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 (Kds = 0.3 ± 0.1, 0.2 ± 0.2 and 5.4 ± 1.1 µM, 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 (Ψ1911, Ψ1915, Ψ1917) occurring in the loop of the E. coli H69 affected the dissociation constant, but not the stoichiometry for the binding of paromomycin (Kd = 2.6 ± 0.1 µM). 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.

New trends in the use of aminoglycosides

“…Instead, some AGs, including NEO, PAR, and TOB, have been identified to have a secondary binding site at the major groove of h69 of the 23S rRNA of the 50S ribosomal subunit. 64, 72 Crystallography data revealed that h69 forms direct contact with the A-/P-site tRNAs, as well as the decoding center, by looping around the interface of the two subunits and forming an inter-subunit bridge (B2). Due to the vital position of h69 in the 3D structure, the binding of AGs to h69 hinders the ribosomal movement/global conformational rearrangement when the 30S rotates around the 50S ribosomal subunit in racquet-like movement around the L1 stalk domain.…”

Nucleotide modifications and tRNA anticodon–mRNA codon interactions on the ribosome

“…We use the most exact computational method, free energy perturbation, to achieve this to the accuracy of the interaction model (i.e., the force field). Linear interaction energy calculations on tRNA Phe binding to different codons in the ribosomal decoding center (Almlöf et al 2007) and free energy calculations on the formation (Scheunemann et al 2010) of modified base pairs in RNA double helices in solution (Scheunemann et al 2010) have shown that current force fields and simulation protocols are capable of providing accurate results for this kind of system.…”

Detection and Quantification of Ribosome Inhibition by Aminoglycoside Antibiotics in Living Bacteria Using an Orthogonal Ribosome-Controlled Fluorescent Reporter

“…8-10 , translocation 2,3,11,12 , and ribosome recycling 8 and by inducing translational miscoding 2-4,13 through specific interactions with the decoding site (A-site) of the 16S ribosomal RNA (rRNA) in helix 44 (h44) of the small (30S) ribosomal subunit 14-17 (Figure 1b), and in the case of some aminoglycosides, with helix 69 (H69) of the large (50S) ribosomal subunit. 8,10,12,15,18 Aminoglycosides also show promise as treatments for other diseases, including HIV 2 ; human genetic disorders, where their ability to induce miscoding has been used to suppress disease-associated premature termination codons 2,19 ; and fungal infection, where amphiphilic aminoglycoside analogs have been shown to perturb the function of the fungal plasma membrane. 20 …”