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. 2017 Dec 19;114(51):E10899-E10908.
doi: 10.1073/pnas.1715501114. Epub 2017 Dec 5.

Aminoglycoside Interactions and Impacts on the Eukaryotic Ribosome

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Free PMC article

Aminoglycoside Interactions and Impacts on the Eukaryotic Ribosome

Irina Prokhorova et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Aminoglycosides are chemically diverse, broad-spectrum antibiotics that target functional centers within the bacterial ribosome to impact all four principle stages (initiation, elongation, termination, and recycling) of the translation mechanism. The propensity of aminoglycosides to induce miscoding errors that suppress the termination of protein synthesis supports their potential as therapeutic interventions in human diseases associated with premature termination codons (PTCs). However, the sites of interaction of aminoglycosides with the eukaryotic ribosome and their modes of action in eukaryotic translation remain largely unexplored. Here, we use the combination of X-ray crystallography and single-molecule FRET analysis to reveal the interactions of distinct classes of aminoglycosides with the 80S eukaryotic ribosome. Crystal structures of the 80S ribosome in complex with paromomycin, geneticin (G418), gentamicin, and TC007, solved at 3.3- to 3.7-Å resolution, reveal multiple aminoglycoside-binding sites within the large and small subunits, wherein the 6'-hydroxyl substituent in ring I serves as a key determinant of binding to the canonical eukaryotic ribosomal decoding center. Multivalent binding interactions with the human ribosome are also evidenced through their capacity to affect large-scale conformational dynamics within the pretranslocation complex that contribute to multiple aspects of the translation mechanism. The distinct impacts of the aminoglycosides examined suggest that their chemical composition and distinct modes of interaction with the ribosome influence PTC read-through efficiency. These findings provide structural and functional insights into aminoglycoside-induced impacts on the eukaryotic ribosome and implicate pleiotropic mechanisms of action beyond decoding.

Keywords: PTC read-through; aminoglycosides; protein synthesis; ribosome; translation.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Aminoglycosides target the decoding center of the 80S ribosome in a different ways. (A) Secondary structure of h44 of the small ribosomal subunit from bacteria (E. coli) and eukaryotes (identical in S. cerevisiae and Homo sapiens). Substituted nucleotides implicated in the selectivity of aminoglycosides are marked in red. (B) Binding of paromomycin (PAR-1) to h44 in the 80S ribosome from S. cerevisiae. Paromomycin is colored violet, and rings I, II, III, and IV of paromomycin are marked. Ring I is in stacking with A1754. Residues A1754, and G1645 are colored pink. h44 is shown in orange; H69 of the large ribosomal subunit is shown in light blue; and the eukaryote-specific protein eS30 is shown in green. Oxygen atoms are colored red, and nitrogen atoms are colored blue. (C) Comparison of PAR-1 binding to h44 in the 70S ribosome from T. thermophilus (PDB ID code 5EL6) and the 80S ribosome from S. cerevisiae. Paromomycin in complex with 70S is shown in yellow; residues of 16S rRNA of 70S are in green; other color-coding is as in B. The shift in the position of A1754 and the movement of 5′-OH group in ring III of paromomycin are marked with arrows. (D) Binding of gentamicin (GENT-1) to h44 in the 80S ribosome from S. cerevisiae. Gentamicin is shown in green; other color-coding is as in B. Rings I, II, and III of gentamicin are marked, and atoms located at a hydrogen bonding distance are connected by dashed lines.
Fig. 2.
Fig. 2.
Interactions of the aminoglycoside derivative TC007 with the 80S ribosome. (A, Left) The view from the A-site of the ribosome is indicated. (Right) Enlarged view showing binding sites of TC007-1 and TC007-2 in close proximity to h44 of the small ribosomal subunit and H69 of the large ribosomal subunit, respectively. The regions of h44 of the small ribosomal subunit and H71 of the large ribosomal subunit, which form intersubunit bridge B3, are colored violet. The 40S subunit is shown in orange. Contacts of TC007 and rRNA residues are depicted by dashed lines. The 60S subunit is shown in blue, and TC007 is in magenta. Oxygen atoms are colored red, and nitrogen atoms are colored blue. (B) Close-up view of the second binding site of TC007-2 located between helices H68, H69, and H70 of the large ribosomal subunit. Local alignment demonstrates that TC007-2 would clash with the phosphate group of C2248 if H69 adopted the conformation characterized by the different rotational state of the ribosomal subunits as observed in the 80S–gentamicin structure. The 60S subunit from the 80S–gentamicin structure is colored gray; residue C2248 is colored lime and is marked with spheres; other color-coding is as in A.
Fig. 3.
Fig. 3.
Aminoglycoside-induced changes in human 80S PRE complex conformation. (A) Schematic showing classic (C) and hybrid (H2 and H1) states of the human PRE ribosome complex. Large (60S) and small (40S) subunits (unrotated, gray; rotated, pink), tRNAs (orange), and sites of donor (Cy3, green) and acceptor (Cy5, red) fluorophore labeling on tRNA in the P- and A-sites, respectively, are indicated. (BE) Population FRET histograms showing the impact of paromomycin (B), G418 (C), gentamicin (D), and TC007 (E) on the equilibrium distribution of FRET states exhibited by the 80S PRE complex. The concentration of antibiotic is indicated; n, number of single-molecule observations made in each experiment.
Fig. 4.
Fig. 4.
Aminoglycoside-induced miscoding during tRNA selection on the human ribosome. (A) Schematic showing the process of tRNA selection, in which the ternary complex of eEF1A (blue), GTP, and aa-tRNA (orange) enters the A-site of the 80S ribosome. The process of tRNA selection proceeds through the A/T state in which codon–anticodon pairing on the small subunit occurs while the 3′-aminoacylated CCA-end of tRNA remains bound to eEF1A. GTP hydrolysis by eEF1A facilitates the aa-tRNA release from eEF1A and the accommodation of its 3′CCA end into the large subunit A-site on the classically (C) configured (unrotated) PRE complex. Peptidyl- and aminoacyl-tRNAs in the P- and A-sites, respectively, then undergo peptide bond formation (PBF), enabling deacylated P-site tRNA and peptidyl-tRNA to achieve hybrid (H2 and H1) states. Large (60S) and small (40S) subunits (unrotated, gray; rotated, pink), tRNAs (orange), and sites of donor (Cy3, green) and acceptor (Cy5, red) fluorophore labeling on tRNA in the P- and A-sites, respectively, are indicated. (B) Population FRET histograms showing that aminoglycoside-induced errors in tRNA selection lead to the accumulation of PRE ribosome complexes bearing near-cognate tRNA in the A-site. The concentration of antibiotic is indicated; n, number of single-molecule observations made in each experiment.
Fig. 5.
Fig. 5.
Overview of the secondary binding sites of aminoglycosides in 80S ribosome. (A) Binding sites of gentamicin (GENT), G418, TC007, and paromomycin (PAR) in the 80S ribosome from S. cerevisiae. All structures were aligned either on 18S rRNA or on 28S rRNA, for small and large subunits, respectively, in the 80S–gentamicin structure. The ribosome is colored light gray; elements of the binding pockets of aminoglycosides are colored light orange; gentamicin is colored green; G418 is colored yellow; TC007 is colored magenta; and paromomycin is colored violet. (B) Binding of GENT-7, G418-2, and TC007-3 to the peptide exit tunnel of the 80S ribosome. G418 is colored yellow; gentamicin is colored light green; TC007 is colored magenta; the large ribosomal subunit is colored blue; and the eukaryote-specific protein eL37 is colored green. Similar poses are observed for G418-2 and GENT-7, but the orientation of TC007-3 is different. Oxygen atoms are colored red, and nitrogen atoms are colored blue. (C) Binding sites of PAR-2, GENT-2, and G418-3 in the E-site of the small ribosomal subunit overlapping the position of the mRNA. Structures of the 80S ribosome in complex with paromomycin, gentamicin, and G418 were locally aligned on the structure of the 70S ribosome from T. thermophilus in complex with tRNAs and mRNA (PDB ID code 5EL6). mRNA is colored red; elements of the 70S ribosome are omitted for clarity. The 40S subunit is colored orange; paromomycin is colored violet; the universal protein uS11 is colored green; and other color-coding is as in A. (D) Interactions of PAR-3 with the elements of the intersubunit bridge B2c. Contacts made between paromomycin and G984 of the 40S subunit, A2152 of the 60S subunit, and Asp176 of uL2 are marked with dashed lines. The 40S subunit is colored light pink; paromomycin is colored violet; the 60S subunit is colored blue; and the universal protein L2 (uL2) is colored blue. Oxygen atoms are colored red, and nitrogen atoms are colored blue. (E) GENT-4 and GENT-5 stabilize particular conformations of bridge B2c. The 80S–paromomycin structure was aligned on the 80S–gentamicin structure based on the 28S rRNA. The alignment demonstrates that rearrangement of the bridge B2c would be blocked by gentamicin due to a clash with h24 of the 40S subunit in the 80S–paromomycin structure (colored in pink). 80S–gentamicin contacts are marked with dashed lines. The 40S subunit from 80S–gentamicin structure is colored lime; other color-coding is as in AC. (F) GENT-6 targets bridge B4 formed by the universal protein uS15 protein and H34 of the large subunit. uS15 is shown in yellow; other color-coding is as in D. Glutamine 142, which interacts with GENT-6, is depicted as spheres. (G) Interactions of GENT-8 and TC007-4 with the elements of the ribosomal P-stalk. Different conformation of the helices 42 and 97 of the P-stalk are shown in blue for 80S-GENT and 80S-TC007 structures and in cyan for the 80S-apo structure (PDB ID code 4V88). H42 in the apo conformation would clash with GENT-8. Arg62 in the uL6 protein approaching TC007 is shown as spheres. Color-coding is as in AE.

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References

    1. Fosso MY, Li Y, Garneau-Tsodikova S. New trends in aminoglycosides use. MedChemComm. 2014;5:1075–1091. - PMC - PubMed
    1. Davies J. Inactivation of antibiotics and the dissemination of resistance genes. Science. 1994;264:375–382. - PubMed
    1. Borovinskaya MA, et al. Structural basis for aminoglycoside inhibition of bacterial ribosome recycling. Nat Struct Mol Biol. 2007;14:727–732. - PubMed
    1. Feldman MB, Terry DS, Altman RB, Blanchard SC. Aminoglycoside activity observed on single pre-translocation ribosome complexes. Nat Chem Biol. 2010;6:244. - PMC - PubMed
    1. Youngman EM, He SL, Nikstad LJ, Green R. Stop codon recognition by release factors induces structural rearrangement of the ribosomal decoding center that is productive for peptide release. Mol Cell. 2007;28:533–543. - PubMed

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