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

Bertrand Beckert,Daniel N Wilson,Shanmugapriya Sothiselvam,Maxim S Svetlov,Lars V Bock,Michael Graf,Nora Vázquez-Laslop,C Axel Innis,Maha Abdelshahid,Britta Seip,Stefan Arenz,Alexander S Mankin,Helmut Grubmüller,Elodie C Leroy

doi:10.1038/s41467-021-24674-9

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.

Highlights

Macrolides and ketolides comprise a family of clinically important antibiotics that inhibit protein synthesis by binding within the exit tunnel of the bacterial ribosome
We have employed the ErmDL leader uORF that bears an internal Arg6-Leu7-Arg[8] (+X+) motif as a model to investigate the molecular basis of translation arrest at these sequences by the macrolide ERY and the ketolide TEL
We have demonstrated that in the presence of TEL, an intact +X+ motif is critical since substitution of Arg[6] or Arg[8] to any other amino acid, except Lys, abolished the translational arrest (Fig. 2b, c, f)

Summary

Introduction

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. 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). One important family of ribosome-targeting antibiotics are the macrolides, which display broad-spectrum activity against many Gram-positive bacteria, and have been in clinical usage since the discovery of the founding member erythromycin (ERY) in the 1950s3,4. Structures of macrolides and ketolides in complex with the ribosome revealed that these compounds bind in a similar fashion within the nascent polypeptide exit tunnel (NPET)[7,8,9,10]

Methods

Results

Conclusion