Molecular mechanism of viomycin inhibition of peptide elongation in bacteria Mikael Holma, Anneli Borga, Måns Ehrenberga, and Suparna Sanyala,1 aDepartment of Cell and Molecular biology, Uppsala University, 75124 Uppsala, Sweden Edited by Joseph D. Puglisi, Stanford University School of Medicine, Stanford, CA, and approved December 16, 2015 (received for review September 2, 2015) Viomycin is a tuberactinomycin antibiotic essential for treating multi- activity of viomycin in genetic code translation (6) and its stabilization drug-resistant tuberculosis. It inhibits bacterial protein synthesis by of peptidyl tRNA in the A site (24). blocking elongation factor G (EF-G) catalyzed translocation of messen- In this study we used rapid kinetics methods in a state-of-the-art ger RNA on the ribosome. Here we have clarified the molecular aspects in vitro translation system with in vivo-like rates (25, 26) to charac- of viomycin inhibition of the elongating ribosome using pre-steady- terize the mechanism of viomycin inhibition of the peptide elongation state kinetics. We found that the probability of ribosome inhibition by cycle. We constructed a quantitative model for viomycin action with viomycin depends on competition between viomycin and EF-G for precise estimates of the model parameters. This model quantifies the binding to the pretranslocation ribosome, and that stable viomycin functional aspects of viomycin inhibition of translocation. It paves the binding requires an A-site bound tRNA. Once bound, viomycin stalls the way for deeper understanding of the basis of the antimicrobial activity ribosome in a pretranslocation state for a minimum of ∼45 s. This of viomycin and the evolution of viomycin resistance, thereby stalling time increases linearly with viomycin concentration. Viomycin facilitating further development of the chemically malleable inhibition also promotes futile cycles of GTP hydrolysis by EF-G. Finally, tuberactinomycin class of antibiotics. we have constructed a kinetic model for viomycin inhibition of EF-G catalyzed translocation, allowing for testable predictions of tuberacti- Results nomycin action in vivo and facilitating in-depth understanding of re- The Probability That a Ribosome Becomes Inhibited by Viomycin During sistance development against this important class of antibiotics. an Elongation Cycle Is Determined by Competition Between Viomycin and EF-G for Ribosome Binding. To study the effects of viomycin on protein synthesis | viomycin | ribosome | antibiotics | tuberculosis translating ribosomes we carried out tripeptide formation experi- ments at varying concentrations of the drug using a quench-flow in- uberculosis (TB) is a global threat to human health, and over strument. In these experiments 70S ribosomes programmed with an T9 million people contracted the disease in 2013 (1). The mRNA encoding Met-Phe-Thr-Stop(MFTmRNA)andwithP-site emergence of strains of Mycobacterium tuberculosis, the causative bound f[3H]Met-tRNAfMet were rapidly mixed with a factor mixture agent of TB, resistant to several first- and second-line antitubercular containing Phe-tRNAPhe and Thr-tRNAThr in ternary complex with drugs is a significant concern. The tuberactinomycin antibiotics are EF-Tu·GTP, EF-G, and varying concentrations of viomycin. The bacterial protein synthesis inhibitors, commonly used as second-line reactions were stopped at different incubation times by formic acid drugs against multidrug-resistant TB. Viomycin was the first drug of and the relative amount of each peptide was determined by reversed- this class to be discovered (2, 3). It is a cyclic pentapeptide that phase HPLC (RP-HPLC) with on-line radiation detection. contains several nonstandard amino acids (Fig. 1A) and is produced In the absence of viomycin (red trace in Fig. 1D) tripeptide by a nonribosomal peptidyl transferase (4). formation displayed biphasic kinetics with a fast phase accounting Viomycin affects bacterial protein synthesis by inhibiting mRNA for ∼85% of the total amplitude. The mean times of tripeptide translocation (5) and causing misreading of the genetic code (6). The formation in the fast phase were 240, 180, and 150 ms at 2.5, 5, and present study focuses on viomycin inhibition of translocation. During 10 μM EF-G, respectively. The slow phase (∼15%), probably translocation the mRNA moves through the ribosome by one base caused by a small fraction of partially active ribosomes, had a mean triplet (codon), the peptidyl transfer RNA (tRNA) moves from the ribosomal A to P site, and the deacylated tRNA moves from the P to Significance E site. Translocation is catalyzed by elongation factor G (EF-G) in a GTP hydrolysis-dependent manner (7) and occurs via formation of Antibiotics are widely used to treat bacterial infections, but their tRNA hybrid states (8), relative rotation of the ribosomal subunits mechanisms of action are often poorly understood. Viomycin is a (9), and movement of the L1 stalk (10). Bulk FRET, chemical tuberactinomycin antibiotic used for treating multidrug-resistant footprinting, and single-molecule FRET experiments have shown tuberculosis. Using an in vitro translation system that displays ki- that viomycin stabilizes the ribosome in a pretranslocation state with netic rates comparable to those in living cells we have characterized tRNAs in hybrid A/P and P/E configurations, rotated ribosomal the mechanism of action of viomycin and constructed a kinetic subunits, and the L1 stalk in a closed conformation interacting with – model for how viomycin inhibits the translocation step of the theP-sitetRNA(1116); this structure has been visualized by cryo- peptide elongation cycle. Our results are vital for understanding the EM (17). However, a crystal structure of the viomycin-bound ribo- mechanism of the antimicrobial activity of viomycin and its sister some (18) and one single-molecule FRET study (19) have suggested drugsinlivingbacteriaaswellas resistance mechanisms against stabilization of the tRNAs in the classical state. them, which can have strong implications for global health. Recent crystal (18, 20) and cryo-EM (17) structures of the vio- mycin-bound ribosome have shown that the drug binds adjacent to Author contributions: M.H. and S.S. designed research; M.H. and S.S. performed research; the ribosomal A site, in a pocket between helix 44 of the 16S rRNA M.H., A.B., and M.E. contributed new reagents/analytic tools; M.H. and S.S. analyzed data; in the small subunit and helix 69 of the 23S rRNA in the large subunit and M.H., A.B., M.E., and S.S. wrote the paper. (Fig. 1 B and C). In these structures the monitoring bases A1492 and The authors declare no conflict of interest. A1493 are in their active, flipped-out conformation, where they in- This article is a PNAS Direct Submission. teract with the codon–anticodon helix in the A site (21, 22). In fact, Freely available online through the PNAS open access option. the bound viomycin molecule sterically blocks the flipped-in confor- 1To whom correspondence should be addressed. Email: [email protected]. mation of these bases observed in the apo ribosome (23). This steric This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. restriction of the monitoring bases may explain the error-inducing 1073/pnas.1517541113/-/DCSupplemental. 978–983 | PNAS | January 26, 2016 | vol. 113 | no. 4 www.pnas.org/cgi/doi/10.1073/pnas.1517541113 Downloaded by guest on September 26, 2021 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. 18, 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 BIOCHEMISTRY 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. time in the seconds range and was treated as a background reaction MFT mRNA in the absence of class-1 release factor. This af- (SI Materials and Methods). fected the amplitude of the slow but not the fast phase (SI Materials When viomycin was included in the factor mixture, so that all and Methods). Therefore, the fraction of viomycin-inhibited ribo- ribosomes were viomycin-free before mixing in the quench-flow in- somes was estimated from the degree of reduction of the fast phase strument, we observed a large effect on the kinetics of tripeptide amplitude by viomycin and not from the amplitude of the slow phase. formation (orange through violet traces in Fig. 1D), whereas the rate The fraction of viomycin-inhibited ribosomes increased with vio- and amplitude of dipeptide formation remained unaffected (Fig. mycin concentration in a near hyperbolic manner (Fig.