Fusidic acid targets elongation factor G in several stages of translocation on the bacterial ribosome

Abstract

The antibiotic fusidic acid (FA) targets elongation factor G (EF-G) and inhibits ribosomal peptide elongation and ribosome recycling, but deeper mechanistic aspects of FA action have remained unknown. Using quench flow and stopped flow experiments in a biochemical system for protein synthesis and taking advantage of separate time scales for inhibited (10 s) and uninhibited (100 ms) elongation cycles, a detailed kinetic model of FA action was obtained. FA targets EF-G at an early stage in the translocation process (I), which proceeds unhindered by the presence of the drug to a later stage (II), where the ribosome stalls. Stalling may also occur at a third stage of translocation (III), just before release of EF-G from the post-translocation ribosome. We show that FA is a strong elongation inhibitor (K50% ≈ 1 μm), discuss the identity of the FA targeted states, and place existing cryo-EM and crystal structures in their functional context.

Keywords: Antibiotic Action; Elongation Factor G; Fusidic Acid; Kinetics; Protein Synthesis; Slow Inhibitors; Translation Elongation Factor; Translocation.

FIGURE 1.

General scheme for peptide elongation cycle “i” during ribosomal synthesis of a protein and its inhibition by FA. In the special case that i = 1, Fig. 1 describes the first elongation cycle in the synthesis of a protein, as in Fig. 4. The scheme contains parameters obtainable from the present experiments, also relevant for FA inhibition of protein synthesis in the living cell. Subscheme A shows the inhibition of peptide bond formation by EF-G and FA.

FIGURE 2.

Dipeptide formation after preincubation of initiation complexes with EF-G and FA. A, time traces for dipeptide formation obtained after preincubation of initiation complexes (1 μm) with EF-G(GDP) (20 μm) and FA at varying concentration and subsequent mixing with EF-Tu(GTP)·Leu-tRNA^Leu ternary complexes (2 μm). B, fraction of ribosomes bound in EF-G·FA complexes as a function of FA concentration in the presence of GDP (black trace) or GTP (purple trace). C, variation in the time of release of FA from the complex at 20 and 5 μm EF-G (slow phase in Fig. 2A). D, total time of dipeptide formation as a function of the FA concentration at 5 and 20 μm EF-G. E, schematic of dipeptide formation in the presence of EF-G and FA. Upon preincubation with FA and EF-G, the initiation complexes partitioned between the free state (R¹) and the EF-G- and FA-bound state R_I3⁰. EF-G binds to the initiated ribosome R¹ with rate constant k_G3⁰ to form R_G3⁰, from which it dissociates with rate constant q_G3⁰. FA binds to R_G3⁰ with rate constant k_F3⁰ to form R_I3⁰, from which it dissociates slowly with rate constant q_F3⁰. Upon mixing of the pre-equilibrated reaction mixture with ternary complex, the free initiation complexes, R¹, rapidly form dipeptide, whereas the bound ribosomes slowly release FA before they can bind ternary complex and form dipeptide. Error bars, S.E.

FIGURE 3.

Dipeptide formation in the absence of preincubation of initiation complexes with EF-G and FA. Time traces were obtained after mixing of initiated ribosomes (0.5 μm) with EF-G (20 μm) and EF-Tu(GDP)·tRNA^Leu ternary complexes (1 (circles) or 2 μm (squares)) in the presence (2 mm; red traces) or absence (black traces) of FA. (The concentrations are the effective concentrations after mixing.)

FIGURE 4.

Schematic of FA inhibition of the first peptide elongation steps (see also Fig. 1). The FA inhibition modes I, II, and III (IIIA and IIIB) are enclosed in red boxes. The 70S initiation complex (R¹) with fMet-tRNA^fMet in the P site may bind ternary complex and form ribosomal complex R_pep¹ by peptide bond formation. Alternatively, EF-G may bind to R¹ with the rate constant k_G3⁰ and form complex R_G3⁰, from which EF-G dissociates with the rate constant q_G3⁰. FA may bind to R_G3⁰ with the rate constant k_F3⁰ to form the stable complex R_I3⁰, from which it slowly dissociates with the rate constant q_F3⁰. These reactions define the third inhibition mode of FA (III). Ribosomal complex R_pep¹ may, via the complexes R_G0¹, R_G1¹, R_G2¹, and R_G3¹, be rapidly translocated by EF-G to ribosomal complex R² with Michaelis-Menten parameters k_G¹ and K_G¹. Alternatively, FA may bind to R_G1¹ with association rate constant k_F1¹ and form R_I1¹, which is rapidly transformed to R_I2¹ in which the ribosome is stalled until FA release with rate constant q_F2¹. After this, FA may rebind to R_G2¹, or the reaction may continue downstream to the post-translocation state R_G3¹ with the rate constant k_G2¹. The post-translocation state may inefficiently bind FA with rate constant k_F3¹ or release EF-G with rate constant q_G3¹. The first binding of FA to the translocating ribosome with rate constant k_F1¹ and subsequent dissociation of FA from R_I2¹ define the first and strongest mode of action of FA. After the first FA dissociation from R_I2¹, multiple rebinding of FA to R_G2¹ defines the second inhibition mode of FA.

FIGURE 5.

Single round tripeptide formation. A, time traces for tripeptide formation in the presence of varying concentrations of FA and 20 μm EF-G. B, the fraction of ribosomes bound by FA during single turnover tripeptide formation plotted as a function of FA concentration. C, variation in the average time of release of the FA complex in presence of 20 (black) or 5 μm (green) EF-G compared with the average time of release from the post-translocation complex (R_I3¹) measured in a separate set of experiments (purple). D, total time of tripeptide formation as a function of the FA concentration in the presence of 20 or 5 μm EF-G. Error bars, S.E.

FIGURE 6.

A, translocation of mRNA monitored with fluorescent 3′-pyrene labeled mRNA at 20 μm EF-G and varying FA concentration. From fluorescence time traces F_[FA](t), where F is fluorescence intensity, [FA] is fusidic acid concentration, and t is incubation time, normalized intensity ratios Δ_[FA](t) = (F_[FA](t) − F₀(∞))/(F₀(0) − F₀(∞)) were formed. Final time was defined as the time at which the mRNA movement had been completed. The intensity ratio at final time is given by Δ_[FA](∞) = (F_[FA](∞) − F₀(∞))/(F₀(0) − F₀(∞)). By definition, it was zero in the absence of FA and increased with increasing FA concentration due to increasing values of F_[FA](∞). Solid lines, double exponential functions fitted to Δ_[FA](t). A digital filter was used to reduce the noise and the number of data points in the traces. B, the intensity ratio Δ_[FA](∞) and the fraction of FA-bound ribosomes estimated from tripeptide experiments (Fig. 5B) were plotted as functions of the FA concentration. The Δ_[FA](∞) values were normalized for correspondence with quench flow data at 400 μm FA. Solid lines, hyperbolic functions fitted to both data series. Error bars, S.E.

FIGURE 7.

Cycling of EF-G in multiple turnover translocation. A small amount of EF-G (0.05 μm) was allowed to cycle on in situ-formed pretranslocation complexes (2 μm) translocating the fMet-Leu dipeptidyl-tRNA into the ribosomal P site enabling formation of fMet-Leu-Phe tripeptide. Tripeptide formation was followed over time in the absence (black trace) or presence of FA (20 μm; gray trace).