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. 2013 Oct 24;155(3):636-646.
doi: 10.1016/j.cell.2013.09.022. Epub 2013 Oct 24.

The ClpXP Protease Unfolds Substrates Using a Constant Rate of Pulling but Different Gears

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The ClpXP Protease Unfolds Substrates Using a Constant Rate of Pulling but Different Gears

Maya Sen et al. Cell. .
Free PMC article

Abstract

ATP-dependent proteases are vital to maintain cellular protein homeostasis. Here, we study the mechanisms of force generation and intersubunit coordination in the ClpXP protease from E. coli to understand how these machines couple ATP hydrolysis to mechanical protein unfolding. Single-molecule analyses reveal that phosphate release is the force-generating step in the ATP-hydrolysis cycle and that ClpXP translocates substrate polypeptides in bursts resulting from highly coordinated conformational changes in two to four ATPase subunits. ClpXP must use its maximum successive firing capacity of four subunits to unfold stable substrates like GFP. The average dwell duration between individual bursts of translocation is constant, regardless of the number of translocating subunits, implying that ClpXP operates with constant "rpm" but uses different "gears."

Figures

Figure 1
Figure 1. Single-molecule Experimental Constructs
(A) Cartoon depicting protein unfolding and polypeptide translocation by ClpXP. (B) Experimental geometry of dual-trap optical tweezers assay (not to scale). Biotinylated ClpX was immobilized on streptavidin-coated beads (SA). The DNA-tethered substrate was immobilized on the surface of beads coated with anti-digoxigenin antibodies (AD). (C-D) Single-molecule trajectories of substrate processing by ClpXP at 1 mM ATP and forces ranging from 6 to 12 pN. GFP unfolding events are indicated by arrows and followed by translocation of unfolded polypeptide. Substrates are composed of GFP moieties (green) fused to titinCM and a C-terminal ssrA tag (black and red, respectively), as well as an N-terminal ybbR tag (light blue) for attachment to the bead. Raw data (2.5 kHz in gray) were filtered and decimated to 100 Hz (green, black, and blue lines).
Figure 2
Figure 2. Effects of ATP, ADP and Pi on translocation
(A) General scheme depicting a motor (M) that binds to one ATP molecule (T), undergoes a tight binding transition, and hydrolyzes ATP, followed by the release of inorganic phosphate (Pi) and ADP. (B) Representative trajectories for translocation of the titinCM moiety of the fusion substrates measured between 6 and 12 pN at different ATP concentrations with ATP regeneration system (ATP/RS). The trajectories are offset for clarity. (C) Pause-free velocity of translocation (mean ± SEM) as a function of external force at 5 mM ATP (red markers) and 35 μM ATP (black markers). (D) Km (blue) and Vmax (green) are plotted against force. Inset: Km/Vmax ratio plotted for forces between 5 to 15 pN. Error bars are from the fits (SEM). (E) Km (blue) and Vmax (green) plotted against ADP concentration at 7.5 pN. (F) Pause-free velocity of translocation (mean ± SEM) plotted as a function of phosphate concentration [Pi] at 7.5 pN with a fixed [ATP] (see also Figure S1).
Figure 3
Figure 3. ATPγS induces long-lived pauses
(A) Representative trajectories for forces between 6 and 12 pN were measured with increasing [ATPγS] at fixed [ATP]. Trajectories are offset for visual clarity. (B) Translocation rate (mean ± SEM) plotted against [ATPγS], with the fit shown in red. (C) Inverse density of ATPγS-induced pauses (mean ± SEM) plotted against the inverse of [ATPγS], with the linear fit shown in red. Inset: pause density plotted as a function of [ATPγS]. (D) Pause duration as a function of [ATPγS] (see also Figure S2).
Figure 4
Figure 4. Effect of ATP concentration on Burst Size and Dwell Duration
(A) Representative trajectories of ClpXP translocating substrate in 3 nm steps at 10-14 pN and different ATP concentrations. Raw data were filtered and decimated to 1250 Hz (in gray) or 50 Hz (in red, blue, green). T-test fits to the data are shown in black. (B) Burst size distributions for ATP concentrations near Km (red) and saturating ATP (blue). (C) Mean dwell duration plotted against [ATP]. Inset: dwell-time distribution for near-Km conditions (red) and saturating ATP (blue) (see also Figure S3).
Figure 5
Figure 5. GFP Unfolding Mechanism
(A) Probability of GFP unfolding as a function of [ATP] and ATPase rate (inset). (B) GFP unfolding events display two intermediates at 300 Hz. (C) Mechanism of GFP unfolding by ClpXP at [ATP] >> Km. (D) Trajectory at [ATP]=200 μM illustrating the ClpXP-induced unfolding and refolding of β11 (see also Figure S4).
Figure 6
Figure 6. Minimal Mechanochemical Model of ClpXP Translocation
(A) The pathway of ATP hydrolysis for a single subunit of ClpX. An empty subunit (E, red sphere) binds ATP (T, orange) and undergoes a tight binding of ATP (T*, green). Then, the subunit hydrolyzes ATP to ADP and Pi (DP, blue), the force-generation step occurs upon phosphate release (D, purple), and ADP dissociates, leaving an empty subunit (E, red sphere). (B) Schematic depiction of inter-subunit coordination at saturating (green box) and limiting ATP concentrations (red box) for one possible scenario depicting sequential ATP binding. The subunits in gray correspond to those that do not bind ATP. During the dwell phase, at least two ATPs are bound to the high-affinity subunits (T, blue outline) and additional ATPs can bind to the low-affinity ClpX subunits (T, green outline), depending on [ATP]. During the burst phase, the motor hydrolyzes all bound ATPs, releases phosphate, and translocates the substrate by 2, 3, or 4 nm into the central pore.

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