Binding Site of Macrolide Antibiotics on the Ribosome: New Resistance Mutation Identifies a Specific Interaction of Ketolides with rRNA

Abstract

Macrolides represent a clinically important class of antibiotics that block protein synthesis by interacting with the large ribosomal subunit. The macrolide binding site is composed primarily of rRNA. However, the mode of interaction of macrolides with rRNA and the exact location of the drug binding site have yet to be described. A new class of macrolide antibiotics, known as ketolides, show improved activity against organisms that have developed resistance to previously used macrolides. The biochemical reasons for increased potency of ketolides remain unknown. Here we describe the first mutation that confers resistance to ketolide antibiotics while leaving cells sensitive to other types of macrolides. A transition of U to C at position 2609 of 23S rRNA rendered E. coli cells resistant to two different types of ketolides, telithromycin and ABT-773, but increased slightly the sensitivity to erythromycin, azithromycin, and a cladinose-containing derivative of telithromycin. Ribosomes isolated from the mutant cells had reduced affinity for ketolides, while their affinity for erythromycin was not diminished. Possible direct interaction of ketolides with position 2609 in 23S rRNA was further confirmed by RNA footprinting. The newly isolated ketolide-resistance mutation, as well as 23S rRNA positions shown previously to be involved in interaction with macrolide antibiotics, have been modeled in the crystallographic structure of the large ribosomal subunit. The location of the macrolide binding site in the nascent peptide exit tunnel at some distance from the peptidyl transferase center agrees with the proposed model of macrolide inhibitory action and explains the dominant nature of macrolide resistance mutations. Spatial separation of the rRNA residues involved in universal contacts with macrolides from those believed to participate in structure-specific interactions with ketolides provides the structural basis for the improved activity of the broader spectrum group of macrolide antibiotics.Macrolide antibiotics inhibit protein synthesis in a wide range of pathogenic bacteria. The drugs bind to a single site in the large ribosomal subunit located near the entrance to the nascent peptide tunnel and are thought to sterically hinder the growth of the polypeptide chain and cause dissociation of peptidyl-tRNA from the ribosome (1, 10, 18, 33). Macrolide antibiotics were also shown to affect ribosome assembly (9).The macrolide binding site is composed primarily of RNA. Two segments of 23S rRNA, the central loop of domain V and the loop of hairpin 35 from domain II, are believed to be the major components of the drug binding site on the ribosome. Mutations of A2058 (Escherichia coli numeration) and several neighboring positions in the central loop of domain V confer resistance to macrolides (27), as reviewed in references 34 and 35. Methylation of A2058 by Erm-type methyl-transferases drastically reduces the affinity of macrolides for the ribosome, suggesting the direct interaction between this RNA residue and the drug (11,1...

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).…”

“…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).…”

Effects of the antimicrobial tylosin on the microbial community structure of an anaerobic sequencing batch reactor

“…Macrolides inhibit protein synthesis by interacting with the 50S subunit of the bacterial ribosome. Although macrolides are not expected to target methanogenic archaea (Auerbach et al, 2004;Garza-Ramos et al, 2001), the inhibition of anaerobic bacteria may result in indirect adverse effects on methanogenesis. Most Gram-negative bacteria are resistant to macrolides due to relative impermeability of the cellular outer membrane and active drug efflux systems (Nikaido, 1996).…”

Macrolide resistance mechanisms inEnterobacteriaceae: Focus on azithromycin

“…In species such as M. pneumoniae or S. pneumoniae it has been found that C2611A and C2611G mutations result in a similar pattern of resistance (Pereyre et al, 2004;Tait-Kamradt et al, 2000), and may thus predict a similar scenario in Enterobacteriaceae. Meanwhile, the U2609C mutation has a differential effect, resulting in slightly higher erythromycin and azithromycin susceptibility levels but an increase in the telithromycin and cethromycin resistance (Garza-Ramos et al, 2001).…”