Differential Effects of Thiopeptide and Orthosomycin Antibiotics on Translational GTPases

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.

Section: Resultsmentioning

confidence: 99%

Section: H89 H89mentioning

confidence: 99%

mentioning

confidence: 99%

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

Arenz¹,

Juette

Graf³

et al. 2016

“…faecalis TetM and E. coli ribosomes were purified as described previously (29), and ribosome binding was verified by using pelleting assays as described for EF-G (30). Cryo-EM data collection on a Titan Krios transmission electron microscope (FEI Company) and processing using the SPIDER software package (31) was as described previously (32).…”

Section: Methodsmentioning

confidence: 99%

Structural basis for TetM-mediated tetracycline resistance

Dönhöfer

Franckenberg

Wickles

et al. 2012

165

141

Ribosome protection proteins (RPPs) confer tetracycline resistance by binding to the ribosome and chasing the drug from its binding site. The current model for the mechanism of action of RPPs proposes that drug release is indirect and achieved via conformational changes within the drug-binding site induced upon binding of the RPP to the ribosome. Here we report a cryo-EM structure of the RPP TetM in complex with the 70S ribosome at 7.2-Å resolution. The structure reveals the contacts of TetM with the ribosome, including interaction between the conserved and functionally critical C-terminal extension of TetM and the decoding center of the small subunit. Moreover, we observe direct interaction between domain IV of TetM and the tetracycline binding site and identify residues critical for conferring tetracycline resistance. A model is presented whereby TetM directly dislodges tetracycline to confer resistance.antibiotic | protein synthesis | RNA | tigecycline | translation

“…Currently, avi is used as growth promoter added to animal food (6), and evn was considered for clinical use for humans by the Schering-Plough Corporation (7,8).…”

mentioning

confidence: 99%

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

Krupkin

Wekselman

Matzov

et al. 2016

Two structurally unique ribosomal antibiotics belonging to the orthosomycin family, avilamycin and evernimicin, possess activity against Enterococci, Staphylococci, and Streptococci, and other Gram-positive bacteria. Here, we describe the high-resolution crystal structures of the eubacterial large ribosomal subunit in complex with them. Their extended binding sites span the A-tRNA entrance corridor, thus inhibiting protein biosynthesis by blocking the binding site of the A-tRNA elbow, a mechanism not shared with other known antibiotics. Along with using the ribosomal components that bind and discriminate the A-tRNA-namely, ribosomal RNA (rRNA) helices H89, H91, and ribosomal proteins (rProtein) uL16-these structures revealed novel interactions with domain 2 of the CTC protein, a feature typical to various Gram-positive bacteria. Furthermore, analysis of these structures explained how single nucleotide mutations and methylations in helices H89 and H91 confer resistance to orthosomycins and revealed the sequence variations in 23S rRNA nucleotides alongside the difference in the lengths of the eukaryotic and prokaryotic α1 helix of protein uL16 that play a key role in the selectivity of those drugs. The accurate interpretation of the crystal structures that could be performed beyond that recently reported in cryo-EM models provide structural insights that may be useful for the design of novel pathogen-specific antibiotics, and for improving the potency of orthosomycins. Because both drugs are extensively metabolized in vivo, their environmental toxicity is very low, thus placing them at the frontline of drugs with reduced ecological hazards. degradable antibiotics | ribosomes | resistance | species-specific antibiotics | Gram-positive