Multifaceted Mechanism of Amicoumacin A Inhibition of Bacterial Translation

Abstract

Amicoumacin A (Ami) halts bacterial growth by inhibiting the ribosome during translation. The Ami binding site locates in the vicinity of the E-site codon of mRNA. However, Ami does not clash with mRNA, rather stabilizes it, which is relatively unusual and implies a unique way of translation inhibition. In this work, we performed a kinetic and thermodynamic investigation of Ami influence on the main steps of polypeptide synthesis. We show that Ami reduces the rate of the functional canonical 70S initiation complex (IC) formation by 30-fold. Additionally, our results indicate that Ami promotes the formation of erroneous 30S ICs; however, IF3 prevents them from progressing towards translation initiation. During early elongation steps, Ami does not compromise EF-Tu-dependent A-site binding or peptide bond formation. On the other hand, Ami reduces the rate of peptidyl-tRNA movement from the A to the P site and significantly decreases the amount of the ribosomes capable of polypeptide synthesis. Our data indicate that Ami progressively decreases the activity of translating ribosomes that may appear to be the main inhibitory mechanism of Ami. Indeed, the use of EF-G mutants that confer resistance to Ami (G542V, G581A, or ins544V) leads to a complete restoration of the ribosome functionality. It is possible that the changes in translocation induced by EF-G mutants compensate for the activity loss caused by Ami.

Keywords: amicoumacin A; antibiotic resistance; elongation factor EF-G; initiation; microscale thermophoresis; rapid kinetics; translocation.

FIGURE 1

The model of Ami arrangement at the E site of the ribosome complex. The model presents Ami interaction (light blue) with the 5′-end of mRNA (green) and the implied interaction of the 16S rRNA (orange) with EF-G (magenta) amino acids (yellow), providing resistance to Ami. The EF-G conserved loops I and II are shown in brown and blue, respectively. The P-site tRNA is shown in light gray (PDB: 4V7D, 4V5F, 4W2F, and 4V9O).

FIGURE 2

The influence of Ami on the initiation in a complete system. (A) Titration of the 30S IC formation with mRNA containing AUG start codon (denoted as AUG, in the presence of Ami, AUG + Ami; Ede, edeine; Ksg, kasugamycin), UUC start codon (denoted as UUC, in the presence of Ami – UUC + Ami), AUG start codon with the participation of the elongator BPY-Phe-tRNA^Phe (denoted as AUG/Phe, in the presence of Ami – AUG/Phe + Ami). (B) Dissociation constants (K_d) calculated from (A). The K_d value for mRNA with AUG in the absence of Ami is very low and practically not visible on the graph; the K_d values for mRNA with UUC start codon or AUG codon with the participation of Phe-tRNA^Phe in the absence of Ami are not shown as they do not form the initiation complex. (C) Association of the 50S subunit (0.3 μM) with the 30S IC (0.1 μM) monitored by light scattering. Time curves are labelled according to the mRNA start codon and presence of Ami, Ede, or Ksg. (D) Association of the 50S subunit (0.3 μM) with the 30S IC (0.1 μM) monitored by fluorescent signal from the BPY reporter of the initiator BPY-Met-tRNA^fMet, labelling as in (C).

FIGURE 3

The influence of Ami on the initiation in a system lacking IF3. (A) Titration of the 30S IC formation in the absence of IF3 with mRNA containing either AUG or UUC start codon. (B) Dissociation constants (K_d) calculated from (A). (C) Association of the 50S subunit (0.3 μM) with the 30S IC in the absence of IF3 (0.1 μM) monitored by light scattering. (D) Association of the 50S subunit (0.3 μM) with the 30S IC in the absence of IF3 (0.1 μM) monitored by fluorescent signal from the BPY reporter of the initiator BPY-Met-tRNA^fMet.

FIGURE 4

The influence of Ami on the aminoacyl-tRNA binding and the stability of peptidyl-tRNA binding to the A site of the ribosome. (A) The pre-steady-state kinetics of the ternary complex EF-Tu⋅GTP⋅Phe-tRNA^Phe (2 μM) interaction with the 70S IC (50 nM) containing fMet-tRNA^fMet(Prf20) at the P site without addition of antibiotic (no antibiotic), in the presence of amicoumacin A (Ami), tetracycline (Tet), and kirromycin (Kirr). The dependence of the k_on (B) and k_off (C) values on the Mg²⁺ ion concentration obtained for the pretranslocation complex containing deacylated tRNA^fMet at the P site and fMet-[¹⁴C]Val-tRNA^Val at the A site. (D) The K_d values calculated from (B) to (C).

FIGURE 5

The pre-steady state kinetics of translocation catalyzed by intact or mutant forms of EF-G. The dependence of the fast translocation phase rate on the concentration of EF-G monitored by the fluorescence change of fMet-Phe-tRNA^Phe(Prf16/17) (A) or BPY-Met-Phe-tRNA^Phe (B). The time courses of single round translocation (at saturating concentration of EF-G (5 μM) monitored by the fluorescence change of fMet-Phe-tRNA^Phe(Prf16/17) (C) or BPY-Met-Phe-tRNA^Phe (D). Viomycin (Vio). Error bars (s.d.) in (A,B) were obtained from at least two independent experiments with 5–7 technical replicates each, however, do not exceed the size of symbols.

FIGURE 6

The influence of Ami on the multiple turnover translocation catalyzed by intact or G542V EF-G. (A) The time courses of fMet-[¹⁴C]Val-puromycin formation. (B) The time courses of fMet-[¹⁴C]Val-Phe tripeptide formation.