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Binding of aminoglycoside antibiotics to helix 69 of 23S rRNA.

Scheunemann AE, Graham WD, Vendeix FA, Agris PF - Nucleic Acids Res. (2010)

Bottom Line: 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).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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695-7622, USA.

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.

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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.
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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.

Mentions: Prior to the above-mentioned titration, the imino protons of the free H69 were identified and assigned by using a combination of 1H 1D and 2D NOESY NMR experiments run at 2°C and in aqueous solvent (see Materials and Methods section). The results of these experiments yielded six imino proton resonances observed between 11.00 and 14.00 ppm (Figure 6A). The identified peaks were sequentially and specifically assigned by using the cross-peaks observed in the same spectral region of the 2D NOESY spectrum (Figure 7). The two imino protons engaged in the wobble base pair G1907–U1923 were readily identified and assigned due to their distinctive chemical shifts found at 11.62 and 12.06 ppm (Table 3). This preliminary result constituted the starting point for the assignment of G1910, G1921 and G1922 imino protons (Figure 7; Table 3). The broad peak observed at 12.83 ppm on the 1D spectrum of the H69 (Figure 6A; Table 3) could not be identified on the NOESY spectrum indicating a fast exchange between the corresponding imino proton and the protons of water. The fast exchange could be due to the fraying of the terminal stem base pair G1906–C1924 and therefore, this resonance was assigned to the G1906 imino proton. The identification and assignment of U1911 imino protons and the non-hydrogen bonded imino protons of the loop residues were prevented by this property of fast exchange (57).Table 3.


Binding of aminoglycoside antibiotics to helix 69 of 23S rRNA.

Scheunemann AE, Graham WD, Vendeix FA, Agris PF - Nucleic Acids Res. (2010)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875026&req=5

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.
Mentions: Prior to the above-mentioned titration, the imino protons of the free H69 were identified and assigned by using a combination of 1H 1D and 2D NOESY NMR experiments run at 2°C and in aqueous solvent (see Materials and Methods section). The results of these experiments yielded six imino proton resonances observed between 11.00 and 14.00 ppm (Figure 6A). The identified peaks were sequentially and specifically assigned by using the cross-peaks observed in the same spectral region of the 2D NOESY spectrum (Figure 7). The two imino protons engaged in the wobble base pair G1907–U1923 were readily identified and assigned due to their distinctive chemical shifts found at 11.62 and 12.06 ppm (Table 3). This preliminary result constituted the starting point for the assignment of G1910, G1921 and G1922 imino protons (Figure 7; Table 3). The broad peak observed at 12.83 ppm on the 1D spectrum of the H69 (Figure 6A; Table 3) could not be identified on the NOESY spectrum indicating a fast exchange between the corresponding imino proton and the protons of water. The fast exchange could be due to the fraying of the terminal stem base pair G1906–C1924 and therefore, this resonance was assigned to the G1906 imino proton. The identification and assignment of U1911 imino protons and the non-hydrogen bonded imino protons of the loop residues were prevented by this property of fast exchange (57).Table 3.

Bottom Line: 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).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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695-7622, USA.

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.

Show MeSH
Related in: MedlinePlus