Negamycin induces translational stalling and miscoding by binding to the small subunit head domain of the Escherichia coli ribosome

Abstract

Negamycin is a natural product with broad-spectrum antibacterial activity and efficacy in animal models of infection. Although its precise mechanism of action has yet to be delineated, negamycin inhibits cellular protein synthesis and causes cell death. Here, we show that single point mutations within 16S rRNA that confer resistance to negamycin are in close proximity of the tetracycline binding site within helix 34 of the small subunit head domain. As expected from its direct interaction with this region of the ribosome, negamycin was shown to displace tetracycline. However, in contrast to tetracycline-class antibiotics, which serve to prevent cognate tRNA from entering the translating ribosome, single-molecule fluorescence resonance energy transfer investigations revealed that negamycin specifically stabilizes near-cognate ternary complexes within the A site during the normally transient initial selection process to promote miscoding. The crystal structure of the 70S ribosome in complex with negamycin, determined at 3.1 Å resolution, sheds light on this finding by showing that negamycin occupies a site that partially overlaps that of tetracycline-class antibiotics. Collectively, these data suggest that the small subunit head domain contributes to the decoding mechanism and that small-molecule binding to this domain may either prevent or promote tRNA entry by altering the initial selection mechanism after codon recognition and before GTPase activation.egamycin, a natural product originally isolated from cultures of Streptomyces purpeofuscus, exhibits broad-spectrum antibacterial activity against key pathogens for which clinical treatment options are dwindling (1, 2). The chemical structure of negamycin, [2-[(3R, 5R)-3,6-diamino-5-hydroxyhexanoyl]-1-methylhydrazino]acetic acid, and synthetic routes for its synthesis were elucidated in the early 1970s ( Fig. 1A) (3,4). Early toxicity studies in dogs revealed that daily administration of negamycin led to the formation of N-methylhydrazinoacetic acid, an inhibitor of glutamate pyruvate transaminase, an outcome that caused reversible hepatic coma. Consequently, further clinical studies were not pursued (5). Although the antimicrobial activity and efficacy of negamycin have since been confirmed, analogs exhibiting an improved therapeutic window have yet to be found (6, 7). Progress on this front has been hampered, at least in part, by the fact that the molecular mechanisms of negamycin-induced cell growth inhibition have yet to be discerned conclusively.Negamycin decreases the viability of Escherichia coli by preferentially targeting protein synthesis (8). Early mechanistic investigations proposed that negamycin may act by inhibiting translation initiation (8), decreasing translational fidelity during the elongation phase of protein synthesis (9, 10), and disrupting proper translation termination (9,(11)(12)(13). Uncertainties regarding negamycin's inhibition mechanism, its highly polar physicochemical properties, and the lack of streptomycin cross-resistance led som...

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

“…This is consistent with single molecule fluorescence resonance energy transfer (smFRET) studies, which suggest that negamycin acts while enabling the binding of a ternary complex with a near-cognate tRNA and a differently ratcheted state of the 30S and 50S ribosomal subunits. 21 The MIC 90 of 31f against a panel of 100 clinical isolates of P. aeruginosa maintained in the AstraZeneca collection improved 4-fold over that of negamycin (16 vs 64 μg/mL). Another 4-fold improvement in the MIC 90 would approximate the range of the recommended clinical breakpoints for aminoglycosides.…”

“…21 To understand the contribution of the aminopropyl group of 31f to binding, multiple attempts were made to soak this compound into the ribosome crystal analogous to negamycin. However, no density for 31f was observed, implying that there is limited physiological relevance of negamycin binding to the ribosome only.…”

“…20,21 Mechanistic studies using smFRET also showed negamycin, unlike tetracycline, to enable accommodation of a ternary complex using near cognate tRNA leading to mistranslation and cellular death. 21 This breakthrough in the understanding of the binding mode and the mechanism of action spurred us to initiate an antibiotic discovery program based on negamycin. Negamycin's route of bacterial entry at the site of infection is similar to that of aminoglycosides in that it is driven by the inner membrane potential seemingly without involvement of a membrane carrier under physiological conditions.…”

Resistance mutations generate divergent antibiotic susceptibility profiles against translation inhibitors

“…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).…”