Structural basis for PoxtA-mediated resistance to Phenicol and Oxazolidinone antibiotics

Abstract

AbstractPoxtA and OptrA are ATP binding cassette (ABC) proteins of the F subtype (ABCF) that confer resistance to oxazolidinone, such as linezolid, and phenicol antibiotics, such as chloramphenicol. PoxtA/OptrA are often encoded on mobile genetic elements, facilitating their rapid spread amongst Gram-positive bacteria. These target protection proteins are thought to confer resistance by binding to the ribosome and dislodging the antibiotics from their binding sites. However, a structural basis for their mechanism of action has been lacking. Here we present cryo-electron microscopy structures of PoxtA in complex with the Enterococcus faecalis 70S ribosome at 2.9–3.1 Å, as well as the complete E. faecalis 70S ribosome at 2.2–2.5 Å. The structures reveal that PoxtA binds within the ribosomal E-site with its antibiotic resistance domain (ARD) extending towards the peptidyltransferase center (PTC) on the large ribosomal subunit. At its closest point, the ARD of PoxtA is still located >15 Å from the linezolid and chloramphenicol binding sites, suggesting that drug release is elicited indirectly. Instead, we observe that the ARD of PoxtA perturbs the CCA-end of the P-site tRNA causing it to shift by ∼4 Å out of the PTC, which correlates with a register shift of one amino acid for the attached nascent polypeptide chain. Given that linezolid and chloramphenicol are context-specific translation elongation inhibitors, we postulate that PoxtA/OptrA confer resistance to oxazolidinones and phenicols indirectly by perturbing the P-site tRNA and thereby altering the conformation of the attached nascent chain to disrupt the drug binding site.