Binding of aminoglycoside antibiotics to helix 69 of 23S rRNA

Abstract

Aminoglycosides antibiotics negate dissociation and recycling of the bacterial ribosome's subunits by binding to Helix 69 (H69) of 23S rRNA. The differential binding of various aminoglycosides to the chemically synthesized terminal domains of the Escherichia coli and human H69 has been characterized using spectroscopy, calorimetry and NMR. The unmodified E. coli H69 hairpin exhibited a significantly higher affinity for neomycin B and tobramycin than for paromomycin (K(d)s = 0.3 +/- 0.1, 0.2 +/- 0.2 and 5.4 +/- 1.1 microM, respectively). The binding of streptomycin was too weak to assess. In contrast to the E. coli H69, the human 28S rRNA H69 had a considerable decrease in affinity for the antibiotics, an important validation of the bacterial target. The three conserved pseudouridine modifications (Psi1911, Psi1915, Psi1917) occurring in the loop of the E. coli H69 affected the dissociation constant, but not the stoichiometry for the binding of paromomycin (K(d) = 2.6 +/- 0.1 microM). G1906 and G1921, observed by NMR spectrometry, figured predominantly in the aminoglycoside binding to H69. The higher affinity of the E. coli H69 for neomycin B and tobramycin, as compared to paromomycin and streptomycin, indicates differences in the efficacy of the aminoglycosides.

Figure 1.

Structures of the terminal stems and loops of the E. coli and human helix 69, and of the aminoglycosides. (A) Terminal hairpin sequence and secondary structure of helix 69 (H69) from the E. coli ribosome. The RNA was synthesized with and without the pseudouridine (Ψ) modifications at the 23S rRNA positions of 1911, 1915 and 1917. (B) Terminal hairpin sequence and secondary structure of helix 69 from the human ribosome. The RNA was synthesized without the pseudouridine (Ψ) modifications which occur naturally at the 28S rRNA positions of 3727, 3731 3733, 3737 and 3739. The chemical structures of the aminoglycosides used in the reported experiments: (C) Neomycin B; (D) Tobramycin; (E) Paromomycin; (F) Streptomycin.

Figure 2.

Thermal denaturations of the unmodified and modified E. coli H69 and the human H69. The unmodified (blue) and modified (red) E. coli H69 and the human H69 (green) RNA hairpins were subjected to repeated thermal denaturations and renaturations in the absence of aminoglycosides. The graphs are the results of point-by-point averages of nine transitions. The thermodynamic parameters obtained from the curves are found in Table 1.

Figure 3.

(A) Effect of neomycin B on the thermal stability of the unmodified E. coli H69. The unmodified E. coli H69 (1 µM) was titrated with increasing amounts of neomycin B (0–10 µM) and the complex RNA–neomycin B subjected to repeated thermal denaturations and renaturations. Each curve is the result of point-by-point averages of either nine or 12 transitions for each concentration: 0 µM (blue square), 0.75 µM (purple square), 1.50 µM (aqua square), 2.25 µM (yellow triangle), 3.00 µM (pink square), 4.50 µM (dark red circle), 5.25 µM (dark red square), 6.75 µM (small blue square) and 9.00 µM (dark cyan square). With increasing amounts of aminoglycoside, the T_m of the RNA increases. (B) Binding of neomycin B, tobramycin, paromomycin and streptomycin to the unmodified E. coli H69 and to the human H69. The differences in melting temperature (ΔT_m) of the H69 with the addition of increasing concentrations of aminoglycoside provided binding curves for each of the aminoglycosides. Tobramycin bound to the unmodified E. coli H69 (filled triangle) and to the human H69 (cross mark); neomycin bound to the unmodified E. coli H69 (filled circle) and to the human H69 (filled rhombus); paromomycin bound to the unmodified E. coli H69 (filled square) and to the unmodified human H69 (inverted filled triangle). Streptomycin was not bound by the unmodified E. coli H69 and thus, was assessed at two concentrations only (open square). The binding of paromomycin to the Ψ-modified E. coli H69 is also shown (double dagger). Addition of streptomycin did not increase the T_m. The binding affinities (K_d) and free energy of binding (ΔG°) for each aminoglycoside and for the different H69 are shown in Table 2. (C) The binding of paromomycin to the unmodified and Ψ-modified E. coli H69 and human H69. The unmodified (red square) and Ψ-modified (blue triangle) E. coli and human H69 (light-blue inverted triangle) (1 µM) were titrated with paromomycin (0–10 µM) and the RNA was thermally denatured and renatured, repeatedly. Binding curves were extracted from the differences in melting temperature (ΔT_m) of the two H69 RNAs with the addition of increasing concentrations of aminoglycoside. The binding affinities (K_d) and free energy of binding (ΔG°) paromomycin by the unmodified and Ψ-modified E. coli H69 are shown in Table 2.

Figure 4.

Paromomycin binding to the unmodified E. coli H69 detected by changes in CD spectra. CD spectra were collected of the unmodified E. coli H69 in absence of aminoglycoside (black line) and in the presence of increasing concentrations of paromomycin (0.75–9.00 µM). In this example, the ellipticity increased with increasing concentration of paromomycin at low concentrations (0.75, 1.50 and 2.25 µM; arrow up). At higher concentrations of paromoycin (3.00 4.50, 5.25, 6.00, 6.75, 7.50, 8.25 and 9.00 µM; arrow down), the ellipticity of the RNA decreased.

Figure 5.

ITC of neomycin B binding to the unmodified E. coli and human H69 hairpins. The unmodified E. coli and human H69 hairpins were titrated with neomycin and the change in heat capacity monitored by ITC. (A) Titration of the unmodified E. coli H69 with neomycin B as monitored by ITC. The ITC profile for the titration of the unmodified E. coli H69 with neomycin B was conducted at 37°C. Each heat burst is the result of a 10 μL injection of 250 μM neomycin. (B) Binding of neomycin by the unmodified E. coli (filled square) and human H69 RNAs (filled circle). The corrected injection heats (kcal/mol) are derived by integration of the corresponding heat burst curves (A) and corrected for background. The data for the titration of the human H69 has been normalized to that of the E. coli H69 in order to plot them on the same graph.

Figure 6.

Detection of the based paired imino protons of unmodified E. coli H69 by NMR. The base-paired, imino proton region of the one-dimensional ¹H NMR spectra of H69 exhibited six low field shifted resonances between 11.00 and 14.00 ppm: (A) H69-neomycin B complex. The line broadening of all resonances and the dramatic chemical shift change of G1906 and G1921 could be observed in comparison to the free H69 (B). H69–neomycin B complex. The line broadening of all resonances and the dramatic chemical shift change of G1906 and G1921 could be observed in comparison to the free H69 (B).

Figure 7.

¹H 2D NOESY spectrum of the unmodified E. coli H69. The 2D spectrum is expanded in order to focus on the imino protons resonance region (F₁ = 11.00–13.90 ppm; F₂ = 11.00–13.50). The sequential NOE connectivities between imino protons in H69 are shown and were used to unambiguously assign the base paired protons.

Figure 8.

Escherichia coli unmodified H69 ¹H 1D NOE difference NMR experiments. (A) The G1906 imino proton resonance of the free H69 is irradiated. G1906 and G1910 respective imino proton resonances were found to be in close proximity and as a consequence the irradiation of G1906 imino proton resonance expanded to G1910 and this, in addition to spin diffusion, resulted in the observation of very weak NOE effects between G1906 and all residues. (B) The G1906 imino proton resonance of the free H69 is irradiated in the presence of neomycin. Strong NOE effects are observed between G1906 and G1907, U1923 and G1921. Knowing that U1923 is adjacent to G1906, the NOE effect detected between these residues could be partially due to the spillage of G1906 resonance irradiation over the peak of U1923. The observation of an intense NOE between G1906 and the distant peak of G1907 imino proton allow us to conclude that neomycin could have restricted the dynamics of H69 upon H69–neomycin B complex formation. Arrow denotes the irradiated peak and asterisks denote the resulting NOE effects.