A ketolide resistance mutation in domain II of 23S rRNA reveals the proximity of hairpin 35 to the peptidyl transferase centre

Abstract

Ketolides represent a new generation of macrolide antibiotics. In order to identify the ketolide-binding site on the ribosome, a library of Escherichia coli clones, transformed with a plasmid carrying randomly mutagenized rRNA operon, was screened for mutants exhibiting resistance to the ketolide HMR3647. Sequencing of the plasmid isolated from one of the resistant clones and fragment exchange demonstrated that a single U754A mutation in hairpin 35 of domain II of the E. coli 23S rRNA was sufficient to confer resistance to low concentrations of the ketolide. The same mutation also conferred erythromycin resistance. Both the ketolide and erythromycin protected A2058 and A2059 in domain V of 23S rRNA from modification with dimethyl sulphate, whereas, in domain II, the ketolide protected, while erythromycin enhanced, modification of A752 in the loop of the hairpin 35. Thus, mutational and footprinting results strongly suggest that the hairpin 35 constitutes part of the macrolide binding site on the ribosome. Strong interaction of ketolides with the hairpin 35 in 23S rRNA may account for the high activity of ketolides against erythromycin-resistant strains containing rRNA methylated at A2058. The existence of macrolide resistance mutations in the central loop of domain V and in hairpin 35 in domain II together with antibiotic footprinting data suggest that these rRNA segments may be in close proximity in the ribosome and that hairpin 35 may be a constituent part of the ribosomal peptidyl transferase centre.

Binding and Action of CEM-101, a New Fluoroketolide Antibiotic That Inhibits Protein Synthesis

“…However, the conclusions about the contribution of the 11,12 side chain to the drug activity should be drawn only cautiously. Although original studies suggested that interactions of the side chain of ketolides with A752 in the loop of helix 35 in 23S rRNA significantly contribute to the drugs' affinity (9,17,49), more recent genetic studies questioned that conclusion because mutations at and around A752 or at U2609, with which the side chains interact, had only a minor effect upon drug inhibitory action (14,21,30,49). In agreement with these observations, the additional interaction afforded by the extended side chains of telithromycin and CEM-101 or, for that matter, the fluorine atom at the C-2 position of the CEM-101 lactone did not translate in our study to an improved ability of either of the ketolides compared with that of azithromycin in inhibiting erythromycin binding to the ribosome (Table 1).…”

“…Various ketolides differ from CEM-101 in the chemical nature of the alkyl-aryl side chain and the site of its attachment to the lactone scaffold. Nevertheless, in the RNA probing experiments, all these drugs afford a nearly complete protection of A752 in the E. coli ribosome from chemical modification (9,17,48,49), indicating that the interaction of the alkyl-aryl side chain with the A752-U2609 base pair is important for binding of a range of clinically relevant ketolides to the ribosome. Despite a generally similar orientation of the side chains of CEM-101 and telithromycin, the variation in their chemical structures produces an important difference in the mode of binding.…”

“…Early biochemical and genetic studies showed that the extended side chain of ketolides establishes important new interactions with the ribosome that might account for the increased efficacy of these drugs. Specifically, chemical probing and resistance mutations pointed to interactions of the 11,12 side chain of telithromycin with the rRNA residues in the loop of helix 35 of the E. coli 23S rRNA and with U2609 (14,17,49). However, subsequent crystallographic studies of the first clinically approved ketolide telithromycin bound to the bacterial (Deinococcus radiodurans) or archaeal (Haloarcula marismortui) ribosome showed the placement of the 11,12 side chain in a position that was hardly compatible with rRNA protections and mutations observed in the E. coli ribosome (3,43).…”

The structure of ribosome-lankacidin complex reveals ribosomal sites for synergistic antibiotics

“…Most of these compounds inhibit cell growth by interfering with peptide bond formation (15). The second major antibiotic binding site in the large ribosomal subunit is located at the upper segment of the nascent peptide exit tunnel (NPET), adjacent to the PTC, and is used by macrolides and type B streptogramins (3,7,(16)(17)(18)(19)(20)(21). Binding to this site impedes progression of the nascent proteins toward the tunnel exit.…”

Synthesis and antibacterial activity of novel 11,12-cyclic carbonate azithromycin 4″-O-carbamate derivatives

“…7 Their mechanism of action has been reported to interact with nucleotide A752 directly in domain II of the 23S rRNA in addition to the main interaction of the drugs in domain V and inhibit protein synthesis by blocking elongation. 8 This results in tighter binding to ribosomes and imparts some activity against methylated ribosomes in some species. 9,10 The study of the high-resolution X-ray co-crystal structures has revealed that macrolides bind at the entrance to the peptide tunnel in the 23S rRNA, and the cladinose group in their structures is located at and fits with the cavity formed by G2505, C2610 and C2611 in domain V. 11 On the basis of the results of the X-ray co-crystal structure study, many new derivatives of macrolides for the effective management of erythromycin resistance have been investigated by different research groups.…”