A novel site of antibiotic action in the ribosome: interaction of evernimicin with the large ribosomal subunit

Abstract

Evernimicin (Evn), an oligosaccharide antibiotic, interacts with the large ribosomal subunit and inhibits bacterial protein synthesis. RNA probing demonstrated that the drug protects a specific set of nucleotides in the loops of hairpins 89 and 91 of 23S rRNA in bacterial and archaeal ribosomes. Spontaneous Evn-resistant mutants of Halobacterium halobium contained mutations in hairpins 89 and 91 of 23S rRNA. In the ribosome tertiary structure, rRNA residues involved in interaction with the drug form a tight cluster that delineates the drug-binding site. Resistance mutations in the bacterial ribosomal protein L16, which is shown to be homologous to archaeal protein L10e, cluster to the same region as the rRNA mutations. The Evn-binding site overlaps with the binding site of initiation factor 2. Evn inhibits activity of initiation factor 2 in vitro, suggesting that the drug interferes with formation of the 70S initiation complex. The site of Evn binding and its mode of action are distinct from other ribosome-targeted antibiotics. This antibiotic target site can potentially be used for the development of new antibacterial drugs.

Figure 1

Structure of Evn.

Figure 2

Footprinting of Evn on the ribosome. (A) The primer extension gel that illustrates the effect of Evn on DMS modification of nucleotides in hairpins 89 and 91 of E. coli 23S rRNA. Lane 1, unmodified control; lane 2, ribosomes modified with DMS in the absence of Evn; lanes 3 and 4, ribosomes modified with DMS in the presence of 10 μM or 50 μM Evn, respectively. Nucleotides protected by Evn are indicated by lines. (B) Secondary structure of a segment of domain V of E. coli 23S rRNA containing hairpins 89 and 91 (28, 50). Nucleotides protected by Evn are indicated by arrowheads.

Figure 3

H. halobium mutations conferring resistance to Evn. (A) Secondary structure of the segment of H. halobium 23S rRNA encompassing the central loop of domain V and the neighboring regions (28, 50). Nucleotide substitutions conferring Evn resistance are shown in red. (B) Three-dimensional arrangement of Evn^r mutations in the ribosome (28). The rRNA region 2454–2585 is shown in a space-fill (Upper) or backbone (Lower) representation. Coloring of the hairpins is the same as in A. The nitrogen bases of the nucleotides, whose mutation confer Evn resistance, are shown in red. (C) Spatial arrangement of Evn resistance mutations in both rRNA and ribosomal protein L10e. The large ribosomal subunit is shown in a crown projection with the interface side of the 50S subunit toward the reader (28). rRNA and ribosomal proteins are outlined in gray. Hairpins 89 and 91 are shown blue in a “stick” representation, and the α-carbon atoms of the protein L10e chain are shown in cyan in space-fill representation. The sites corresponding to the location of mutations that confer Evn resistance are shown in red.

Figure 4

Confirmation that single nucleotide substitutions in the 23S rRNA are sufficient to confer Evn resistance. Single base pair changes in the 23S rRNA were introduced into a wild-type H. halobium strain via linkage to a single base pair change conferring resistance to Ani. After transformation and selection of Ani resistance colonies, the susceptibility of the transformants to Evn was assayed by E test (see Materials and Methods). Ani^r indicates cells that contained an Ani resistance mutation C2452T.

Figure 5

The regions of similarity between S. pneumoniae L16 and H. marismortui L10e sequences. Identical amino acids in L16 and L10e sequences are indicated by colons, and chemically similar residues are marked by single dots. Amino acid positions in protein L16 where Evn^r mutations were found in S. pneumoniae, S. aureus, and E. faecium are shown in bold and marked by *, and the corresponding positions in H. marismortui L10e are shown in bold.

Figure 6

(A) Effect of Evn on IF2-dependent formation of 70S initiation complex. [³H]fMet-tRNA was bound to the mRNA-programmed 30S subunits in the presence (●) or absence (○) of IF2 and varying concentration of Evn. After addition of 50S subunits and puromycin, the amount of fMet-puromycin formed was determined by ethyl acetate extraction and liquid scintillation counting.