Stepwise unfolding of a β barrel protein by the AAA+ ClpXP protease

Abstract

In the AAA+ ClpXP protease, ClpX uses the energy of ATP binding and hydrolysis to unfold proteins before translocating them into ClpP for degradation. For proteins with C-terminal ssrA tags, ClpXP pulls on the tag to initiate unfolding and subsequent degradation. Here, we demonstrate that an initial step in ClpXP unfolding of the 11-stranded β barrel of superfolder GFP-ssrA involves extraction of the C-terminal β strand. The resulting 10-stranded intermediate is populated at low ATP concentrations, which stall ClpXP unfolding, and at high ATP concentrations, which support robust degradation. To determine if stable unfolding intermediates cause low-ATP stalling, we designed and characterized circularly permuted GFP variants. Notably, stalling was observed for a variant that formed a stable 10-stranded intermediate but not for one in which this intermediate was unstable. A stepwise degradation model in which the rates of terminal-strand extraction, strand refolding or recapture, and unfolding of the 10-stranded intermediate all depend on the rate of ATP hydrolysis by ClpXP accounts for the observed changes in degradation kinetics over a broad range of ATP concentrations. Our results suggest that the presence or absence of unfolding intermediates will play important roles in determining whether forced enzymatic unfolding requires a minimum rate of ATP hydrolysis.

Fig. 1

(A) Cartoon showing ClpXP after engaging the ssrA tag of ^SFGFP-ssrA but before unfolding, translocation, and degradation. The GFP chromophore is shown in CPK representation (nitrogen, blue; oxygen, red; carbon, green). (B) Diagram of the secondary structure of ^SFGFP-ssrA. β strands are the same color as in the structure in panel A. (C) Absorption of 400-nm light results in excited state proton transfer (ESPT) in which the phenolic proton moves to Glu²²² on strand 11. Return to the ground state is accompanied by fluorescence emission at 511 nm. Mutation of Ser²⁰⁵ or Glu²²² prevents ESPT and fluorescence after excitation with 400-nm light. Fluorescence arising from excitation of the deprotonated chromophore with 467-nm light does not depend on Ser²⁰⁵ or Gly²²².

Fig. 2

(A) A thrombin cleavage sequence (LVPRGS) was inserted between strands 10/11 or 9/10 in the ^SFGFP-10/11-ssrA and ^SFGFP-9/10-ssrA proteins (NCBI accession codes JF951868 and JF951869, respectively), allowing creation of split proteins. (B) SDS-PAGE showing ^SFGFP-10/11-ssrA or ^SFGFP-9/10-ssrA (10 μM each) before and after thrombin cleavage and ClpXP (0.3 μM ClpX₆; 0.9 μM ClpP₁₄) extraction/degradation. The gel is a composite, with the lower portion taken from a gel containing 8-fold more sample than the upper portion. CPK, creatine phosphokinase. (C) Absorbance spectra of ^SFGFP-10/11-ssrA before (closed circles) or after (open circles) thrombin cleavage. (D) Fluorescence emission spectrum of ^SFGFP-10/11-ssrA before (closed circles) or after (open circles) thrombin cleavage. (E) Incubation of 10 μM thrombin-cleaved ^SFGFP-10/11-ssrA with 1 μM ClpXP (1 μM ClpX₆; 2 μM ClpP₁₄) and 4 mM ATP resulted in loss of 400-nm (open circles) but not 467-nm (closed circles) fluorescence. (F) Incubation of 10 μM thrombin-cleaved ^SFGFP-9/10-ssrA with 1 μM ClpXP and 4 mM ATP resulted in loss of 400-nm (open circles) and 467-nm (closed circles) fluorescence. The experiments in panels E and F contained an ATP-regeneration system.

Fig. 3

(A) Changes in 400-nm fluorescence (open circles) or 467-nm fluorescence (closed circles) following incubation of ^SFGFP-ssrA (10 μM) with ClpXP (1 μM ClpX₆; 2 μM ClpP₁₄) and 50 μM ATP. (B) Same proteins as in panel A but using 4 mM ATP. The inset shows the concentration of the strand-extracted intermediate after 250 s as a function of ClpXP concentration from 4 mM ATP experiments like the one in the main panel. Values plotted are averages (n=4) ± 1 standard deviation. (C) Changes in 400-nm fluorescence (open diamonds) or 467-nm fluorescence (closed diamonds) following incubation of H148D-^SFGFP-ssrA (10 μM; NCBI accession code JF951865) with ClpXP (1 μM ClpX₆; 2 μM ClpP₁₄) and 50 μM ATP. (D) Same proteins as in panel B but using 4 mM ATP. An ATP-regeneration system was used in all experiments.

Fig. 4

(A) Cartoon representation of the order of β strands in ^SFGFP-ssrA and circularly permuted variants. (B) Permuted variants (1 μM) were incubated overnight with ClpXP (1.25 μM ClpX₆; 2.5 μM ClpP₁₄), the SspB adaptor (1 μM), and 0, 50, or 300 μM ATP before assaying degradation by SDS-PAGE. (C) End-point experiments like those in panel B were performed but degradation was assayed by reduced 467-nm fluorescence. GFP-ssrA (circles); ^SFGFP-ssrA (squares); cp6-^SFGFP-ssrA (diamonds); cp7-^SFGFP-ssrA (triangles). The lines are fits to a modified form of the Hill equation. In the panel B and C experiments, an ATP-regeneration system was used.

Fig. 5

(A) The cp6-^SFGFP-5/6-ssrA protein (10 μM; NCBI accession code JF951870) was cleaved with thrombin and incubated with ClpXP (1 μM ClpX₆; 2 μM ClpP₁₄), 4 mM ATP, and an ATP-regeneration system. ClpXP extraction of the terminal β strand resulted in time-dependent loss of 467-nm fluorescence and 400-nm fluorescence (data not shown). The initial rate of this reaction (0.2 min⁻¹ enz⁻¹) was slow, but within error of the rate of ClpXP degradation of uncleaved cp6-^SFGFP-5/6-ssrA (data not shown). (B) Absorbance spectra of thrombin-cleaved cp6-^SFGFP-5/6-ssrA (closed triangles), the cp6-^SFGFP-5 protein after ClpXP strand extraction and purification by S200 gel filtration (open triangles), and cp6-^SFGFP-ssrA denatured by incubation at pH 2 (squares).

Fig. 6

(A) Michaelis-Menten plots of ClpXP degradation (0.1 μM ClpX₆; 0.2 μM ClpP₁₄) of ssrA-tagged variants of GFP and ^SFGFP in the presence of 4 mM ATP and an ATP-regeneration system. Initial degradation rates were calculated from changes in 467-nm fluorescence. The lines are fits to the equation rate = V_max•[S]/(K_M + [S]). Error bars (± 1 SD) based on four independent replicates. K_M and V_max values for each substrate are listed in Table 1. (B) Proteins (0.5 μM) were incubated with different concentrations of GuHCl for 2 weeks, and denaturation was assayed by 467-nm fluorescence. The solid lines are fits to a two-state unfolding model. cp7-^SFGFP-ssrA has an unusual native baseline and higher m_eq value than the other proteins (Table 1). These properties could reflect an increase in the solvent accessibility of the unfolded protein and/or the presence of a populated unfolding intermediate. (C) Rates constants for unfolding (k_u) were determined by single-exponential fits of changes in 467-nm fluorescence after jumps to different concentrations of GuHCl. The values plotted are averages of three independent experiments ± 1 standard deviation. The final protein concentration in each assay was 0.5 μM.

Fig. 7

Sequences corresponding to the 6^th β-strand (TLVNRIELKGI) or the 11th β-strand strand (HMVLLEFVTAA) of ^SFGFP were inserted between the titin^I27 protein and the ssrA tag (NCBI accession codes JF951871 and JF951872, respectively). ClpXP (1 μM ClpX₆; 2 μM ClpP₁₄) degradation of each substrate (10 μM) was monitored by SDS-PAGE, staining with Coomassie blue and densitometry. Reactions contained 4 mM ATP and an ATP-regeneration system.

Fig. 8

(A) Fractional rates of ATP hydrolysis (α = v/V_max) by ClpXP (1 μM ClpX₆; 2 μM ClpP₁₄) at different concentrations of ATP in the presence of 10 μM GFP-ssrA (squares), ^SFGFP-ssrA (circles), cp6-^SFGFP-ssrA (triangles), or cp7-^SFGFP-ssrA (diamonds). The solid line is a fit of the ^SFGFP-ssrA data to α = 1/(1+(K_M/[ATP])ⁿ), the Hill form of the Michaelis-Menten equation. K_M, V_max, and n values for each protein substrate are listed in Table 1. (B) Fractional degradation rates (v/V_max) for ClpXP (1 μM ClpX₆; 2 μM ClpP₁₄) proteolysis of 10 μM GFP-ssrA (diamonds), ^SFGFP-ssrA (closed circles), cp6-^SFGFP-ssrA (triangles), and cp7-^SFGFP-ssrA (downward triangles) are plotted as a function of the fractional rate of ATP hydrolysis (α). Solid lines are fits to the equation k_a•α²/(k_b•(1−α)+α) for GFP-ssrA and cp6-SFGFP-ssrA, where k_a = k₁k₂/(k₁+k₂) and k_b = k_-1/(k₁+k₂). (C) Single-intermediate model for enzymatic unfolding, where ES represents the complex of ClpXP with intact GFP and EI represents a complex in which the terminal β strand of the substrate has been extracted, leaving a 10-stranded β barrel.