 3a). Lactimidomycin nucleotides and interacts with eL42 in a manner resembling the tRNA a b Peptidyl transferase center (PTC) Central tRNA E-site protuberance * Cycloheximide * Lycorine * T-2 toxin * Lactimidomycin * Narciclasine * Deoxynivalenol * Phyllanthoside * Nagilactone C * Verrucarin A * Anisomycin * Homoharringtonine tRNA E-site Blasticidin S E-site tRNA A-site tRNA PTC 60S P-site tRNA 60S Head 40S Decoding centre Body mRNA tunnel Decoding centre * Cryptopleurine Geneticin G418 mRNA tunnel Pactamycin 40S Edeine mRNA Figure 2 | Binding sites of inhibitors on the yeast ribosome. a, The binding the subunit interface. b, All inhibitors target mRNA and tRNA binding sites can be grouped in four functional regions: the tRNA E-site and the sites. The tRNAs and mRNA (white) structures were taken from the PDB peptidyl transferase centre (PTC) on the large subunit (60S) and the decoding (Protein Data Bank) entries 3I8I and 3I8H. Eukaryotic-specific inhibitors are centre (DC) and the mRNA channel on the small subunit (40S). View from marked with an asterisk. 518 | NATURE | VOL 513 | 25 SEPTEMBER 2014 ©2014 Macmillan Publishers Limited. All rights reserved ARTICLE RESEARCH abU2763 the first peptide bond, whereas cycloheximide stalls ribosomes during C2764 U2763 C93 C2764 C93 ongoing translation18–20. 25S rRNA 25S rRNA eL42 G2793 To better understand this difference, we used a rapid kinetic approach uL15 G2793 to study the effects of both inhibitors on tRNA binding to the E-site with * a fluorescently labelled tRNA (proflavin (Prf) tRNAPhe)21. Binding of G92 tRNAPhe(Prf) to the 80S or 70S ribosomes resulted in an almost iden- Cycloheximide Y43 G2794 tical rapid fluorescence change, reflecting the recruitment of the tRNA to the E-site, whereas transition from the E-site to the P-site was slow K55 G2794 22 eL42 P56 and not monitored (Extended Data Fig.
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