Regulation and mechanistic basis of macrolide resistance by the ABC-F ATPase MsrD

Antibiotic resistance ABC-Fs (ARE ABC-Fs) are translation factors currently proliferating among human pathogens that provide resistance against clinically important ribosome-targeting antibiotics. Here, we combine genetic and structural approaches to determine the activity of the streptococcal ARE ABC-F protein MsrD on the ribosome and its regulation in response to macrolide exposure. We show that cladinose-containing macrolides lead to insertion of MsrDL leader peptide into a conserved crevice of the ribosomal exit tunnel, which remained thus far undocumented, concomitantly with 23S rRNA rearrangements that preclude proper accommodation of release factors and inhibits termination. The stalled ribosome obstructs formation of a Rho-independent terminator which prevents msrD transcriptional attenuation. This stalled ribosome is rescued by MsrD powered by its two functionally asymmetric ATPase sites, but not by MsrD mutants which do not provide antibiotic resistance, showing evidence of equivalence between MsrD function in antibiotic resistance and its action on this complex.