Structures of the orthosomycin antibiotics avilamycin and evernimicin in complex with the bacterial 70S ribosome

Abstract

The ribosome is one of the major targets for therapeutic antibiotics; however, the rise in multidrug resistance is a growing threat to the utility of our current arsenal. The orthosomycin antibiotics evernimicin (EVN) and avilamycin (AVI) target the ribosome and do not display cross-resistance with any other classes of antibiotics, suggesting that they bind to a unique site on the ribosome and may therefore represent an avenue for development of new antimicrobial agents. Here we present cryo-EM structures of EVN and AVI in complex with the Escherichia coli ribosome at 3.6- to 3.9-Å resolution. The structures reveal that EVN and AVI bind to a single site on the large subunit that is distinct from other known antibiotic binding sites on the ribosome. Both antibiotics adopt an extended conformation spanning the minor grooves of helices 89 and 91 of the 23S rRNA and interacting with arginine residues of ribosomal protein L16. This binding site overlaps with the elbow region of A-site bound tRNA. Consistent with this finding, single-molecule FRET (smFRET) experiments show that both antibiotics interfere with late steps in the accommodation process, wherein aminoacyl-tRNA enters the peptidyltransferase center of the large ribosomal subunit. These data provide a structural and mechanistic rationale for how these antibiotics inhibit the elongation phase of protein synthesis.

Avilamycin and evernimicin induce structural changes in rProteins uL16 and CTC that enhance the inhibition of A-site tRNA binding

“…These two independent studies show similar modes of action, namely, inhibition of A-tRNA accommodation. Moreover, a comparison of the avi and evn bound to D. radiodurans 50S subunit (this study) and to E. coli 70S ribosome (28) reveals that in both bacterial species the binding site of these antibiotic spans uL16, H89, and H91 in a similar way. However, there is a major difference between the two species: protein CTC second domain, or its homolog second domain of bL25, which participates in blocking A-tRNA accommodation by avi and evn, exists in many Gram-positive pathogens and in D50S but does not exist in the E. coli ribosome (Fig.…”

“…Our study demonstrates that avi and evn bind at a unique site of the ribosome that is not targeted by any other class of antibiotics. After submitting an abstract describing our results* and while our manuscript was in preparation, cryo-EM reconstructions of E. coli 70S (E70S) ribosomes in complexes with avi and evn were published (28). In this study (28), the low resolution and the quality of the EM maps did not enable a precise description of the interactions of avi and evn with the ribosome and consequently did not reveal the detailed mechanisms of resistance and selectivity.…”

“…After submitting an abstract describing our results* and while our manuscript was in preparation, cryo-EM reconstructions of E. coli 70S (E70S) ribosomes in complexes with avi and evn were published (28). In this study (28), the low resolution and the quality of the EM maps did not enable a precise description of the interactions of avi and evn with the ribosome and consequently did not reveal the detailed mechanisms of resistance and selectivity. Conversely, our high-resolution crystal structures of (uniform at 3.35 and 3.58 A) provide findings regarding the binding, modes of action, resistance, and selectivity of avi and evn.…”

“…After submitting a conference abstract † and while writing this manuscript, a study describing the cryo-EM single-particle reconstructions of E70S in complex with avi and evn was reported (28). These two independent studies show similar modes of action, namely, inhibition of A-tRNA accommodation.…”

A Bright Future for Antibiotics?

“…One was performed by 3D cryo-EM using two complexes of E. coli ribosome each with one of them (119), which indicated binding to the large ribosomal subunit at the entrance of the A-site tRNA corridor. The second (50) was performed by X-ray crystallography at somewhat higher resolution and showed a similar binding location at higher details, thus revealing a network of additional, crucial interactions with a ribosomal component, namely the CTC middle domain.…”