Structural and mechanistic basis for translation inhibition by macrolide and ketolide antibiotics

Abstract

Macrolides and ketolides comprise a family of clinically important antibiotics that inhibit protein synthesis by binding within the exit tunnel of the bacterial ribosome. While these antibiotics are known to interrupt translation at specific sequence motifs, with ketolides predominantly stalling at Arg/Lys-X-Arg/Lys motifs and macrolides displaying a broader specificity, a structural basis for their context-specific action has been lacking. Here, we present structures of ribosomes arrested during the synthesis of an Arg-Leu-Arg sequence by the macrolide erythromycin (ERY) and the ketolide telithromycin (TEL). Together with deep mutagenesis and molecular dynamics simulations, the structures reveal how ERY and TEL interplay with the Arg-Leu-Arg motif to induce translational arrest and illuminate the basis for the less stringent sequence-specific action of ERY over TEL. Because programmed stalling at the Arg/Lys-X-Arg/Lys motifs is used to activate expression of antibiotic resistance genes, our study also provides important insights for future development of improved macrolide antibiotics.

Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling

“…Only a handful of other ligand-dependent arrest peptides have been structurally characterized to date 23 , 38 – 42 , including only one other bacterial amino acid-sensing peptide 23 . The best-characterized group comprises drug-dependent arrest peptides belonging to the Erm family, which undergo translational arrest in response to macrolide antibiotics to regulate the expression of macrolide-resistance genes.…”

“…The best-characterized group comprises drug-dependent arrest peptides belonging to the Erm family, which undergo translational arrest in response to macrolide antibiotics to regulate the expression of macrolide-resistance genes. These arrest peptides, among which ErmBL 41 , ErmCL 40 , and ErmDL 42 have been characterized structurally, constitute a special case in that the antibiotic ligand binds to the ribosome with high affinity even in the absence of nascent peptide 28 . As a result, macrolide-dependent arrest peptides are akin to ligand-independent arrest peptides like SecM 43 , MifM 44 , or VemP 24 , with the difference that the shape and dimensions of the exit tunnel are altered by the bound ligand.…”

Cryo-EM structure of Mycobacterium tuberculosis 50S ribosomal subunit bound with clarithromycin reveals dynamic and specific interactions with macrolides

“…The major population of A2062 prefers non-swayed conformation in the vacant ribosome-TEL complex, but tends to rotate about 90° upon the nascent polypeptide chain (NPC or ErmDL) binding, while A2062 remains non-swayed conformation in ERY binding form even the ErmDL is present. The minor alternative conformation exists in both ribosome-TEL structure forms [ 44 ], Figure S7.…”

“…The additional simulation and experiments show that the swayed conformation of A2062 in ErmDL-TEL-stalled ribosomal complex (SRC) is also correlated to the induced state of U2585, but no induced state of U2585 is found in the ERY bound form with the unrotated A2062 [ 44 ]. In the case of ERY binding with A2062, the ribosomal base A2062 has a high probability of contact with ERY in simulation, but the strong interaction and stable conformation may restrict its flexibility and dynamics, which indicates the important role in inhibiting protein synthesis [ 43 ].…”

“…Kannan et al showed that the mutation A2062U confers resistance to ERY derivatives [ 46 ], instead of ketolide (TEL/SOL). The above discoveries may imply that translation modulation may be influenced by the affinity advances of alkyl-aryl side chain as well as the dynamic contributions of 6-methoxy of TEL [ 44 ].…”