Molecular basis of the pleiotropic effects by the antibiotic amikacin on the ribosome

Abstract

Aminoglycosides are a class of antibiotics that bind to ribosomal RNA and exert pleiotropic effects on ribosome function. Amikacin, the semisynthetic derivative of kanamycin, is commonly used for treating severe infections with multidrug-resistant, aerobic Gram-negative bacteria. Amikacin carries the 4-amino-2-hydroxy butyrate (AHB) moiety at the N¹ amino group of the central 2-deoxystreptamine (2-DOS) ring, which may confer amikacin a unique ribosome inhibition profile. Here we use in vitro fast kinetics combined with X-ray crystallography and cryo-EM to dissect the mechanisms of ribosome inhibition by amikacin and the parent compound, kanamycin. Amikacin interferes with tRNA translocation, release factor-mediated peptidyl-tRNA hydrolysis, and ribosome recycling, traits attributed to the additional interactions amikacin makes with the decoding center. The binding site in the large ribosomal subunit proximal to the 3'-end of tRNA in the peptidyl (P) site lays the groundwork for rational design of amikacin derivatives with improved antibacterial properties.

Fig. 1. Overview of the Thermus thermophilus 70S ribosome bound to amikacin.

a Overview of the 70S ribosome complexed with mRNA (cyan), tRNA^Phe in the aminoacyl (A) site (green), tRNA_i^fMet in the peptidyl (P) site (pink), tRNA^Phe in the exit (E) site (orange), and AMK bound to three potentially relevant sites. (Inset 1) AMK bound near the decoding center in the small subunit, (inset 2) AMK bound in the large subunit P site, and (inset 3) AMK bound at inter-subunit bridge B5. Chemical structures of the antibiotics AMK with the amino-hydroxy butyrate (AHB) moiety at the N¹ position of the central 2-deoxystreptamine (2-DOS) ring II (b), and KAN (c).

Fig. 2. Canonical binding site of amikacin near the decoding center.

a Simplified representation of the 70S ribosome with the AMK binding site indicated with the blue star. b The unbiased (F_o – F_c) difference electron density map of AMK bound near the decoding center is contoured at 2.3σ. c AMK binds within helix h44 of the decoding center where the AHB moiety forms three unique interactions. d Time courses of f[³H]Met-Phe-Phe tripeptide formation with EF-Tu ternary complex (TC) (5 μM) and EF-G (5 µM) in the absence (black) and presence of 20 μM AMK (red). Solid lines represent the double exponential fit of the data with SEM from n = 3 independent experiments. e Time evolution of fluorescence traces obtained for the EF-G (5 μM) catalyzed movement of pyrene-labeled mRNA on 70S ribosomes (0.5 μM) in the presence of various concentrations (0-5 µM) of AMK. The inhibition of mRNA movement by AMK was estimated from amplitudes of the slow phase of fluorescence traces relative to the total transition (normalized to 1) indicative of inhibited fraction of the ribosomes. f The fraction of AMK-inhibited pre-TC plotted against AMK concentration. Data were fitted with hyperbolic function (solid line) and half-inhibitory concentration (K_I) of AMK on the inhibition of translocation was estimated from mid-point of transition. Experiments were conducted in triplicates and error bars indicate the SEM of data.

Fig. 3. Amikacin binding site in the large subunit proximal to the P-site tRNA.

a Cartoon representation of the 70S ribosome carrying three tRNAs with the AMK binding site indicated with the yellow star. b The unbiased (F_o – F_c) difference electron density map of AMK bound to the large subunit P site is contoured at 2.3σ. c In the large subunit AMK (yellow) binds near the CCA-end of the P-site tRNA (pink), the conserved 23S rRNA P-loop (white), and helix H93 (white). d Interactions between AMK (yellow) and the Watson-Crick base pair G2252-C74 formed by the P-loop (white) and the P-tRNA CCA-end (pink).

Fig. 4. Amikacin binding site at inter-subunit bridge B5.

a Simplified representation of the 70S ribosome with the AMK binding site indicated with the green star. b The unbiased (F_o – F_c) difference electron density map of AMK bound at the inter-subunit space is contoured at 2.3σ. c AMK interacts with elements of inter-subunit bridge B5, helix h44 of 16S rRNA, helix H64 of 23S rRNA, and ribosomal protein uL14. The amine group of the AHB group forms water-mediated (cyan) H-bonds with the phosphate backbone of h44.

Fig. 5. Effects of amikacin on the kinetics of peptide release and ribosome recycling.

a Time courses of BOP-Met-Phe-Leu release from the P site of the ribosomes in pre-TC (0.1 μM) upon mixing with RF2 (1 μM) in the presence of various concentrations of AMK (0-1 μM). The near monophasic curves are fitted with double exponential function (solid lines) and the rates and amplitudes of the predominant fast phase (> 99%) were determined. The fraction inhibited was estimated from the fractional loss in fluorescence amplitude for a given AMK concentration considering the total amplitude of fluorescence transition (without AMK) as 1. b Fraction inhibition of RF2-mediated peptide release as the function of increasing concentrations of AMK. Solid line is the hyperbolic fit of data from which half-maximal inhibitory concentration (K_I) of AMK for peptide release was estimated. c Time traces for Rayleigh light scattering upon splitting of post-TC ribosomes (0.5 μM) into subunits by the concerted action of RRF (20 μM) and EF-G (10 μM) in the presence of various concentrations of AMK (0–20 μM). The scattering traces were fitted with double exponential function and the rates and amplitudes of both the fast and slow phases were determined. d Fraction inhibition of RRF and EF-G-mediated ribosome splitting was estimated from the fractional loss of the amplitude of the fast phase considering amplitude of the entire transition without AMK as 1. The solid line represents the hyperbolic fit of the fraction inhibition plotted against AMK concentration from which the half-maximal concentration (K_I) of AMK to inhibit ribosome recycling was estimated. Experiments were conducted in triplicates, data were fitted in Origin(Pro), Version 2016 (OriginLab Corp.), and error bars indicate the SEM of data.