Molecular Mechanism of Viomycin Inhibition of Peptide Elongation in Bacteria

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 experiments 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 viomycin-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. 1E). For a S1). The relative amplitude of the fast phase gradually decreased given viomycin concentration the fraction of inhibited ribosomes was from around 85% in the absence of viomycin to essentially 0 at much larger at low than at high EF-G concentration (Fig. 1E). This 50 μM viomycin, whereas its mean time remained virtually un- observation suggested competition between EF-G and viomycin for altered, indicating that the fast phase represents tripeptide forma- binding to the pretranslocation ribosome. If EF-G bound first, the tion on viomycin-free ribosomes. A new slow phase appeared with a ribosome proceeded unhindered to tripeptide formation, resulting in mean time of several tens of seconds, likely representing tripeptide the fast phase. Alternatively, if viomycin bound first, the ribosome formationbyviomycininhibitedribosomes after drug dissociation stalled and the mean time of tripeptide formation was greatly pro- (Fig. 1D). The total amount of radiodetectable peptide decreased longed, resulting in the slow phase (Fig. 1D). with time in a viomycin concentration-dependent manner (Fig. 1D). When viomycin was allowed to equilibrate with the 70S initiation This behavior is an artifact, caused by viomycin-induced read- complex by adding it to both the ribosome and the factor mixtures, through of the stop codon following the threonine codon in the we observed tripeptide formation kinetics identical to those when

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 (τi) 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 (τavg) 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.

Holm et al. PNAS | January 26, 2016 | vol. 113 | no. 4 | 979 Downloaded by guest on September 26, 2021 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 pre- sentation. (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.

the drug was included only in the factor mixture. Notably, the ability to slow down peptide elongation. Viomycin is a “slow” fraction of viomycin-inhibited ribosomes was exactly the same (Fig. inhibitor of mRNA translation that stalls the ribosome for a 1F), meaning that preincubation of viomycin with the initiation time, τi, substantially longer than that required to carry out an complex did not confer any advantage to the drug in its competi- uninhibited elongation cycle, τ0 (Fig. 2B). Therefore, its effect tion with EF-G for ribosome binding. It also implies that ribosomes on the average time of one elongation cycle, τavg,canbecon- with an empty A-site bind only a negligible amount of viomycin at veniently written as (27) the concentrations used, indicating that stable viomycin binding requires an A-site bound tRNA. τavg = τ0 + Pi · τi. [1]

The Time a Ribosome Remains Inhibited After Viomycin Binding Here, Pi is the probability that the ribosome is stalled by viomycin Depends Only on the Viomycin Concentration. To estimate the during one elongation cycle (Fig. 1E). The average time increased mean time for translocation after viomycin binding to the pre- nonlinearly with increasing viomycin concentration (Fig. 2C)ina translocation ribosome we designed a two-step tripeptide for- manneralsoobservedinthecaseoffusidicacidinhibitionofelon- mation assay. First, viomycin-stalled pretranslocation ribosomes gating ribosomes (27). The average time (τavg) increased rapidly at were formed by incubating initiated 70S ribosomes programmed low viomycin concentration as a consequence of the rapidly increas- with a truncated MFT mRNA and P-site bound f[3H]Met- ing probability that the ribosome becomes stalled (Fig. 1E). In con- tRNAfMet for 5–10 s at 37 °C with Phe-tRNAPhe–containing trast, at high viomycin concentration where the ribosome is virtually ternary complex and varying concentrations of viomycin. The guaranteed to stall τavg increased slowly due to viomycin rebinding resulting viomycin-stalled pretranslocation complexes were then events (Fig. 2B). This nonlinear response of the average elongation mixed with EF-G, Thr-tRNAThr–containing ternary complex, and cycle time to viomycin concentration confirms that viomycin can bind viomycin, leading to slow translocation of f[3H]Met-Phe-tRNAPhe to two ribosomal states during the elongation cycle. First, the drug followed by rapid formation of f[3H]Met-Phe-Thr tripeptide. binds with high efficiency to a transient state, likely the EF-G–free An mRNA truncated directly after the Thr codon was used to pretranslocation ribosome (Fig. 4). This first binding event is rapidly avoid viomycin-mediated read-through of the stop codon (Fig. followed by transition to a downstream stalled state, likely the EF- S2). The reactions were stopped at different incubation times by G–bound pretranslocation ribosome (Fig. 4). In this state, the stalling formic acid and the relative amount of each peptide was de- time increased linearly with viomycin concentration due to repeated termined as described above. slow dissociation from and fast reassociation of viomycin to the In these experiments we observed a complete absence of the fast stalled ribosome. phase of tripeptide formation (Fig. 2A), implying that virtually all ribosomes were viomycin-bound even at the lowest viomycin con- Viomycin Inhibition Leads to Multiple Rounds of Futile GTP Hydrolysis by centration (1 μM). In contrast, when 70S initiation complex with EF-G Before Translocation. EF-G can bind to the viomycin-stalled empty A-site and P-site bound f[3H]Met-tRNAfMet was preequili- pretranslocation ribosome (12, 14, 17). We wished to know brated with viomycin in the previous section, the fraction of whether such binding leads to multiple cycles of GTP hydrolysis inhibited ribosomes did not change (Fig. 1F), indicating negligible before successful translocation. Therefore, we designed an exper- drug binding. Taken together these results demonstrate that the iment to follow multiple turnover GTP hydrolysis by EF-G in the affinity of viomycin to the ribosome greatly increases upon A-site presence of viomycin-stalled pretranslocation ribosomes. For that, binding of a tRNA. initiated 70S ribosomes programmed with MFT mRNA and P-site fMet The mean time of tripeptide formation on viomycin-stalled ri- bound fMet-tRNA were rapidly mixed in a quench-flow in- Phe bosomes was estimated by fitting a single exponential function to strument with a mixture of Phe-tRNA containing ternary com- 3 each of the curves in Fig. 2A. The mean time increased linearly with plex, viomycin, [ H]GTP, and varying concentrations of EF-G. viomycin concentration from around 45 s at 1 μM to around 120 s at Viomycin-stalled pretranslocation complex was formed within 100 μM(Fig.2B), indicating that after dissociation the drug could 20 ms (Fig. S1). The reactions were stopped at different incubation 3 reassociate to the pretranslocation ribosome and prolong the stall- times by formic acid and the relative amounts of [ H]GTP and 3 ing. In contrast to the fraction of viomycin-inhibited ribosomes, [ H]GDP were determined by anion exchange chromatography which displayed strong negative correlation with EF-G concentra- with on-line radiation detection. 3 tion (Fig. 1E), the mean times displayed no EF-G concentration At all EF-G concentrations [ H]GDP accumulated linearly with dependence in the 2.5–10 μMrange(Fig.2B). This suggests that time (Fig. 3A), showing that steady-state cycling of EF-G on the viomcyin dissociation and reassociation occur on an EF-G bound ribosome was established rapidly. The turnover rate of GTP hy- pretranslocation ribosome. drolysis, zero in the absence of EF-G, saturated already at the lowest EF-G concentration used (0.625 μM), as expected from the The Average Time of an Elongation Cycle Increases Nonlinearly with lack of an EF-G concentration dependence of the stalling time Viomycin Concentration. A major determinant of the growth in- observed above (Fig. 2B). We observed a small linear increase in the − hibitory effect of ribosome targeting antibiotic drugs is their GTP hydrolysis rate at higher EF-G concentrations from 3.9 s 1 at

980 | www.pnas.org/cgi/doi/10.1073/pnas.1517541113 Holm et al. Downloaded by guest on September 26, 2021 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 kV1 or EF-G can bind with rate constant kG . The ratio of these two rate constants defines the first viomycin inhibition constant, KI1. Binding of EF-G brings the ribosome to the second viomycin-sensitive state; here viomycin can bind with rate constant kV2 or the ribosome can escape this state and continue with translocation with rate constant ktrans. The ratio of these two rate constants defines the second viomycin inhibition constant KI2. Ribosomes that become viomycin-bound can still bind EF-G, which will cycle on and off these ribosomes and hydrolyze GTP with turnover rate kGTP. Once viomycin dissociates with rate constants qV1 or qV2 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. BIOCHEMISTRY − 0.625 μMto6.6s1 at 7.5 μM EF-G (Fig. 3B), likely due to EF-G elementary rate constants of the translocation process in the reacting with free 50S subunits. Accounting for this side reaction the presence of viomycin as maximal rate of GTP hydrolysis by EF-G on viomycin-stalled pretranslocation ribosomes, kGTP, was estimated as 3.75 ± 1 ½Vio ½Vio − τ = τ + · τ = τ + · + [4] 0.20 s 1 from the y axis intercept of the straight line in Fig. 3B. avg 0 Pi i 0 . qV2 ½Vio + KI1 · ½EF‐G KI2 This estimate corresponds to a dwell time of 270 ± 15 ms for

EF-G on the viomycin-stalled ribosome, in good agreement The parameters KI1, KI2,andqV 2 that define the concentration de- with previously published dwell time estimates based on sin- pendence of the inhibition of translocation by viomycin were esti- gle-molecule FRET experiments (12, 28). mated by fitting Eqs. 2–4 tothedatainFigs.1E and 2 B and C.The inhibition constant KI1 is 0.55 ± 0.03, meaning that viomycin binds to A Kinetic Model for Viomycin Action on Translating Ribosomes. The the pretranslocation ribosome roughly twice as fast as EF-G. The simplest model for viomycin inhibition of protein synthesis that inhibition constant KI2 is 66 ± 5 μM. This is the viomycin concen- accounts for all of the in vitro results described in the present tration required to double the time a ribosome remains stalled due work is shown in Fig. 4. The total probability that a ribosome to successive viomycin rebinding events. The rate constant for vio- becomes inhibited by viomycin during an elongation cycle is a mycin dissociation from the EF-G–bound ribosome, qV2,is0.022± − combination of the probabilities that viomycin binds either to the 0.0005 s 1. The inverse of this rate constant estimates the residence first or to the second sensitive state and is given by timeforviomycinonthiscomplexas44± 1s.FromEq.4 we determined IC values for translocation inhibition by viomycin, · ½ ‐ 50 = − KI1 EF G · KI2 [2] defined as the viomycin concentration required to double the du- Pi 1 · ½ ‐ + ½ + ½ . KI1 EF G Vio KI2 Vio ration of an average elongation cycle. IC50 values at 2.5, 5, and 10 μM EF-G are 5, 6, and 9 nM viomycin, respectively. The second term on the right side is the product of the Using Eq. 4 it is possible to account for the futile cycles of GTP probabilities that the ribosome escapes inhibition in the first hydrolysis by EF-G on viomycin-stalled ribosomes. The average and the second state. The inhibition constant, KI1,istheratio number of GTP molecules hydrolyzed per elongation cycle as a between the binding rate constants of EF-G and viomycin to function of the viomycin concentration is given by the pretranslocation ribosome (kG and kV1 in Fig. 4). The inhibition constant, K , is the ratio between the rate constants I2 GTP = GTP + k · P · τ = for the forward step required for the ribosome to escape from avg 0 GTP i i [5] the second viomycin-sensitive state and for viomycin binding kGTP ½Vio ½Vio 2 + + . to that state (ktrans and kV2 in Fig. 4). qV2 ½Vio + KI1 · ½EF‐G KI2 The time that a ribosome remains inhibited by viomycin is given by (SI Materials and Methods) Here GTP0 is the number of GTP molecules hydrolyzed during an uninhibited elongation cycle, that is, 2, kGTP is the (saturated) ½ τ = 1 · + Vio [3] turnover rate of GTP hydrolysis by EF-G on the viomycin-stalled i 1 . · τ qV 2 KI2 ribosome and Pi i is the average time per elongation cycle the ribosome is viomycin-bound and stimulating futile GTP hydrolysis Here qV2 is the rate constant for viomycin dissociation from the by EF-G. Considerably more viomycin is required to double the second viomycin-sensitive state, the EF-G–bound ribosome. The GTP cost of an elongation cycle than to double its duration (Fig. average elongation cycle time can now be written in terms of the 3C):15,30,and60nMat2.5,5,and10μM EF-G, respectively.

Holm et al. PNAS | January 26, 2016 | vol. 113 | no. 4 | 981 Downloaded by guest on September 26, 2021 Discussion parameters, KI1, KI2,andqV2. Here it is relevant to consider the From the in vitro results presented here we constructed a simple clinical drug target M. tuberculosis, which is known to be signifi- yet powerful model for viomycin inhibition of translocation and cantly more sensitive to viomycin and its sister drug capreomycin estimated its three kinetic parameters, KI1, KI2, and qV2 (Fig. 4 than E. coli (32). Slow-growing mycobacteria such as M. tuberculosis and Eq. 4). According to this model there are two viomycin- maintain a much lower intracellular ribosome concentration than sensitive states in the elongation cycle. Elongating ribosomes the fast-growing E. coli (33). This by itself would increase the drug first become viomycin-sensitive upon aminoacyl tRNA delivery susceptibility of M. tuberculosis because at any intracellular drug to the ribosomal A site by EF-Tu. This EF-G–free pre- concentration a larger fraction of the ribosomes would be disabled. translocation state is the most viomycin-sensitive state. Viomycin However, the drug susceptibility of individual ribosomes does play a and EF-G compete for first binding to this state such that the major role, as evidenced by multiple resistance mutations present probability of a successful viomycin attack is 50% when the con- either in ribosomal RNA (34) or in the rRNA methylase TlyA (32, centration of viomycin is equal to the product of the inhibition 35). In our model such resistance mutations would lead to changes in the numerical values of the parameters K , K ,andq .De- constant KI1 and the EF-G concentration (Eq. 2). Whether or not I1 I2 V2 the first state becomes viomycin-bound, EF-G binding brings the stabilization of drug binding on the ribosome would lead to a larger ribosome to the second viomycin-sensitive pretranslocation state, dissociation rate constant, qV2, whereas decreased drug binding rate where viomycin-bound ribosomes stall for a minimum time of ∼45 s. or decreased lifetimes of the two drug sensitive states would be In this state, rebinding of viomycin prolongs the stalling time, reflected by larger KI1 and KI2 constants. Other members of the which increases linearly with viomycin concentration and is dou- tuberactinomycin family such as capreomycin and enviomycin share the same binding site on the bacterial ribosome and have bled when the viomycin concentration reaches KI2, here estimated as 66 μM(Eq.3). We note that the minimal stalling time of ∼45 s effects on bacterial cells similar to those of viomycin. Thus, our after a successful viomycin attack is equivalent to the time re- model likely describes translocation inhibition by the entire quired to translate roughly 900 codons in rapidly growing tuberactinomycin class of antibiotics, and differences in drug ef- Escherichia coli cells with an average codon translation time of ficacy are likely caused by different values of the three parameters 50 ms (29). This greatly exceeds the average distance of 14–26 rather than different mechanisms. codons between ribosomes on mRNA (30), and therefore viomycin Our results show that viomycin inhibition confers an extra energy binding to a ribosome is expected to lead to queuing of trailing cost due to the futile cycling of EF-G on viomycin-stalled ribosomes. ribosomes behind it. However, much higher concentrations of viomycin are required to The existence of two viomycin-sensitive states during the elon- increase the GTP cost of an elongation cycle than are required to gation cycle, one before and one after binding of EF-G to the reduce the elongation rate (Fig. 3C). The in vivo implication of this ribosome, is supported by two recent structures of the viomycin- extra energy loss by viomycin-bound ribosomes is hard to estimate, bound pretranslocation ribosome. One is a 3.3-Å crystal structure but it could be significant under energy-limited conditions. (18) containing three tRNAs and no EF-G, and the other is a 7-Å In summary, we have characterized the mechanism of viomycin cryo-EM structure (17) containing two tRNAs and EF-G. In the inhibition of the elongation step of bacterial protein synthesis. The former structure the ribosome is observed in the nonrotated state kinetic model we present here along with estimates of its three key and the bound tRNAs are in the classical A, P, and E sites, whereas parameters provides a quantitative basis for understanding the in the latter structure the ribosome is in the rotated state and the antimicrobial activity of viomycin, also applicable in vivo. We are tRNAs are in the hybrid A/P and P/E states. Because FRET ex- optimistic that our model is general enough to be instrumental also periments suggested that viomycin binding induces rotation of the for characterization of other tuberactinomycin antibiotics and re- ribosomal subunits (11), the functional significance of the non- sistance mechanisms that have evolved also against these viomycin- rotated viomycin-bound structure is unclear. Further structural related drugs. Finally, our model will aid characterization of more work is required to understand the significance of different vio- effective elongation inhibitors, which is one of the main goals of mycin-bound states of the ribosome and also to clarify how the global antibiotics research. drug affects large-scale ribosome dynamics during translocation. Materials and Methods Our conclusion that viomycin binds tightly to the ribosome only Reagents and Buffers. All experiments were performed at 37 °C in Hepes- after a tRNA has been delivered to the A site by EF-Tu also finds polymix buffer [95 mM KCl, 5 mM NH Cl, 0.5 mM CaCl , 8 mM putrescine, 1 mM support in the crystal structure of the viomycin-bound ribosome 4 2 spermidine, 5 mM potassium phosphate, 5 mM Mg(OAc)2,1mMdithioery- (18). Here viomycin occupies the space between H69 and h44 that thritol, and 5 mM Hepes, pH 7.5]. All reaction mixes except those for the GTP is vacated by the monitoring bases A1492 and A1493 when they hydrolysis experiments contained 1 mM ATP, 1 mM GTP, 10 mM phospho- engage with the codon–anticodon minihelix during mRNA enolpyruvate (PEP), 1 μg/mL pyruvate kinase (PK), and 0.1 μg/mL myokinase decoding (21, 22, 31). This suggests that A-site binding of a tRNA (MK). In the GTP hydrolysis experiments the reaction mixes contained 1.5 mM liberates the viomycin binding site, which would otherwise be oc- ATP, 0.5 mM [3H]GTP, and 10 mM PEP but no PK or MK. His-tagged initiation cupied by the monitoring bases. Further evidence that A-site factors IF1, IF2, and IF3; elongation factors EF-G, EF-Tu, and EF-Ts; and amino- binding of tRNA increases viomycin affinity to the ribosome comes acyl tRNA-synthetases (ThrRS and PheRS) were purified using nickel-affinity chromatography (HiTrap; GE Healthcare). All protein concentrations were de- from data presented in a recent single-molecule study (13). From termined using the Bradford assay. Ribosomes (E. coli MRE600) and f[3H]Met- their data we estimate a dissociation constant for viomycin binding tRNAfMet were prepared according to ref. 36, and ribosome concentration was to 70S ribosomes with an empty A site as large as 20 μM. In determined spectrophotometrically. XR7 mRNA with the coding sequence Met- contrast, we observe that when viomycin is equilibrated with ri- Phe-Thr-Stop (AUG-UUU-ACG-AUU) and the truncated mRNA coding MFT bosomes with peptidyl tRNA in the A site, drug binding is fully (AUG-UUU-ACC) were was prepared according to ref. 27 and the truncated saturated already at 1 μM, implying a dissociation constant much mRNA was purchased from IBA. Bulk tRNA was prepared from E. coli MRE600 Phe smaller than 1 μM. according to ref. 37. tRNA was overexpressed in E. coli MRE600 from a plasmid with a T7 promoter and prepared from these cells as in ref. 37. [3H]Met The free concentration of viomycin required to significantly re- 3 duce the average peptide elongation rate is very low, in the nano- and [ H]GTP were from Perkin-Elmer, viomycin was from USP, and all other chemicals were from either Merck or Sigma-Aldrich. molar range under in vivo-like conditions (Fig. 3C). In addition to cell permeability factors, drug efflux pumps and drug degradation Quench-Flow Tripeptide Formation Experiments. Two mixtures were prepared. pathways the sensitivity of bacteria to viomycin will depend on the The initiation mixture contained 70S ribosomes (0.3 μM), IF1, IF2, and IF3 concentration of intracellular ribosomes and their sensitivity to (1 μM each), f[3H]Met-tRNAfMet (1.5 μM), and MFT mRNA (0.7 μM). The the drug, as determined by the numerical values of the three elongation mixture contained EF-Tu (12 μM), EF-Ts (2 μM), EF-G (10, 20, or

982 | www.pnas.org/cgi/doi/10.1073/pnas.1517541113 Holm et al. Downloaded by guest on September 26, 2021 40 μM), threonine (200 μM), phenylalanine (200 μM), ThrRS (0.5 μM), PheRS the resulting mixture was incubated for 5–10 s and then one volume of the (0.5 μM), bulk tRNA (150 μM, of which tRNAThr1 and tRNAThr2 made up 4 μMand second elongation mixture was added. The reaction was quenched at different tRNAPhe 1.7 μM), and 2.3 μM of overexpressed tRNAPhe.Viomycin(1–100 μM) time points after the addition of the second elongation mixture using formic was added either to the elongation mixture or to both mixes as indicated. acid (17% final). The samples were treated identically to the quench-flow After 15-min incubation at 37 °C equal volumes of the two mixes were samples above. rapidly mixed and the reaction quenched at different time points with formic acid (17% final concentration) using a quench-flow instrument (RQF-3; GTP Hydrolysis Experiments. Two mixtures were prepared. The initiation mix- × KinTek Corp.). After quenching the samples were centrifuged at 20,800 g ture contained 70S ribosomes (0.5 μM), fMet-tRNAfMet (0.8 μM), and MFT mRNA μ and the supernatant discarded. The pellet was resuspended in 165 L0.5M (0.8 μM). The elongation mixture contained phenylalanine (200 μM), PheRS KOH to cleave the peptides from the tRNA. After 10 min 13 μL of 100% (0.5 μM), overexpressed tRNAPhe (4 μM), EF-Tu (8 μM), EF-Ts (1 μM), EF-G (1–40 μM), formic acid was added, the samples were centrifuged at 20,800 × g, and the andviomycin(400μM). Both reaction mixes contained 0.5 mM [3H]GTP, 1.5 mM radioactive peptides in the supernatant were analyzed by RP-HPLC using a ATP, and 10 mM PEP. After 15-min incubation at 37 °C equal volumes of H O/MeOH/trifluoroacetic acid (62/38/0.1 by volume) mobile phase and a 2 the two mixes were rapidly mixed and the reaction quenched at differ- C-18 column (Merck) with on-line scintillation counting (β-RAM model 3; IN/US ent time points with formic acid (17% final concentration) using the Systems) to quantify the relative amounts of f[3H]Met, f[3H]Met-Phe, and f[3H]Met-Phe-Thr peptides. quench-flow instrument. The acid-quenched samples were centrifuged at 20,800 × g. The supernatant containing the [3H]GTP and [3H]GDP was analyzed by anion exchange chromatography with on-line scintillation Sequential Tripeptide Formation Experiments. Three mixtures were prepared. β The initiation mixture contained ribosomes (0.9 μM),IF1,IF2,andIF3(1μM counting ( -RAM model 3; IN/US Systems). A Mono-Q GL column (GE each), f[3H]Met-tRNAfMet (0.8 μM), and truncated MFT mRNA (1 μM). Two elon- Healthcare) was used and the mobile phase was a multistep gradient of – gation mixes were prepared. The first contained phenylalanine (200 μM), PheRS 0 2 M NaCl in 20 mM Tris (pH 7.5). (0.5 μM), tRNAPhe (4 μM), EF-Tu (5 μM), EF-Ts (2 μM), and viomycin (2–200 μM). The second contained threonine (200 μM), ThrRS (0.5 μM), EF-Tu (8 μM), Data Analysis and Curve Fitting. All curve fitting was done in MATLAB R2014b EF-Ts (2 μM), bulk tRNA (225 μM, of which tRNAThr1 and tRNAThr2 made up (MathWorks) using the Leonardt–Marquardt algorithm as implemented in the 6 μM), viomycin (1–100 μM), and EF-G (15–60 μM). All three mixes where in- curve-fitting toolbox. Detailed descriptions of curve-fitting procedures and cubated for 15 min at 37 °C. During the experiment, one volume of the initi- derivations of the equations in the main text can be found in SI Materials ation mixture was mixed with one volume of the first elongation mixture, and and Methods.

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