Stochastic but highly coordinated protein unfolding and translocation by the ClpXP proteolytic machine

Abstract

ClpXP and other AAA+ proteases recognize, mechanically unfold, and translocate target proteins into a chamber for proteolysis. It is not known whether these remarkable molecular machines operate by a stochastic or sequential mechanism or how power strokes relate to the ATP-hydrolysis cycle. Single-molecule optical trapping allows ClpXP unfolding to be directly visualized and reveals translocation steps of ∼1-4 nm in length, but how these activities relate to solution degradation and the physical properties of substrate proteins remains unclear. By studying single-molecule degradation using different multidomain substrates and ClpXP variants, we answer many of these questions and provide evidence for stochastic unfolding and translocation. We also present a mechanochemical model that accounts for single-molecule, biochemical, and structural results for our observation of enzymatic memory in translocation stepping, for the kinetics of translocation steps of different sizes, and for probabilistic but highly coordinated subunit activity within the ClpX ring.

Fig. 1

Single-molecule unfolding and translocation of substrates. (A) Cartoon structure of titin¹²⁷ (pdb code, 1tit), colored from the N terminus (blue) to the C terminus (red). Spheres show α carbons for residues 13, 15, and 87. CIpXP pulling on a C-terminal ssrA tag is resisted by local structure, including β-sheet hydrogen bonding between the C-terminal β strand and the β strand with residues 13 and 15. (B) The V13P and V15P mutations disrupt hydrogen bonds that directly or indirectly stabilize the titin¹²⁷ domain. (C) Experimental setup for single-molecule assays of CIpXP unfolding and translocation. CIpXP is attached to one laser-trapped bead and has engaged the ssrA tag of a multi-domain substrate consisting of four titin domains and a Halo domain, which is attached to a second laser-trapped bead via a DNA linker. (D) Trajectories for CIpXP unfolding and translocation of multi-domain substrates. Unfolding of individual domains increases bead-bead distance (upward movement), whereas translocation decreases bead-bead distance (downward movement). After completed translocation of one domain, there is a variable dwell time before CIpXP unfolds the next domain. The dwell baselines before and after titin unfolding events are spaced as expected for the end-to-end distance of a native titin domain (4.4 nm) or native titin plus the linker to the Halo domain.

Fig. 2

CIpXP unfolding of domains in multi-domain substrates. (A–C) Distributions of pre-unfolding dwell times for the V13P, V15P, and Halo domains. In each plot, the solid line is a non-linear-least-squares fit to y=A*(1-exp(−t/τ_unf)). (D) For the Halo-WT-V13P-V13P-V13P-ssrA substrate, long “terminal” dwells were often observed following unfolding and translocation of the V13P titin domains. (E) CIpXP unfolding of wild-type titin¹²⁷ domains. Black symbols are pre-unfolding dwells; gray symbols are “terminal” dwells. The line is a fit to y=A*(1-exp(−t/τ_unf)). (F) Plots of average force versus average pre-unfolding dwell times (calculated over a moving 50-point window) for the V13P and V15P domains. (G) Plot of average force versus average pre-unfolding dwell times (calculated over a moving 40-point window) for the Halo domain. See also Figs. S1, S4, and S7.

Fig. 3

Translocation and pausing. (A) V13P translocation traces proceeding with approximately constant velocity (left panel) or with a pause (right panel). (B) Plots of average force versus average translocation velocity were calculated over a moving 50-point window for the V13P and V15P domains and over a 40-point window for the Halo domain. The lines are fits to a single-barrier Boltzmann equation v = v₀•(1.05)/(1+0.05•exp(F•0.7/kT)) where F is the average force and kT is 4.1 pN•nm at room temperature. (C) Probability of pausing of CIpXP along the length of a titin domain (N = 25). (D) Probability of pausing of CIpXP along the length of a Halo domain (N = 24). Secondary structure in the native structure is indicated schematically in panels C and D (arrows represent β strands; zigzag lines represent α helices).

Fig. 4

Physical steps during titin translocation. (A) Representative stepping in CIpXP translocation trajectories. Raw data were decimated to 500 Hz (gray) or 50 Hz (orange). Chi-square fits to the 50 Hz data are shown in black. (B) Distribution of physical steps sizes during titin translocation. (C) Mean physical step size during titin translocation as a function of force. X- and Y-error bars are ± 1 SD (N = 70–221). (D) Mean number of physical steps required to translocate an 89-residue titin domain and 4-residue linker as a function of force (black squares). X- and Y-error bars are ± 1 SD (N = 6– 20). Gray X’s are step numbers from individual translocation trajectories. (E) Mean dwell times ± SEM (N = 45–236) before (red) or after (green) physical steps of 1–4 nm (pre- and post-step values are offset slightly on the x-axis for clarity). (F) Distribution of dwell times preceding steps of all sizes during titin translocation. (G) Occurrence of steps of different size either before (N−1) or after (N+1) physical steps of 1–4 nm. (H) Distribution of times required to complete translocation of 89-residue titin domains and subsequent 4-residue linkers after subtracting pauses. See also, Figs. S2, S3, S5, S6, and S8.

Fig. 5

Unfolding and translocation by RWERWE CIpXP. (A) V13P unfolding and translocation traces for RWERWE CIpXP (top) and CIpXP with six active subunits (bottom). (B) Distributions of RWERWE CIpXP pre-unfolding dwell times for the V13P domain. The line is a non-linear-least-squares fit to y = A*(1-exp(−t/τ_unf)). (C) Representative stepping in titin V13P translocation by CIpXP (orange) and RWERWE CIpXP (green). Decimation and fits (black) are described in Fig. 4A. (D) Distribution of RWERWE physical step sizes. Inset- Cumulative frequency distributions of dwell times preceding steps for CIpXP (orange) or RWERWE CIpXP (green). See also Fig. S8.

Fig. 6

Solution degradation times are poorly predicted by single-molecule unfolding and translocation times. (A) Plot of average times required for solution degradation (τ_deg) of titin-ssrA (WT), V15P-titin-ssrA (V15P), V13P-titin-ssrA (V13P), carboxymethylated titin-ssrA (CM), GFP-ssrA (GFP), and Halo-ssrA (Halo) versus τ_unf + τ_trans times from single-molecule experiments (Kim et al., 2000; Kenniston et al., 2003; Sen et al., 2013; this work). Times for titin and GFP degradation were determined at 30 °C, whereas single-molecule experiments and Halo-ssrA degradation were performed at room temperature. Degradation is slower at lower temperatures (see panel C), which would increase the discrepancy between the solution and single-molecule results. τ_unf values were determined under load and could be different at zero force, but V13P and V15P τ_unf values (Fig. 2E) would not increase 4-fold and the Halo τ_unf value appears to decrease (Fig. 2F). (B) τ values for single-turnover binding and unfolding of GFP-ssrA (0.5 µM) by CIpXP (10 µM CIpX^ΔN; 20 µM CIpP) at different temperatures. (C) τ_deg values (1/V_max) at different temperatures determined from Michaelis-Menten plots of steady-state rates of degradation of different concentrations of GFP-ssrA by CIpX^ΔN (0.3 µM) and CIpP (0.9 µM). (D) K_M values for GFP-ssrA degradation at different temperature (conditions as in panel C). (E) Rates of CIpXP ATP hydrolysis at different temperatures by CIpX^ΔN (0.3 µM) in the presence of CIpP (0.9 µM) and GFP-ssrA (20 µM). (F) Fractional activity of CIpXP at different temperatures calculated as (τ_c + τ_unf + 5 s)/τ_deg, where the τ_c + τ_unf value is taken from panel B and 5 s is the estimated time for translocation of GFP-ssrA.

Fig. 7

Mechanochemical models for CIpXP function. X4 and X3 rings are hydrolytically and mechanically active. iX3, iX2, iX1, and iX0 rings are inactive. Numbers after the X are bound ATPs. In the cartoons of the CIpX hexamer, dark red subunits bind ATP tightly, red subunits bind ATP weakly, and light gray subunits do not bind ATP. (A) Unfolding model. ATP hydrolysis in the X4 or X3 rings results in an unfolding power stroke, which allows translocation to begin, or in a futile power stroke. ATP-binding reactions are represented by green arrows and conformational changes by dark red arrows. For simplicity, ATP binding to iX0 or iX1 rings is not shown in this panel, ATP-dissociation reactions are not included, and different configurations of nucleotide-bound subunits in the X3 ring are not considered. Pseudo first-order rate constants for ATP-association reactions are for saturating concentrations of ATP. The mechanical stability of a native protein determines the rate of the unfolding reaction; other rates are determined by the properties of CIpXP. The rate constants in parenthesis give exponential unfolding kinetics (τ_unf ~6 s). (B) Translocation model. Depending on which ATP-bound subunit in the X4 or X3 rings hydrolyzes ATP first, physical translocation steps of 1, 2, 3, or 4 nm are taken (black arrows). A physical step of N nm is associated with N hydrolysis/release events. Numbers in parentheses are rate constants that were adjusted to provide a reasonable fit to experimental data. (C) In the cartoons shown, initial ATP hydrolysis in subunits of X4 or X3 rings (labeled d, c, b, or a) result in very fast ATP hydrolysis/release events that generate power strokes (arrows) in the ATP-bound counter-clockwise subunits, generating physical translocation steps of 4, 3, 2, or 1 nm, respectively. (D) As shown on the left, if wild-type (W) subunits occupy the d and a positions in X4 rings of RWERWE CIpX, then translocation steps of 1 nm (subunit a fires first) or 4 nm (subunit d fires first) are taken. When subunit d fires first, ATP is released from the counter-clockwise inactive c (R) and b (E) subunits to generate power strokes (crooked arrows). If wild-type (W) subunits occupy the b or c positions in the X4 ring (center and right, respectively), then initial hydrolysis in these subunits results in steps of 2 or 3 nm, respectively, again with ATP release from counter-clockwise inactive subunits generating power strokes (crooked arrows). See also Figs. S2, S5, and S6.