Negamycin Interferes with Decoding and Translocation by Simultaneous Interaction with rRNA and tRNA

Abstract

SUMMARY Negamycin (NEG) is a ribosome targeting antibiotic which exhibits clinically promising activity. Its binding site and mode of action have remained unknown. We solved the structure of the Thermus thermophilus ribosome bound to mRNA and three tRNAs, in complex with NEG. The drug binds to both small and large ribosomal subunits at 9 independent sites. Resistance mutations in the 16S rRNA unequivocally identified the binding site in the vicinity of the conserved helix 34 (h34) in the small subunit as the primary site of antibiotic action in the bacterial and, possibly, eukaryotic ribosome. At this site, NEG contacts 16S rRNA as well as the anticodon loop of the A-site tRNA. Although the NEG site of action overlaps with that of tetracycline (TET), the two antibiotics exhibit different activities: while TET sterically hinders binding of aminoacyl-tRNA to the ribosome, NEG stabilizes its binding thereby inhibiting translocation and stimulating miscoding.

Structural Insights Lead to a Negamycin Analogue with Improved Antimicrobial Activity against Gram-Negative Pathogens

“…5-Fluoro analogue 17 also showed moderately weaker biochemical and antimicrobial activities compared to negamycin, whereas the 5S fluoro derivative 18, similar to the 5S negamycin 6, was considerably less active. Our observations with compounds substituted on the C-terminal end of negamycin (12,19,20) were similar to previous studies; all changes led to abolishment …”

Resistance mutations generate divergent antibiotic susceptibility profiles against translation inhibitors

“…Two previously identified negamycin resistance mutations, U1052G and U1060A, localize to the head domain of the 30S subunit positioned within helix 34 (h34) and are in close proximity to the negamycin binding site ( Fig. 1 A and B) (11,12). These mutations cause high levels of negamycin resistance, with minimum inhibitory concentration (MIC) values that are approximately eight-(U1052G) and fourfold (U1060A) greater than wild-type susceptibility levels ( Table 1).…”

“…Data deposition: The crystallographic structures and structure factor amplitudes have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 5IT8, 5J5B, 5J91, 5JC9, 5J7L, 5J8A, and 5J88). susceptibility profiles have recently been observed for negamycinresistant mutants (11,12). One of these resistance mutants displays negamycin resistance and tetracycline hypersusceptibility that cannot be reconciled with any currently available structural data.…”

“…Negamycin is a natural bactericidal antibiotic that displays activity against a broad spectrum of Gram-negative pathogens, including Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii (11,12). Tetracycline is broad spectrum antibiotic with bacteriostatic activity that is used to treat a variety of bacterial infections (1).…”

“…Tetracycline is broad spectrum antibiotic with bacteriostatic activity that is used to treat a variety of bacterial infections (1). The mechanism of action for both antibiotics is to interfere with tRNA binding to the aminoacyltRNA (aa-tRNA) site of the small 30S ribosomal subunit (11,12). The clinical utility of tetracycline has been reduced because of the rise of antibiotic resistance, providing incentive for the introduction of tigecycline into the clinic (13).…”