Structure of Erm-modified 70S ribosome reveals the mechanism of macrolide resistance

Abstract

Many antibiotics inhibit bacterial growth by binding to the ribosome and interfering with protein biosynthesis. Macrolides represent one of the most successful classes of ribosome-targeting antibiotics. The main clinically-relevant mechanism of resistance to macrolides is dimethylation of the 23S rRNA nucleotide A2058 located in the drug binding site, a reaction catalyzed by the Erm-type rRNA-methyltransferases. Here, we present the crystal structure of the Erm-dimethylated 70S ribosome at 2.4Å resolution together with the structures of unmethylated 70S ribosome functional complexes alone and in combination with macrolides. Altogether, our structural data do not support the previous models and, instead, suggest a principally new explanation of how A2058-dimethylation confers resistance to macrolides. Moreover, high-resolution structures of two macrolide antibiotics bound to the unmodified ribosome revealed a previously unknown role of desosamine moiety in drug binding, laying a foundation for the rational knowledge-based design of macrolides that can overcome Erm-mediated resistance.

Binding and Action of Triphenylphosphonium Analog of Chloramphenicol upon the Bacterial Ribosome

“…This interaction results in rotation of the nucleobase of A2062 by ~160° around its N-glycosidic bond into a position where it forms a Hoogsteen base pair with the residue m 2 A2503 (Figure 4C). Similar re-orientation of this nucleotide was observed previously in the ribosome-CHL structures [2-4], as well as in structures of ribosome-bound amino acid derivatives of CHL [13] and macrolides [2,28]. These additional interactions of the TPP moiety of the CAM-C4-TPP The overall binding position of the amphenicol moiety of ribosome-bound CAM-C4-TPP is identical to those observed previously for the parent CHL compound (in complex with either Escherichia coli [4] or Tth [3] 70S ribosomes in the absence of mRNA and tRNAs or in the complex with mRNA and all three tRNAs (Figure 4B, green; Figure S2A) [2]) as well as for the CHL analog histidyl-CAM (Figure 4B, blue) [13].…”

“…This interaction results in rotation of the nucleobase of A2062 by~160 • around its N-glycosidic bond into a position where it forms a Hoogsteen base pair with the residue m 2 A2503 (Figure 4C). Similar re-orientation of this nucleotide was observed previously in the ribosome-CHL structures [2][3][4], as well as in structures of ribosome-bound amino acid derivatives of CHL [13] and macrolides [2,28]. These additional interactions of the TPP moiety of the CAM-C4-TPP likely account for its increased affinity to the ribosome and better inhibitory properties compared to those of CHL.…”

“…The amphenicol moieties of CAM-C4-TPP, its parent CHL, and the fluorescent derivative BODIPY-CAM are expected to have identical ribosome binding sites and, therefore, these compounds are expected to compete with each other for binding to the ribosome. Although CHL binds in the A site of the PTC [2][3][4]27] and ERY binds in the upper part of the NPET [2,3,28], their binding sites sufficiently overlap resulting in competition for binding to the ribosome between CHL-and ERY-based compounds [2,29]. The apparent dissociation constants K Dapp of CHL determined from the displacement of BODIPY-CAM (2.6 ± 1.5 µM) or BODIPY-ERY (2.8 ± 0.5 µM) are consistent with each other and with the previously published data [13], including data determined by direct [ 14 C]-CHL binding (2.3 µM [30]).…”

“…To understand the functional relevance of the introduced TPP moiety of CAM-C4-TPP (Figure 3A) in the context of the bacterial ribosome, we have determined the crystal structure of CAM-C4-TPP bound to the Thermus thermophilus (Tth) 70S ribosome at 2.80Å resolution (Table S1). In this experiment, we used Tth 70S ribosomes complexed with E. coli protein Y (PY) as a tool to obtain structures of higher resolution [13,28,31,32]. This approach is based on our previous finding that binding of PY to a vacant 70S ribosome stabilizes it by locking the head of the 30S subunit in an unrotated state, which leads to overall better diffraction and, subsequently, improved structural resolution [31,32].…”

“…Note that nucleotide A2062 of the 23S rRNA is pointed toward the viewer and is not involved in Hoogsteen-edge base pairing with the nucleotide m 2 A2503 in the NPET wall. (B-F) Occlusion of the nascent peptide exit tunnel by chloramphenicol (B, green, PDB entry 6ND5 [2]), CAM-C4-TPP (C, yellow), macrolide erythromycin (D, red, PDB entry 6XHX [28]), lincosamide clindamycin (E, magenta, PDB entry 4V7V [4]), and type B streptogramin quinupristin (F, blue, PDB entry 4U26 [38]). Note that, unlike A-site binding chloramphenicol, the NPET-binding antibiotics, such as macrolides, lincosamides, or streptogramins B, as well as CAM-C4-TPP partially obstruct the lumen of the NPET.…”