Chemically related 4,5-linked aminoglycoside antibiotics drive subunit rotation in opposite directions

Abstract

Dynamic remodelling of intersubunit bridge B2, a conserved RNA domain of the bacterial ribosome connecting helices 44 (h44) and 69 (H69) of the small and large subunit, respectively, impacts translation by controlling intersubunit rotation. Here we show that aminoglycosides chemically related to neomycin-paromomycin, ribostamycin and neamine-each bind to sites within h44 and H69 to perturb bridge B2 and affect subunit rotation. Neomycin and paromomycin, which only differ by their ring-I 6'-polar group, drive subunit rotation in opposite directions. This suggests that their distinct actions hinge on the 6'-substituent and the drug's net positive charge. By solving the crystal structure of the paromomycin-ribosome complex, we observe specific contacts between the apical tip of H69 and the 6'-hydroxyl on paromomycin from within the drug's canonical h44-binding site. These results indicate that aminoglycoside actions must be framed in the context of bridge B2 and their regulation of subunit rotation.

Figure 1. Investigation of the neomycin family of aminoglycosides.

(a) 4,5-linked 2-deoxystreptamine (2-DOS) aminoglycosides in the neomycin family. (b) Overview of neomycin-binding sites in the small-subunit decoding site (h44, dark green) and in the large subunit H69 (light blue). 16S rRNA (30S) is shown in blue, 23S rRNA (50S) in grey, P-site tRNA in green and mRNA in magenta. (c) Cartoon illustrating the ribosome labelling strategy used for monitoring intersubunit rotation via single-molecule FRET, which has previously been shown to be affected by neomycin. (d) Aminoglycoside-induced inhibition of in vitro translation. IC₅₀ values are indicated. Experiments were performed in triplicate and the mean±s.d. is plotted.

Figure 2. Neomycin-induced effects on intersubunit rotation in wild-type and A1408G aminoglycoside-resistant ribosomes.

(a) (Left) Single-molecule fluorescence (donor—green; acceptor—red) and FRET (blue) trajectories illustrating typical conformational changes in ribosomes labelled as shown in Fig. 1c imaged in the absence of drug. FRET idealization is overlaid in red. (Right) A zoomed-in view highlights the transient nature of the intermediate-FRET state. (b,c) (top panels) smFRET trajectories summed into FRET histograms reveal the population behaviours across a range of neomycin concentrations in (b) wild-type and (c) A1408G ribosomes. (Bottom panels) Initial and final FRET values for each transition summed into two-dimensional histograms (transition density plots). Experiments were performed in triplicate on three separate days.

Figure 3. Paromomycin effects on intersubunit rotation in wild-type and A1408G aminoglycoside-resistant ribosomes.

(a,b) (Top panels) smFRET trajectories summed into FRET histograms reveal the population behaviours across a range of paromomycin concentrations in (a) wild-type and (b) A1408G ribosomes. (Bottom panels) Initial and final FRET values for each transition summed into two-dimensional histograms (transition density plots). Experiments were performed in triplicate on three separate days.

Figure 4. Crystal structure of the paromomycin-bound partially rotated ribosome.

(a) Paromomycin binding within the (left) h44-decoding site and (right) within H69. 16S rRNA (blue), 23S rRNA H69 (grey) and paromomycin (h44—green; H69—teal) are shown, along with a (2F_obs−F_calc) electron density map, calculated in PHENIX and contoured at 1.4 s.d. from the mean. Paromomycin and H69 rRNA contacts <3.5 Å are shown as dashed lines. (Inset) Paromomycin contacts within H69 are indistinguishable from those formed by neomycin (r.m.s.d.=0.412 Å). (b) Paromomycin induces global rearrangements of the 70S ribosome that are indistinguishable from those stabilized by neomycin. (Inset) View of the 30S subunit from the perspective of the 50S subunit. (Left) Difference in the vector shifts between equivalent RNA phosphorus atoms and protein Cα atoms in the unrotated compared with the partially rotated paromomycin-bound ribosome; (middle) superposition of the partially rotated neomycin-bound and partially rotated paromomycin-bound ribosomes. (Right) The fully rotated compared with the partially rotated paromomycin-bound ribosome. The vectors are colour coded as indicated in the scale. Ribosomes were superimposed using the 50S subunit as the frame of reference. 30S head domain: H; 30S body: B; 30S platform: P; 30S spur: Sp.

Figure 5. Paromomycin contacts the universally conserved A1913 residue of H69 from its canonical h44 site of binding.

(a) The decoding site region of the paromomycin- (left) and neomycin- (right) bound ribosome (unrotated) exhibit strong and weak electron density for residue A1913 of H69, respectively. The 16S rRNA (blue), 23S rRNA (grey), h44-bound paromomycin (green) and h44-bound neomycin (light blue) are overlaid with feature-enhanced electron density maps, calculated in PHENIX and contoured at 1.4 s.d. from the mean. The temperature factors for residue A1913 are as follows. Unrotated paromomycin-bound: ∼90 Ų; unrotated neomycin-bound: ∼430 Ų. The lack of density for A1913 in the neomycin-bound structure is highlighted with an asterisk. (b) Paromomycin (green) bound within the canonical small-subunit h44-decoding site (blue) contacts the apical tip of H69 via paromomycin ring I (6′-O) and the universally conserved base A1913 (N6) of H69 (grey).

Figure 6. Neomycin and paromomycin rinse off of h44 but not H69.

Aminoglycoside real-time deliveries and rinse-outs were performed on wild-type ribosomes. For both (a) neomycin and (b) paromomycin, five population FRET histograms are shown representing the chronological order of the experiment. Left to right: (1) no drug; (2) real-time delivery of 10 μM aminoglycoside; (3) aminoglycoside equilibrium; (4) real-time rinse-out; (5) 90 s following rinse-out.