Single-molecule protein unfolding and translocation by an ATP-fueled proteolytic machine

Abstract

All cells employ ATP-powered proteases for protein-quality control and regulation. In the ClpXP protease, ClpX is a AAA+ machine that recognizes specific protein substrates, unfolds these molecules, and then translocates the denatured polypeptide through a central pore and into ClpP for degradation. Here, we use optical-trapping nanometry to probe the mechanics of enzymatic unfolding and translocation of single molecules of a multidomain substrate. Our experiments demonstrate the capacity of ClpXP and ClpX to perform mechanical work under load, reveal very fast and highly cooperative unfolding of individual substrate domains, suggest a translocation step size of 5-8 amino acids, and support a power-stroke model of denaturation in which successful enzyme-mediated unfolding of stable domains requires coincidence between mechanical pulling by the enzyme and a transient stochastic reduction in protein stability. We anticipate that single-molecule studies of the mechanical properties of other AAA+ proteolytic machines will reveal many shared features with ClpXP.

Figure 1. Experimental Design

(A) Cartoon of the ClpXP protease midway through degradation of a multidomain substrate. The substrate is threaded through the pore of the ClpX hexamer (yellow-brown). Loops in the pore grip the substrate, and downward loop movements, powered by ATP binding and hydrolysis, unfold each domain and translocate the denatured polypeptide into the lumen of ClpP (green), where degradation to small fragments occurs (Ortega et al., 2000; Siddiqui et al., 2003; Martin et al., 2008a; 2008b; Glynn et al., 2009). (B) Representation of an optical double-trap in a passive force-clamp geometry. ClpXP was attached to one polystyrene bead, a multidomain substrate was tethered to a second bead by double-stranded DNA, and connectivity between the beads was maintained by interactions between ClpXP and the substrate. (C) Structural representation of the multidomain substrate. (D) ClpXP unfolding of a domain of the substrate increases the bead-bead distance, whereas translocation of the unfolded polypeptide decreases this distance. The dwell is the time required to unfold the next domain.

Figure 2. Single-Molecule Unfolding and Translocation by ClpXP

(A) Representative extension-versus-time traces of unfolding and translocation events, accompanying ClpXP degradation of the multidomain substrate at different forces using 2 mM ATP. Traces are offset on the y-axis to avoid overlap. Gray lines represent raw data following decimation; colored lines represent averages over a 10-point sliding window. Red segments represent FLN domains; blue segments represent the HaloTag domain; green segments represent translocation of the 47-residue linker between the HaloTag domain and FLN1. Dwells between FLN subunits unfolding in the top 8 pN trace were spaced at ~4 nm (insert), which is the length of a folded FLN domain (Furuike et al., 2001). No traces contained eight FLN unfolding events, presumably because C-terminal FLN domains were unfolded and translocated before measurements began. (B) Plots of force versus unfolding extension minus native length (NL) for FLN domains (red symbols; mean ± SEM) or the HaloTag domain (blue symbols). The red line is a WLC force-extension model (red line) with a contour length (37.3 nm) and a fitted persistence length (0.61 nm) consistent with reported values for FLN domains (Furuike et al., 2001). For the HaloTag domain, NL was 3.3 nm, the native distance between Asp106 and the HaloTag C-terminus; Asp106 forms a covalent bond with the HaloTag ligand (Los and Wood, 2007) on the DNA linker. The blue WLC curve was calculated with a persistence length of 0.61 nm and a fitted contour length of 62.1 nm, a value within 7% of previous measurements (Taniguchi and Kawakami, 2010). (C) Raw data showing four independent FLN-unfolding transitions (offset on the x-axis for clarity). The three transitions on the left occurred without detectable intermediates and within 1 ms, as did ~85% of all unfolding transitions. The rightmost transition occurred in two stages, as expected for an unfolding intermediate, but was still complete within 5 ms; ~15% of FLN unfolding events behaved similarly.

Figure 3. ClpX Unfolding and Translocation

Experiments with ClpX^SC alone showed some upward steps consistent with single-domain FLN unfolding and subsequent translocation (red regions). Highly cooperative unfolding transitions were observed (insert) in these regions. In the black regions, many upward steps were too large to represent single-domain unfolding, were frequently not followed by translocation, and probably correspond to load-induced backward slipping of substrate segments that had already been unfolded/translocated by ClpX. Following translocation through the ClpX pore, portions of the substrate may interact with the bead, refold, or be engaged by another ClpX^SC enzyme, further complicating analysis. The combined forward and backward motions result in a slow rate of net tracking along the substrate (e.g., in both middle traces there is no net forward movement between 150 and 500 s). Mean velocities from Gaussian fits (± 95% confidence interval) of the distribution of translocation velocities for ClpX^SC were 21.1 ± 2.9 aa s⁻¹ at 4–8 pN and 18.3 ± 2.2 aa s⁻¹ at 8–12 pN.

Figure 4. Distribution of Dwell Times Preceding FLN-Unfolding Events

(A) Pre-unfolding dwell times (n=154) for ClpXP varied widely, irrespective of the force regime. (B) Steady-state rates of ATP hydrolysis by ClpX^SC/ClpP were determined at different ATP concentrations in the presence of the multidomain substrate (20 μM) or carboxymethylated titin_I27-LAA (30 μM), a model unfolded substrate (Kenniston et al., 2003). Symbols are averages (± SD) based on independent replicates (n ≥ 3). The solid lines are non-linear least-squares fits to the Hill equation (rate = V_max/(1+(K_M/[ATP])^h). Multidomain substrate; V_max = 2.2 0.1 enz⁻¹ sec⁻¹; K_M = 16.8 0.1 μM; h = 1.3 ± 0.1. Unfolded substrate; V_max = 3.6 0.1 enz⁻¹ sec⁻¹; K_M = 29.8 2.2 μM; h = 1.4 ± 0.1). No protein substrate; V_max = 1.2 0.1 enz⁻¹ sec⁻¹; K_M = 10.1 1.5 μM; h = 1.4 ± 0.3. Positive cooperativity (h > 1) in ATP hydrolysis by ClpX and ClpXP has been reported (Burton et al., 2003; Hersch et al., 2005). (C) Dwell times were combined, plotted as cumulative frequencies, and fitted to a triple-exponential function, yielding rate constants of 3.4 ± 0.16 s⁻¹ (amplitude 35 ± 1%), 0.23 ± 0.02 s⁻¹ (amplitude 47 ± 2%) and 0.03 ± 0.01 s⁻¹ (amplitude 18 ± 2%). The inset shows the residuals for single, double, and triple exponential fits. (D) Single-exponential fits of cumulative frequencies versus pre-unfolding dwell times for FLN2 events (filled squares; k = 0.20 ± 0.04 s⁻¹), FLN1 events (open squares; k = 0.04 ± 0.01 s⁻¹), and HaloTag events (circles; k = 0.04 ± 0.02 s⁻¹).

Figure 5. Translocation Velocities at Different Forces and Nucleotide Concentrations

(A) Histograms with distributions of ClpXP translocation velocities, weighted by the length translocated at that velocity, are shown for experiments using 2 mM ATP in four force regimes. The solid lines are Gaussian fits. (B) Red symbols are mean velocities from Gaussian fits (± 95% confidence interval), calculated from the panel-A data. The solid line is a non-linear least-squares fit of the force dependence to a single-barrier Boltzmann model (see text). The blue bar represents a range of unloaded velocities from single-molecule and ensemble experiments using different substrates (Martin et al., 2008c; Shin et al., 2009). (C) Translocation-velocity distributions at 8–12 pN for experiments performed using 75 μM ATP (frequent rupture of bead linkage occurred when less ATP was used) or 1 mM ATP plus 1 mM of ATP S. The dashed vertical line represents the mean velocity for 8–12 pN using 2 mM ATP. Solid lines are Gaussian fits.

Figure 6. Visualization of Individual ClpXP Translocation Steps

(A)–(G) For the y-axis of each panel, the black numbers represent amino acids, assuming a WLC model with a persistence length of 0.61 nm (see Fig. 2B legend), and the red numbers represent changes in bead-bead distance. In panels A–C, the black vertical bars represent the smallest steps resolved. Experiments were performed using 2 mM ATP (A–E), 75 μM ATP (F), or 1 mM ATP plus 1 mM ATP S (G). Asterisks mark events that appear to be minor slipping of the substrate relative to ClpXP, which was more common during translocation under higher loads, at low ATP, or in ATP/ATP S mixtures. (H) Power spectra (Svoboda et al., 1993) of pairwise-distance distribution functions of single translocation curves show main peaks near ~0.08 and ~0.16 aa⁻¹, corresponding to step sizes of ~6.25 (range 5.7–7.1) and ~12.5 (range 10–13) amino acids.

Figure 7. Translocation Steps by ClpX alone

(A)–(G) For the y-axis of each panel, the black numbers represent amino acids, assuming a WLC model with a persistence length of 0.61 nm (see Fig. 2B legend), and the red numbers represent changes in bead-bead distance. (H) Power spectra of pairwise-distance distribution functions of single translocation curves show main peaks near ~0.095 and ~0.15 aa⁻¹, corresponding to step sizes of ~6.8 (range 6.2–7.7) and ~10.5 (range 10–11) amino acids.