Revisiting the structures of several antibiotics bound to the bacterial ribosome

Abstract

The increasing prevalence of antibiotic-resistant pathogens reinforces the need for structures of antibiotic-ribosome complexes that are accurate enough to enable the rational design of novel ribosome-targeting therapeutics. Structures of many antibiotics in complex with both archaeal and eubacterial ribosomes have been determined, yet discrepancies between several of these models have raised the question of whether these differences arise from species-specific variations or from experimental problems. Our structure of chloramphenicol in complex with the 70S ribosome from Thermus thermophilus suggests a model for chloramphenicol bound to the large subunit of the bacterial ribosome that is radically different from the prevailing model. Further, our structures of the macrolide antibiotics erythromycin and azithromycin in complex with a bacterial ribosome are indistinguishable from those determined of complexes with the 50S subunit of Haloarcula marismortui, but differ significantly from the models that have been published for 50S subunit complexes of the eubacterium Deinococcus radiodurans. Our structure of the antibiotic telithromycin bound to the T. thermophilus ribosome reveals a lactone ring with a conformation similar to that observed in the H. marismortui and D. radiodurans complexes. However, the alkyl-aryl moiety is oriented differently in all three organisms, and the contacts observed with the T. thermophilus ribosome are consistent with biochemical studies performed on the Escherichia coli ribosome. Thus, our results support a mode of macrolide binding that is largely conserved across species, suggesting that the quality and interpretation of electron density, rather than species specificity, may be responsible for many of the discrepancies between the models.

Fig. 1.

Interaction between chloramphenicol and the ribosome. (A) An unbiased 3.2-Å-resolution Fo-Fc difference electron density map contoured at +3σ calculated using amplitudes from T.th. 70S ribosome crystals soaked with 500 μM chloramphenicol. The chloramphenicol moiety is shown in orange, along with a potassium ion mediating the interaction between the drug and the ribosome. (B) The same electron density as shown in A, with the chloramphenicol moiety and two magnesium ions modeled in the D.ra. 50S structure (11) shown in red. The T.th. 70S ribosome and the D.ra. 50S subunit structures were superimposed using equivalent phosphate atoms in the 23S rRNA and the program Lsqman (54). The superimposed structures had an rmsd of 1.21 Å for 2,800 atoms. (C) Overlay of the chloramphenicol molecules modeled in the T.th. 70S (orange) and D.ra. 50S (red) complex structures. Both the model coordinates and the view are the same as in A and B. (D) Overlay of the chloramphenicol molecule modeled in the T.th. 70S complex (orange) and the anisomycin molecule modeled in H.ma. 50S complex (blue). The superimposed structures had an rmsd of 1.53 Å for 2,800 atoms. (E) Interactions between chloramphenicol and the T.th. 70S ribosome. The drug is shown as green sticks, and key interacting residues in the ribosome are shown in white. The potassium ion is displayed as a gray sphere. (F) A surface representation of the binding pocket for chloramphenicol in the T.th. 70S ribosome, showing surface complementarity between the drug (orange) and the A-site crevice in the ribosome as well as the aminoacyl group (blue) of bound phe-tRNA (37).

Fig. 2.

Erythromycin and azithromycin, (A and D) Unbiased 3.2-Å Fo-Fc difference electron density maps contoured at +3σ calculated using amplitudes from T.th. 70S ribosome crystals soaked with 500 μM erythromycin (A) or azithromycin (D). (B, C, E, and F) Overlay of erythromycin or azithromycin molecules from the T.th. 70S ribosome (orange; B, C, E, and F), D.ra. 50S subunit (red; B and E) and H.ma. 50S subunit (blue; C and F) complex structures. The superimposed structures had an rmsd between 1.23 Å and 1.55 Å for 2,800 atoms.

Fig. 3.

Binding of telithromycin to the ribosome. (A) An unbiased 3.2-Å-resolution Fo-Fc difference electron density map contoured at +3σ calculated using amplitudes from T.th. 70S ribosome crystals soaked with 500 μM telithromycin. The drug is shown as orange sticks. (B) Overlay of the telithromycin moieties in the T.th. 70S (orange), D.ra. 50S (red), and H.ma. 50S (blue) complex structures. The superimposed H.ma. 50S and D.ra. 50S rRNA structures had an rmsd of 1.54 Å and 1.23 Å atoms for 2,800 atoms relative to the T.th. 70S structure, respectively. (C) Positioning of the alkyl-aryl moiety of telithromycin (green) relative to U2609 and A752 (E.coli numbering) from T.th. 23S rRNA (white).