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

Abstract

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

Elongation factor-Tu can repetitively engage aminoacyl-tRNA within the ribosome during the proofreading stage of tRNA selection

“…4A) in the presence of the peptide antibiotic evernimicin (EVN). EVN binds Helices 89 and 91 adjacent to the GAC to sterically inhibit aa-tRNA elbow movement through the accommodation corridor (53), while having no detectable impact on the GTPase activities of translational GTPases (58,59). Positioning the donor fluorophore in the P site in this manner enabled measurement of aa-tRNA elbow conformation during navigation of the accommodation corridor.…”

Ribosome-Targeting Antibiotics: Modes of Action, Mechanisms of Resistance, and Implications for Drug Design

“…During the past two decades, the structural details of key ribosome complexes have been elucidated with the use of X-ray crystallography and cryo-electron microscopy (cryo-EM) reconstructions, providing important insights into the process of protein synthesis and its inhibition. The first highresolution views of the ribosomal subunits (5)(6)(7)(8) provided a wealth of structural information and laid the basis for understanding the mechanisms of action of many ribosome-targeting antibiotics P a g e | 4 of 36 (Figure 1a,b) (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). Subsequently, multiple crystal structures of the 70S ribosome were obtained (28)(29)(30), which opened further possibilities for understanding the mechanism of protein synthesis and the action of ribosome-targeting inhibitors.…”

“…They bind in a cleft formed between ribosomal protein uL11 and helices H43 and H44 of the 23S rRNA. In this site, thiopeptides overlap with the location of domain V of EF-G explaining how these compounds interfere with the binding of translation factors to the ribosome(Figure 6a).A new antibiotic binding site on the ribosome has recently been discovered from the structures of the 70S ribosome in complex with the orthosomycin antibiotics evernimicin and avilamycin(9,121). Both antibiotics adopt elongated conformations spanning the minor grooves of helices H89 and H91 of the 23S rRNA, contacting few arginine residues of ribosomal protein uL16 (Figure 6b).…”

Microbial Oligosaccharides with Biomedical Applications

“…It is generally acknowledged that the rarely observed orthoester linkage is necessary for the antibiotic properties of the orthosomycins. Although the orthosomycin family includes hygromycin B, everninomicin, avilamycin, flambamycin, curamycin, etc., the former three members have been extensively studied, especially in the aspect of their bioactivity and biosynthesis [ 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 ]. Herein, restricted by the space, everninomicin (EVN) and avilamycin (AVI) will be mainly discussed in this review.…”