Abstract

Target protection proteins confer resistance to the host organism by directly binding to the antibiotic target. One class of such proteins are the antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F-subtype (ARE-ABCFs), which are widely distributed throughout Gram-positive bacteria and bind the ribosome to alleviate translational inhibition from antibiotics that target the large ribosomal subunit. Here, we present single-particle cryo-EM structures of ARE-ABCF-ribosome complexes from three Gram-positive pathogens: Enterococcus faecalis LsaA, Staphylococcus haemolyticus VgaALC and Listeria monocytogenes VgaL. Supported by extensive mutagenesis analysis, these structures enable a general model for antibiotic resistance mediated by these ARE-ABCFs to be proposed. In this model, ABCF binding to the antibiotic-stalled ribosome mediates antibiotic release via mechanistically diverse long-range conformational relays that converge on a few conserved ribosomal RNA nucleotides located at the peptidyltransferase center. These insights are important for the future development of antibiotics that overcome such target protection resistance mechanisms.

Highlights

  • Target protection proteins confer resistance to the host organism by directly binding to the antibiotic target

  • The AREABCFs co-migrated with the 70S fraction through sucrose gradients—with the complex further stabilized in the presence of ATP in the case of LsaA and VgaALC—and co-eluted with ribosomal proteins after affinity purification (Supplementary Figs. 1–3)

  • The resulting in vivo formed complexes were characterized by single-particle cryo-electron microscopy (cryo-EM), yielding antibiotic resistance (ARE)-70S complexes with average resolutions of 2.9 Å for E. faecalis LsaA, 3.1 Å for S. aureus VgaALC and 2.9 Å for L. monocytogenes VgaL (Fig. 1a–c, Supplementary Table 4 and Supplementary Figs. 4–6)

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Summary

Introduction

Target protection proteins confer resistance to the host organism by directly binding to the antibiotic target One class of such proteins are the antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F-subtype (ARE-ABCFs), which are widely distributed throughout Gram-positive bacteria and bind the ribosome to alleviate translational inhibition from antibiotics that target the large ribosomal subunit. In contrast to the macrolides and streptogramin B antibiotics that predominantly inhibit translation during elongation[7], the PLSA antibiotics overlap with the amino acids attached to the CCAends of the A- and/or P-site of tRNAs and trap ribosomes during or directly after initiation[8,9,10]. A lincomycin-resistance ABCF that belongs to this group, termed Lmo0919, has been reported in Listeria monocytogenes[34,35,36]

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