The oxazolidinone antibiotics perturb the ribosomal peptidyl-transferase center and effect tRNA positioning

Abstract

The oxazolidinones represent the first new class of antibiotics to enter into clinical usage within the past 30 years, but their binding site and mechanism of action has not been fully characterized. We have determined the crystal structure of the oxazolidinone linezolid bound to the Deinococcus radiodurans 50S ribosomal subunit. Linezolid binds in the A site pocket at the peptidyltransferase center of the ribosome overlapping the aminoacyl moiety of an A-site bound tRNA as well as many clinically important antibiotics. Binding of linezolid stabilizes a distinct conformation of the universally conserved 23S rRNA nucleotide U2585 that would be nonproductive for peptide bond formation. In conjunction with available biochemical data, we present a model whereby oxazolidinones impart their inhibitory effect by perturbing the correct positioning of tRNAs on the ribosome.

Fig. 1.

The binding site of oxazolidinones. (A) Secondary structure of the peptidyltransferase ring of the 23S rRNA from D. radiodurans with the mutation sites in bacteria (blue) and archaea (purple) that confer resistance to oxazolidinones indicated with E. coli numbering. Nucleotides that directly interact with linezolid are shaded light blue, and the mutations sites associated with resistance for chloramphenicol (cam) (45, 46), anisomycin (aniso) (47, 48), and pleuromutilins (pleuro) (49) are shown. (B) Interface view of the D. radiodurans 50S subunit with the binding position of linezolid (red) and landmark proteins L1 and L11 as indicated. (C) Chemical structure of linezolid, highlighting the three aromatic rings (A–C) and the acetamidomethyl tail. (D) View of linezolid (pink) within the binding pocket formed by eight universally conserved nucleotides (blue) of the 23S rRNA. The arrow indicates tunnel direction.

Fig. 2.

Comparison of the linezolid binding pocket. (A) Comparison of linezolid binding pocket from the native D50S structure (PDB ID code 2ZJR; blue) with the D50S-linezolid structure (pink), as well as the native E. coli 70S (E70S; PDB ID code 2AW4; green) (24), the fully refined H. marismortui 50S (H50S; PDB ID code 1S72; orange) (25) and T. thermophilus 70S (T70S; PDB ID code 2J01; teal) (26). (B) Comparison of linezolid binding pocket on D50S (pink), with the model for bound to E. coli 70S (green) (13) and the H. marismortui 50S structure (orange; U.S. patent number6,947,845 B2). (C) Comparison of the position and orientation of linezolid from structures in B.

Fig. 3.

Binding modes of U2585 and resistance to oxazolidinones. (A) Comparison of the position of U2585 between native (blue) and linezolid-bound (pink) D50S with E70S (green), H50S (orange), and T70S (teal) structures (PDB ID codes as in Fig. 2A). (B) Potential hydrogen bond (dashed line) between N3 of U2585 and O4 of ring-C of linezolid. (C) Overview of linezolid (pink) within the binding pocket, with sites of mutation that give rise to oxazolidinone resistance indicated in magenta. Dashed lines indicate hydrogen bonds between U2504 and C2452.

Fig. 4.

Linezolid overlaps A-site ligands at the peptidyltransferase center. (A–F) Comparison of the binding site of linezolid (pink) on D50S (A) with chloramphenicol (PDB ID code 1K01; green) (6) (B), anisomycin (PDB ID code 1K73; olive) (3) (C), tiamulin (PDB ID code 1XBP; teal) (7) (D), clindamycin (PDB ID code 1JZX; magenta) (6) (E), and A- (yellow) and P-site (orange) phenylalanyl-tRNA CCA-end mimics (PDB ID code 1VQN) (27) (F). In all cases, U2585 (blue) from the D50S-linezolid structure is shown for reference.

Fig. 5.

Model for the inhibitory action of oxazolidinones during translation. Model showing events during normal translation (A–D), compared with the effect of the oxazolidinone linezolid (red) during translation (E–H), as described in the text. Small and large subunits are shown in yellow (transparent) and blue, respectively.