ABCE1 Controls Ribosome Recycling by an Asymmetric Dynamic Conformational Equilibrium

Abstract

The twin-ATPase ABCE1 has a vital function in mRNA translation by recycling terminated or stalled ribosomes. As for other functionally distinct ATP-binding cassette (ABC) proteins, the mechanochemical coupling of ATP hydrolysis to conformational changes remains elusive. Here, we use an integrated biophysical approach allowing direct observation of conformational dynamics and ribosome association of ABCE1 at the single-molecule level. Our results from FRET experiments show that the current static two-state model of ABC proteins has to be expanded because the two ATP sites of ABCE1 are in dynamic equilibrium across three distinct conformational states: open, intermediate, and closed. The interaction of ABCE1 with ribosomes influences the conformational dynamics of both ATP sites asymmetrically and creates a complex network of conformational states. Our findings suggest a paradigm shift to redefine the understanding of the mechanochemical coupling in ABC proteins: from structure-based deterministic models to dynamic-based systems.

Keywords: ABC proteins; conformational dynamics; mRNA surveillance; mRNA translation; molecular motors; ribosome recycling; single-molecule FRET; twin ATPases.

Graphical abstract

Figure 1

Biochemical Function of FRET Pair-Labeled ABCE1 (A) Double-cysteine variants probing the conformational states of site I (ABCE1^I124C/K430C) or site II (ABCE1^K177C/T393C) via smFRET are depicted on the crystal structures of ABCE1 (Barthelme et al., 2011, Karcher et al., 2008). Middle: cryo-EM structure of the ribosome-bound (pre-splitting complex) intermediate state of ABCE1 (Becker et al., 2012). Right: cryo-EM structure of the closed (ATP bound) state bound to the small ribosomal subunit (post-splitting complex) (Heuer et al., 2017). (B) ABCE1 wild type and variants were purified to homogeneity by metal affinity and anion exchange chromatography. SDS-PAGE (12.5%, Coomassie and in-gel fluorescence). Donor and acceptor fluorophores are illustrated as D and A, respectively. (C) FRET pair-labeled ABCE1 variants were analyzed by fluorescence-based size-exclusion chromatography (SEC) and subsequently used for smFRET. (D) ATPase activity of ABCE1 before (gray) and after (white) fluorescence labeling. Data represent mean ± SD from three independent experiments.

Figure 2

Conformational States of ATP Sites I and II of ABCE1 Revealed by ALEX-Based smFRET (A and C) 2D-ALEX histogram of site I (A) and site II (C) variants labeled with Cy3B and Atto647N reveals three conformational states (open, intermediate, and closed). 2D-ALEX histogram of free ABCE1 (top panel); ABCE1 with AMP-PNP, 70S, and aRF1/aPelota (middle panel); and AMP-PNP and 30S (bottom panel). Interaction partners (3 μM 70S and aRF1/aPelota or 1 μM 30S ribosome, 2 mM AMP-PNP) affect these states. For clarity, only the double-labeled donor-acceptor species are shown. Cartoons depict the percentage of each state in the conformational equilibrium, with different transparencies as indicated. (B and D) Burst-size histogram of site I (B) and II (D) variants of free ABCE1 and the pre-splitting state and post-splitting state of ABCE1 reveals the diffusion properties of the complexes and allows determination of a relative diffusion coefficient D by fitting the histogram to Equation 1 (Method Details). Errors indicate 95% confidence interval.

Figure 3

Release of ABCE1 from the Small Ribosomal Subunit Is Followed by Site II Opening (A) Cartoon summarizing the experimental settings. (B and C) Release of ABCE1 from the small ribosomal subunit (black) occurring (B) only at the physiological temperature (70°C–80°C) and simultaneous ADP competition or (C) by 30S and AMP-PNP dilution at the indicated time points (manual mixing didn’t allow earlier time points), and its interdependence with site II opening (orange). Data represent mean ± SD from four independent experiments.

Figure 4

Conformational Dynamics at Site II Depend on Binding Partners and Ligands (A) Conformational equilibrium (73°C, 10 min) of the different ABCE1 site II states after incubation with saturating AMP-PNP concentrations (2 mM) and two representative 30S concentrations, as indicated. (B) As in (A) with the full range of 30S concentrations. At each concentration, the percentage of every state was determined and plotted. (C) Influence of different ligands such as nucleotides and ribosomal subunits on the conformational dynamics at site II. Measurements were performed under saturation conditions of nucleotides (2 mM) and 30S ribosomal subunits (1 μM) after 10 min of incubation at 73°C. (D) Burst length distribution of the different conditions (as in C) were analyzed as previously described (Figures 2B and 2D). (E) Time course experiment after incubation with saturating AMP-PNP (2 mM) and 30S (1 μM) concentrations at the representative points in time, as indicated (top 3 panels). Bottom panel: same experiment after pre-incubation with AMP-PNP (73°C, 2 mM, 8 min). (F) As in (E), with the full range of points in time. At each point in time, the percentage of every state was determined and plotted. (G) Cartoon summarizing the findings of (E) and (F). Data represent mean ± SD from 3–5 independent experiments.

Figure 5

Dynamics of ABCE1 in Ribosome Recycling Step 1: free ABCE1 sites are in dynamic equilibrium across three states (open, intermediate, and closed) but predominantly found in the open conformation. ABCE1 displays basal ATPase activity of 5 ATP per minute (see Figure 1D). Step 2: complex with the terminated 70S is mediated by the A-site factors e/aRF1 or e/aPelota and ATP binding. Upon formation of the pre-SC, only site II shifts to the intermediate state, as indicated, and the FeS cluster domain moves toward NBD2 (intermediate; see Figure 2A). Step 3: during splitting, ATP binding and incubation at a physiological temperature trigger the two sites to close, and the FeS cluster domain is repositioned 150° on 30S in the post-SC (closed; see Figures 2A and 2C). Here, either the FeS cluster domain pushes the A-site factor farther into the cleft between the subunits or the domain splits the subunits apart. Step 4: after splitting, bound ABCE1 can build a platform for re-initiation (Heuer et al., 2017). Acquisition of the ADP state triggers dissociation of the post-SC to initiate a new round. ABCE1 is highly dynamic, being at every condition in equilibrium across the indicated conformational states. The percentages of open, intermediate, and closed states for the 2 sites have been experimentally determined for steps 1, 2, and 4. The unstable short-lived step 3 is anticipated to have intermediate values of steps 3 and 4.