Structural Basis for Linezolid Binding Site Rearrangement in the Staphylococcus aureus Ribosome

Abstract

An unorthodox, surprising mechanism of resistance to the antibiotic linezolid was revealed by cryo-electron microscopy (cryo-EM) in the 70S ribosomes from a clinical isolate of Staphylococcus aureus This high-resolution structural information demonstrated that a single amino acid deletion in ribosomal protein uL3 confers linezolid resistance despite being located 24 Å away from the linezolid binding pocket in the peptidyl-transferase center. The mutation induces a cascade of allosteric structural rearrangements of the rRNA that ultimately results in the alteration of the antibiotic binding site.IMPORTANCE The growing burden on human health caused by various antibiotic resistance mutations now includes prevalent Staphylococcus aureus resistance to last-line antimicrobial drugs such as linezolid and daptomycin. Structure-informed drug modification represents a frontier with respect to designing advanced clinical therapies, but success in this strategy requires rapid, facile means to shed light on the structural basis for drug resistance (D. Brown, Nat Rev Drug Discov 14:821-832, 2015, https://doi.org/10.1038/nrd4675). Here, detailed structural information demonstrates that a common mechanism is at play in linezolid resistance and provides a step toward the redesign of oxazolidinone antibiotics, a strategy that could thwart known mechanisms of linezolid resistance.

Keywords: antibiotic resistance; ribosomal mutations; staphylococcus.

FIG 1

Linezolid binding and protein synthesis in S. aureus. (a) The overall structure of the 70S ribosome from S. aureus (Lin^S). (b) (Left panel) The 23S rRNA of the catalytic site is shown in red, the amino-acylated end of the A-site tRNA is shown in green, and the P-site tRNA is shown in orange. The antibiotic linezolid, overlapping the position that the A-site tRNA would otherwise occupy, is shown in yellow. Upon linezolid binding, no amino-acylated tRNAs can enter the active site and form new peptide bonds. The cartoon represents a composite of two PDB entries: 4WFA (24) and 4V5D (38). (Right panel) The chemical structure of linezolid.

FIG 2

A structural clash that prevents linezolid binding in Lin^r MRSA. (a) In vitro (IC₅₀) cell-free transcription-translation assay. Data are plotted as the amount of protein synthesis (measured by luciferase translation) versus the concentration of linezolid (in micrograms per milliliter). (b) Representation of the region of uL3 around the site of the ΔSer145 mutation, viewed in the same orientation. The Lin^s structure is shown on the left (red); the Lin^r structure is shown on the right (green). The portion of uL3 that interacts with rRNA in “helix 90” is shown. The Lin^r structure reveals a contraction in the loop of uL3, visible here by the repositioning of His146 (uL3) to become His145 (uL3) in the LinR ribosome, altering the interaction of this loop with helix 90 of the 23S rRNA. (c) The cryo-EM structure of the 70S ribosome from Lin^s, showing the linezolid position (yellow) and its interaction with 23S rRNA nucleotide G²⁵⁰⁵ (the position of linezolid is from PDB 4WFA). The cryo-EM structure of the 70S ribosome from Lin^r is overlaid in green. Deletion of a single amino acid in the uL3 rProtein of the Lin^r ribosome changes the part of the protein that interacts with an rRNA helix, a helix which in turn makes a direct contact with linezolid. This structural change modifies the position of G²⁵⁰⁵ in the drug binding pocket, contracting this region and providing fewer contacts to stabilize drug binding. (d) Electron density (drawn at 3σ) around the rRNA nucleotides closest to the linezolid binding site in both the Lin^s (red, left panel) and Lin^r (green, right panel), showing the change in orientation of these nucleotides due to the mutation in uL3. (e) Overlay of the cryo-EM structures of the Lin^s and Lin^r 70S ribosomes around the binding cavity of the linezolid antibiotic. The cavity available for linezolid binding is shown in the colored mesh. The remodeled Lin^r binding site (green) is more constricted and less permissive of linezolid binding.