Mechanistic insights into translation inhibition by aminoglycoside antibiotic arbekacin

Figures

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  Figure 1.. ABK chemical structure and the model of its binding to the bacterial ribosome. (A) Chemical structure of ABK with its amino-hydroxy butyrate (AHB) moiety in red. (B) Overview of the bacterial 70S ribosome with Amikacin (structurally similar to ABK) bound at helix 44 of 16S rRNA in the decoding center of 30S subunit. Enlarged image in the box is the detailed view of Amikacin (in blue) in its binding pocket. The conserved nucleobases A1492 and A1493 (orange) are flipped out from the helix 44. Additional interactions of the AHB moiety of the drug with 16S rRNA nucleobases U1495, U1496, G1497 and U1498 (E. coli numbering) can be seen. The figure is based on the cryo-EM maps of PDB IDs 6YPU and 6YHS. (C) Inhibition of in vitro GFP+ protein synthesis in an E. coli based reconstituted translation system induced by ABK added in different concentrations. Experiments were performed in duplicate and the averages are plotted.

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  Figure 2.. Effects of ABK on the fidelity of tRNA selection on the ribosome. (A) Time courses of GTP hydrolysis on EF-Tu ternary complex (T3) with [3H]GTP and Phe-tRNAPhe during initial selection of tRNA on the 70S ribosomes programmed with cognate (UUC, bluish traces) and near-cognate (CUC, reddish traces) mRNA codons. ABK was present in the reaction as indicated. (B) Time courses of f[3H]Met-Phe dipeptide formation with ternary complex (T3) (1 μM) of EF-Tu, Phe-tRNAPhe and GTP reacting to pre-initiated 70S ribosomes (0.5 μM) programmed with either cognate (UUC, bluish traces) or near-cognate (CUC, reddish traces) mRNA codon in their A-site. (C) Kinetic efficiencies (kcat/KM)I of GTP hydrolysis on EF-Tu and (D) Kinetic efficiencies (kcat/KM)D of dipeptide formation with cognate (blue) and near-cognate (red) reactions, estimated from experiments in A and B, respectively). (E) Accuracy of initial selection (I) of tRNA in the presence of ABK; estimated as the ratio between (kcat/KM)I parameters for GTP hydrolysis with cognate and near-cognate codons. (F) Total accuracy (A) of the tRNA selection by the ribosome as estimated from the ratio of (kcat/KM)D parameters for dipeptide formation with…

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  Figure 3.. Effects of ABK on EF-G catalyzed mRNA translocation. (A) Real time fluorescence traces for the EF-G (5 μM) induced translocation of pyrene-labeled mRNA on 70S ribosome (0.5 μM). Ribosomes were pre-incubated with various ABK concentrations as indicated. The amplitudes and rates of fast and slow phases of fluorescence decrease were obtained from double exponential fit (solid lines) of experimental traces. The mean times of the fast and slow phases mRNA movement were estimated from the reciprocal of the rates. (B) The fraction of ABK inhibited ribosomes when ABK was added in the ‘initiation mix’ (IM, blue), and when it was added in the ‘elongation mix’ (EM, red). Solid lines represent hyperbolic fit of the data. (C) Inhibition constants (KI) for the ABK inhibition of translocation with various amounts of EF-G. (D) Time traces for pyrene-labeled mRNA translocation on the ABK (5 μM) saturated ribosomes with increasing concentrations of EF-G (2.5–80 μM). (E) Translocation times of pyrene-labeled mRNA on the ABK-stalled ribosomes at various ABK (0.5–20 μM) and EF-G (2.5–20 μM) concentrations, estimated from multiple experiments as in A. (F) Increase in the rates of mRNA translocation on…

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  Figure 4.. Effects of ABK on the release factor mediated peptide release. (A) BOP-fluorescence traces for the release of BOP-Met-Phe-Leu tripeptide from the ribosome by RF2 (1 μM) at indicated ABK concentrations. Solid lines are double exponential fits of the data. (B) Decrease in the rates of peptide release by RF2 (1 μM) with increasing concentrations of ABK. The rates are estimated from the predominant fast phase of the BOP-fluorescence traces. (C) BOP-fluorescence traces for the release of BOP-Met-Phe-Leu tripeptide from the ABK (10 μM) saturated ribosomes (0.1 μM) at indicated concentrations of RF2. (D) Hyperbolic plot showing increase in the rate of peptide release with increasing RF2 concentrations. The values of kcat and KM were estimated from the double reciprocal plot (inset). Error bars are SEM of the data obtained from at least two independent experiments.

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  Figure 5.. Effect of ABK on ribosome turnover for peptide production and its dependence on EF-G concentration. (A) Time evolution of the fMFFF tetrapeptide production with 70S ribosomes (0.1 μM) in multiple turnover conditions. Black and red traces are in the absence of ABK and blue and green traces in the presence of ABK (1 μM), respectively. The solid lines are single exponential fit of the data. (B) Times for ribosome turnover during tetrapeptide (fMFFF) production at indicated ABK and EF-G concentrations. Error bars represents the SEM of data obtained from at least two individual experiments.

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  Figure 6.. Kinetic model for ABK inhibition of different stages of translation. The RI and RF denote the ribosomal states where the monitoring bases A1492 and A1493 are in the ‘In-helix’ and ‘Flipped-out’ conformations, respectively. ABK binds to RI state with the rate constant k11[A] and induces its transition to RF state. The rate of dissociation of ABK from the RF state is q11. EF-Tu-ternary complex binds to RI and RF states with the rate constant kTu, which leads to AA-tRNA selection and subsequent peptide bond formation. After peptide bond formation, ABK may stall the ribosomes in classical state denoted by RC*A, which hinders EF-G binding and translocation. However, at higher EF-G concentrations, a transient EF-G bound intermediate complex RR*A*G populates via RR*A state, where RR denotes the pre-translocation ribosome in rotated state. The inhibitory effect of ABK is enforced by preferential ‘backward’ dissociation of RR*A*G complex into RR*A and G with the rate \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt}…