Molecular mechanism of viomycin inhibition of peptide elongation in bacteria

Abstract

Viomycin is a tuberactinomycin antibiotic essential for treating multidrug-resistant tuberculosis. It inhibits bacterial protein synthesis by blocking elongation factor G (EF-G) catalyzed translocation of messenger RNA on the ribosome. Here we have clarified the molecular aspects of viomycin inhibition of the elongating ribosome using pre-steady-state kinetics. We found that the probability of ribosome inhibition by viomycin depends on competition between viomycin and EF-G for binding to the pretranslocation ribosome, and that stable viomycin binding requires an A-site bound tRNA. Once bound, viomycin stalls the ribosome in a pretranslocation state for a minimum of ∼ 45 s. This stalling time increases linearly with viomycin concentration. Viomycin inhibition also promotes futile cycles of GTP hydrolysis by EF-G. Finally, we have constructed a kinetic model for viomycin inhibition of EF-G catalyzed translocation, allowing for testable predictions of tuberactinomycin action in vivo and facilitating in-depth understanding of resistance development against this important class of antibiotics.

Keywords: antibiotics; protein synthesis; ribosome; tuberculosis; viomycin.

Fig. 1.

(A) Chemical structure of viomycin. (B) Overview of the bacterial ribosome with a bound viomycin molecule (red) between the two ribosomal subunits (50S in light gray and 30S in dark gray) next to the A-site tRNA (green); the P-site tRNA (orange) is also visible. The figure is based on structural data from ref. , and the PDB ID code is 4V7L. (C) Detailed view of the viomycin binding site showing the flipped-out state of the bases A1492 and A1493. Colors are as in B and the mRNA is visible in purple. (D) Time course of f[³H]Met-Phe-Thr tripeptide formation at varying concentrations of viomycin and 5 µM EF-G. The fast phase is due to tripeptide formation by viomycin-free ribosomes, which escape inhibition. The decrease in the amplitude of this phase with increasing viomycin concentration reflects the increasing fraction of viomycin-bound ribosomes. The overall amplitude decrease with increasing viomycin concentration is due to read through of the stop codon present after the Met-Phe-Thr reading frame. Solid lines represent fits of Eq. S1 to the data. (E) The fraction of ribosomes inhibited by viomycin at different EF-G concentrations as estimated by subtraction of the amplitude of the fast phase with viomycin from that without it, plotted as a function of the viomycin concentration. Solid lines represent fits of Eq. 2 to the data. (Inset) The same data but in the concentration range from 0–10 µM viomycin. (F) Fraction of viomycin-inhibited ribosomes at 10 µM EF-G with and without preincubation of the 70S ribosome with the drug at two viomycin concentrations. All error bars represent SEM.

Fig. S1.

Time courses of f[³H]Met-Phe dipeptide formation in the presence of varying concentrations of viomycin. It shows that the rate of dipeptide formation is not affected by the presence of viomycin and the average time for dipeptide formation is in all cases is 20 ms.

Fig. S2.

Comparison between the regular MFT mRNA and the truncated MFT mRNA in tripeptide formation in the presence of 50 µM viomycin. Whereas all ribosomes programmed with the truncated mRNA are carrying an f[³H]Met-Phe-Thr tripeptide at 400 s, fewer than 60% of the ribosomes programmed with the nontruncated mRNA are doing so. As discussed in Results, this is due to read-through of the stop codon after Thr codon by the ribosome in the presence of viomycin.

Fig. 2.

(A) Time course of f[³H]Met-Phe-Thr tripeptide formation at varying concentrations of viomycin and 5 µM EF-G when viomycin is prebound to the pretranslocation complex. Solid lines represent single exponential fits to the data. (B) The mean time of tripeptide formation (${\tau }_i$) on viomycin-stalled ribosomes at different EF-G concentrations plotted as a function of viomycin concentration. The solid line represents a fit of Eq. 3 to the data. (C) Average elongation cycle time (${\tau }_{avg}$) calculated from the data in Figs. 1E and 2B at different EF-G concentrations plotted as a function of the viomycin concentration. Solid lines represent fits of Eq. 4 to the data. Error bars represent SEM.

Fig. 3.

(A) Time traces of multiple turnover GTP hydrolysis by EF-G on viomycin-stalled pretranslocation complexes at four different EF-G concentrations. A vertical offset of 20 has been added to separate the lines for clarity of presentation. (B) Rate of turnover GTP hydrolysis plotted as a function of EF-G concentration. (C) Fold change in average elongation cycle time and GTP molecules hydrolyzed per elongation cycle calculated using Eqs. 4 and 5 plotted as a function of viomycin concentration. The uninhibited elongation rate is assumed to be 20 amino acids per second and the free EF-G concentration is set to 10 µM; all other model parameters are those from the main text. All error bars represent SEM.

Fig. 4.

Kinetic model for viomycin inhibition of the elongation cycle. A ternary complex binds to the ribosome and brings it to the first viomycin-sensitive state; here viomycin can bind with rate constant $k_{V1}$ or EF-G can bind with rate constant $k_G$ . The ratio of these two rate constants defines the first viomycin inhibition constant, $K_{I1}$. Binding of EF-G brings the ribosome to the second viomycin-sensitive state; here viomycin can bind with rate constant $k_{V2}$ or the ribosome can escape this state and continue with translocation with rate constant $k_{trans}$. The ratio of these two rate constants defines the second viomycin inhibition constant $K_{I2}$. Ribosomes that become viomycin-bound can still bind EF-G, which will cycle on and off these ribosomes and hydrolyze GTP with turnover rate $k_{GTP}$. Once viomycin dissociates with rate constants $q_{V1}$ or $q_{V2}$ and rebinding of the drug is avoided the ribosome will translocate, EF-G will dissociate, and the next ternary complex will bind to begin the cycle anew.