Structural basis for PoxtA-mediated resistance to phenicol and oxazolidinone antibiotics

Abstract

PoxtA and OptrA are ATP binding cassette (ABC) proteins of the F subtype (ABCF). They confer resistance to oxazolidinone and phenicol antibiotics, such as linezolid and chloramphenicol, which stall translating ribosomes when certain amino acids are present at a defined position in the nascent polypeptide chain. These proteins are often encoded on mobile genetic elements, facilitating their rapid spread amongst Gram-positive bacteria, and are thought to confer resistance by binding to the ribosome and dislodging the bound antibiotic. However, the mechanistic basis of this resistance remains unclear. Here we refine the PoxtA spectrum of action, demonstrate alleviation of linezolid-induced context-dependent translational stalling, and present cryo-electron microscopy structures of PoxtA in complex with the Enterococcus faecalis 70S ribosome. PoxtA perturbs the CCA-end of the P-site tRNA, causing it to shift by ∼4 Å out of the ribosome, corresponding to a register shift of approximately one amino acid for an attached nascent polypeptide chain. We postulate that the perturbation of the P-site tRNA by PoxtA thereby alters the conformation of the attached nascent chain to disrupt the drug binding site.

Fig. 1. Alignment of the ARDs from diverse bacterial ABCFs.

The PoxtA and OptrA ARDs are slightly longer (4–5 amino acids) than the equivalent region in EttA, but significantly shorter than in other ARE–ABCFs. The central region with an orange highlight, which includes the ARD loop and some of the adjacent helices in some proteins, was not aligned but simply ordered by length. Sequences were aligned with MAFFT and edited by hand to reduce gap placement. Sequence accession numbers in the title are Uniprot or NCBI protein accessions.

Fig. 2. Context-dependent ribosomal stalling induced by linezolid is countered by PoxtA.

a A heatmap of log₂-relative change in the 5Pseq coverage of wild-type E. faecalis upon linezolid treatment. The distance (in nucleotides, nt) from the 5ʹ of the sequenced mRNA fragments to synonymous codons encoding a specified amino acid is indicated on the x axis. The 0 nt position corresponds to the first nucleotide of the codon. Dark blue signifies decreased 5PSeq coverage upon linezolid treatment, yellow signifies increased coverage upon linezolid treatment. A specific increase in the 5Pseq coverage associated with synonymous codons located at –8 nucleotides is indicative of a ribosomal stall with the corresponding amino acid in the −1 position of the nascent chain, as illustrated schematically in panels b and c (ref. ). b, c Metagene analysis of 5Pseq libraries aligned to alanine codons. Linezolid-induced ribosomal stalling results in a specific increase in 5Pseq read counts 8 nt upstream of alanine codons, indicating a ribosomal stall with alanine residues in the −1 position (see schematics). Samples were prepared with E. faecalis harbouring the empty pCIE_spec vector (a, b) or upon poxtA expression (c, blue line), with and without linezolid treatment. Exponentially growing cells were treated with 4 μg/mL linezolid (final concentration) 10 min before harvest. All analyses were performed on pooled datasets from three biological replicates.

Fig. 3. Cryo-EM structures of PoxtA–70S complexes.

a–c Cryo-EM maps with isolated densities for (a, b) E. faecium PoxtA (red) in complex with the E. faecalis 70S ribosome and a P-tRNA (cyan) or b P-tRNA (cyan) and A-tRNA (tan), c P-tRNA (cyan) only, with small subunit (SSU, yellow) and large subunit (LSU, grey). d Density (grey isosurface) with molecular model of PoxtA from a coloured according to domain as represented in the associated schematics: nucleotide-binding domain 1 (NBD1, tan), antibiotic-resistance domain (ARD, pink), nucleotide-binding domain 2 (NBD2, green) and C-terminal extension (CTE, grey, not modelled). α1 and α2 indicate the two α-helices of the ARD interdomain linker. In d, the ATP nucleotides are coloured blue. e Close view of the ARD tip from state I with sharpened map. f Close view of ATPs bound by PoxtA (state I).

Fig. 4. Interactions between PoxtA and the ribosome-P-tRNA complex.

a Overview of PoxtA interactions with the 23S rRNA (grey), 16S rRNA h41 (yellow), uL1 (gold), uS7 (green), uL5 (pink), bL33 (tan) and the P-tRNA (light blue). The P-tRNA CCA 3ʹ end, acceptor stem (Acc.), and elbow are indicated. b–e Interactions between the P-tRNA elbow (light blue) and the PoxtA NBD2 (b), the P-tRNA acceptor stem (light blue) and the PoxtA ARD (c), the ARD and the P-tRNA CCA-end, including a modelled water molecule (labelled W) (d, e). f An interaction between the PoxtA ARD α2 and the 23S rRNA. The high-resolution model from the combined 70S volume was used.

Fig. 5. PoxtA modulates the conformation of the P-tRNA.

a–c Comparison of of the P-site tRNA from (a) E. faecalis 70S–P-tRNA only complex (grey), (b) PoxtA–70S complex (state I, PoxtA in red and P-tRNA in light blue) and (c) LsaA–70S complex (PDB ID 7NHK, LsaA in green and P-tRNA in light blue). Models of chloramphenicol for PoxtA (PDB ID 6ND5) and lincomycin for LsaA (PDB ID 5HKV) are superimposed for reference. d close-up of (b) showing P-tRNA acceptor stem distortion induced by PoxtA binding. e, f Interaction between the 23S rRNA P-loop (grey) and P-tRNA (light blue) for (e) the P-tRNA-only complex (state IV) and f with bound PoxtA (state I, state IV P-tRNA is overlayed in transparent grey for comparison). g, h Indirect modulation of the chloramphenicol binding site by PoxtA. g The structure of chloramphenicol (cam) stabilised by a nascent peptide chain with alanine in the −1 position. h Modelled shift of the nascent chain shown in g induced by PoxtA displacement of the P-tRNA CCA-end. The P-tRNA from g is overlaid in transparent grey for comparison.

Fig. 6. Model for ribosome protection by PoxtA.

a Elongating ribosomes are stalled by PhO antibiotics with a peptidyl-tRNA in the P-site. The sidechain of the amino acid at position −1 contributes to the PhO-binding site. b Stalled ribosomes are recognised by PoxtA, which induces a shifted P-site tRNA and nascent chain, thereby disrupting the PhO-binding site. c After dissociation of the PhO drug and PoxtA, the P-tRNA and nascent chain return to the regular conformation. Accommodation of an A-tRNA occludes the drug-binding site. d After peptidyl transfer and translocation, amino acid at position −1 would change, thereby resetting the PhO-binding site.