Negamycin interferes with decoding and translocation by simultaneous interaction with rRNA and tRNA

Abstract

Negamycin (NEG) is a ribosome-targeting antibiotic that exhibits clinically promising activity. Its binding site and mode of action have remained unknown. We solved the structure of the Thermus thermophilus ribosome bound to mRNA and three tRNAs, in complex with NEG. The drug binds to both small and large ribosomal subunits at nine independent sites. Resistance mutations in the 16S rRNA unequivocally identified the binding site in the vicinity of the conserved helix 34 (h34) in the small subunit as the primary site of antibiotic action in the bacterial and, possibly, eukaryotic ribosome. At this site, NEG contacts 16S rRNA as well as the anticodon loop of the A-site tRNA. Although the NEG site of action overlaps with that of tetracycline (TET), the two antibiotics exhibit different activities: while TET sterically hinders binding of aminoacyl-tRNA to the ribosome, NEG stabilizes its binding, thereby inhibiting translocation and stimulating miscoding.

Figure 1. Binding of NEG to the T. thermophilus 70S ribosome

(A) Structural formula of NEG. (B) The refined model of NEG at Site 1 is displayed in its respective electron density. The unbiased (F_obs – F_calc) difference Fourier electron density map is contoured at 3σ. Carbon atoms are colored yellow, nitrogens – blue, oxygens – red. (C) NEG binding sites in the small (left) and large (right) subunits of the T. thermophilus 70S ribosome. The refined positions of NEG are shown by yellow spheres. Residues of the 16S rRNA mutated in the NEG-resistant mutants are shown in red. The locations of L1 stalk, central protuberance (CP) and L7/L12 stalk of the 50S subunit are indicated for orientation. (D) NEG^r mutations in the E. coli 16S rRNA (E.coli numbering is used throughout). See also Figures S2, S3 and Movie S1.

Figure 2. NEG Site 1 in the 70S ribosome

(A, B, C) Overview of the NEG binding Site 1 (yellow) in the T. thermophilus 70S ribosome viewed from three different perspectives. The 30S subunit is shown in light yellow, 50S subunit is light blue, mRNA is shown in magenta and tRNAs are displayed in green (A site), dark blue (P-site), and orange (E-site). In (A), the 30S subunit is viewed from the intersubunit interface (50S subunit and A site tRNA were removed for clarity). In (A) and (B), the location of helix 34 of 16S rRNA is highlighted by black outline. (D, E, F) Close-up views of the NEG binding Site 1 shown in panels (A), (B), and (C), respectively. Magnesium ion is shown in green. Nucleotides, whose mutations in E. coli confer NEG resistance, are boxed. See also Figure S3 and Movie S1.

Figure 3. Mechanism of NEG action

(A) Toe-printing analysis of antibiotic effects upon translation of the natural gene osmC or a synthetic gene RST2. Antibiotics thiostrepton (THS), negamycin (NEG), viomycin (VIO), kanamycin (KAN) and tetracycline (TET) were present in the reaction at 100 µM. The antibiotic-induced toe-printing bands corresponding to ribosomes stalled at the 5’-end proximal codons of the genes are indicated by red dots for NEG, green dots for VIO and KAN, and orange dots for TET. The control antibiotic THS arrests the ribosome at the initiator codon. The initiator and subsequent codons are marked by open and filled triangles, respectively. (B) Effect of NEG on translocation of ribosomes from the 1^st (AUG) to the 2^nd (UUU) codon of m291 mRNA (partial relevant sequence shown in the upper panel). Where indicated, ribosomes were incubated with NEG at 10, 100 or 1000 µM prior to the addition of EF-G and GTP. The triangles show the toe-printing bands corresponding to the position of ribosomes at the initiator codon (white triangle – empty A-site; grey triangle – Phe-tRNA^Phe-occupied A site) or ribosomes translocated to the second codon (black triangle). The bar graph shows the translocation efficiency (%) estimated by the ratio of the intensity of the bands indicated by the black and grey triangles. Data are represented as mean +/− SEM. (C) Disk diffusion assay revealing the miscoding activity of NEG. Cells transformed with pBR322bla-stop were plated on LB agar without (bottom) or with 25 µg/mL of ampicillin (top). In the absence of ampicillin, diffusion of antibiotics paromomycin (PAR), spectinomycin (SPC) or NEG inhibits bacterial growth. In the presence of ampicillin, cell growth occurs only with miscoding-inducing antibiotics PAR and NEG which allow expression of functional ß-lactamase, as revealed by nitrocefin staining. The inactivating nonsense mutation in the bla gene of the pBR322bla-stop plasmid is shown above the plates. See also Figure S1.

Figure 4. Superposition of 16S rRNA in the vicinity of NEG binding Site 1 with 18S rRNA

(A) Superposition of h34 of 16S rRNA (light yellow) from current 70S-NEG structure with homologous region of 18S rRNA (cyan) from yeast (PDB entry 3U5C (Ben-Shem et al., 2011)). Superposition is based on the alignment of nucleotides 1029–1050 of T. thermophilus 16S rRNA with nucleotides 1266–1287 of yeast 18S rRNA. (B) Close-up view of NEG Site 1 on the 70S ribosome showing 16S rRNA nucleotides interacting with NEG, superimposed with corresponding nucleotides of the 18S rRNA. In (A) and (B), nucleotides of 16S rRNA are labeled using the E. coli numbering system. In (B), yeast numbering is shown in brackets. Note that particular atoms involved in NEG binding have similar locations in both 16S rRNA and 18S rRNA.

Figure 5. NEG and TET have overlapping binding sites but different mechanisms of action

(A) Overview of the superimposed binding sites of TET (blue) and Site 1 of NEG (yellow) in the 30S subunit. The view and the coloring of 16S rRNA, mRNA and tRNAs are the same as in Figure 2A. Nucleotides 964–966 from h31 of the 16S rRNA, which are involved in NEG binding, are not shown for clarity. NEG structure is from the current work, TET is from PDB entry 4G5K (Jenner et al., 2013). All structures were aligned based on h34 of the 16S rRNA. (B, C, D) Close-up views of the binding sites shown in (A). (B) NEG and TET bind to the overlapping sites but occupy different space. (C) Interaction of NEG with the A-site tRNA possibly leading to tighter binding. (D) Co-occupancy of the ribosome with TET and A-site tRNA is impossible because of the steric clash of the drug with the tRNA anticodon.