Effects of protein stability and structure on substrate processing by the ClpXP unfolding and degradation machine

Abstract

ClpXP is an ATP-dependent protease that denatures native proteins and translocates the denatured polypeptide into an interior peptidase chamber for degradation. To address the mechanism of these processes, Arc repressor variants with dramatically different stabilities and unfolding half-lives varying from months to seconds were targeted to ClpXP by addition of the ssrA degradation tag. Remarkably, ClpXP degraded each variant at a very similar rate and hydrolyzed approximately 150 molecules of ATP for each molecule of substrate degraded. The hyperstable substrates did, however, slow the ClpXP ATPase cycle. These results confirm that ClpXP uses an active mechanism to denature its substrates, probably one that applies mechanical force to the native structure. Furthermore, the data suggest that denaturation is inherently inefficient or that significant levels of ATP hydrolysis are required for other reaction steps. ClpXP degraded disulfide-cross-linked dimers efficiently, even when just one subunit contained an ssrA tag. This result indicates that the pore through which denatured proteins enter the proteolytic chamber must be large enough to accommodate simultaneous passage of two or three polypeptide chains.

Fig. 1. (A) Arc-ssrA variants used for degradation studies. Mutations in residues 1–53 of Arc are indicated by arrows. The st11 and ssrA sequences are H₆KNQHE and AANDENYALAA, respectively. The reported T_m values are taken from the fits in (C). (B) Ribbon model of the Arc dimer. Spheres mark the sites of individual mutations in one subunit. A model of the disulfide bond in the NC11_ox-Arc-ssrA dimer is shown as a ball-and-stick representation. (C) Thermal denaturation of 10 µM Arc-ssrA or variants (monomer equivalents) in 0.1 M NaH₂PO₄ pH 6.8 followed by changes in circular dichroism at 222 nm. The data were fit assuming an equilibrium between unfolded monomers and folded dimers (Bowie and Sauer, 1989).

Fig. 2. (A) ClpXP degrades different concentrations of [³⁵S]Arc-ssrA with linear kinetics as assayed by release of TCA-soluble counts. (B) Variation of ClpXP degradation rates with substrate concentration for Arc-ssrA and four mutant variants. The lines are non-linear least-squares fits to the Michaelis–Menten equation. The fitted values for K_M and k_cat are listed in Table I. All Arc concentrations are calculated in terms of subunit equivalents. The concentrations of ClpX₆ and ClpP₁₄ were 0.1 and 0.5 µM, respectively, except in the NC11_ox-Arc-ssrA degradations, which were performed with 0.05 µM ClpX₆.

Fig. 3. Endoproteinase Arg-C degradation of Arc-ssrA variants assayed by circular dichroism. Reactions were carried out as described in Materials and methods.

Fig. 4. (A) Co-elution of ClpX protein and ATPase activity during cation-exchange chromatography. ClpX (90 µg), purified as described in Materials and methods, was applied to a MonoQ column and eluted with a linear gradient from 150 to 500 mM KCl in buffer pH 7.6 containing 50 mM HEPES-KOH, 15% glycerol, 3 mM DTT, 2 mM MgCl₂, 0.1 mM ATP, 0.1 mM ZnCl₂ and 0.01% Triton. Fractions of 200 µl were collected and assayed for ATPase activity and total protein by the Bradford assay. SDS–PAGE analysis of column fractions is shown in the inset. (B) Correlation between rates of ClpXP-mediated Arc-ssrA degradation and ATP hydrolysis. Data are from Tables I and II, and the error bars reflect the standard deviations of at least three independent measurements. The solid line is a linear fit to the data (slope = 150 ± 20, intercept = 45 ± 39, r² = 0.94).

Fig. 5. (A) ClpXP degrades a disulfide-bonded heterodimer in which just one subunit contains a ssrA tag. Three disulfide-bonded species were present in the experiment, i.e. untagged homodimers, singly tagged heterodimers and tagged homodimers, but only the untagged subunits were ³⁵S-labeled and thus the tagged homodimer was not detected following SDS–PAGE and autoradiography. Lane 4 shows SDS–PAGE of the protein mixture used in the degradation experiment, stained for total protein with SYPRO Orange. (B) Quantitation of the autoradiogram in (A). (C) Specific trapping of the singly tagged heterodimer in ClpP^DFP as measured by gel filtration chromatography The top panel shows the absorbance profile at 214 nm of samples eluting from the column. The elution positions of molecular weight standards are shown above the chromatogram. Fractions 10–35 were analyzed by SDS–PAGE and quantified by PhosphorImager analysis. The ClpP₁₄ and ClpX peaks were identified by staining for total protein with SYPRO Orange. The intensities of the heterodimer band (middle panel) and untagged homodimer band (bottom panel) are shown.

Fig. 6. ClpXP does not degrade untagged ³⁵S-labeled Arc monomers in non-covalent heterodimers with ssrA-tagged monomers. Degradation mixtures were prepared with 2 µM ³⁵S-labeled Arc-st11 plus buffer, 2 µM cold Arc-ssrA or 20 µM unlabeled Arc-ssrA, and proteolysis was followed by the appearance of TCA-soluble counts as described in Materials and methods. The degradation of a sample of ³⁵S-labeled Arc-ssrA is shown for comparison.

Fig. 7. Variation of normalized degradation rates with the rate constants for spontaneous denaturation of Arc-ssrA and variants. ClpXP degradation (squares); Arg-C degradation (circles).

Fig. 8. Models for import into ClpP of denatured proteins either as a single polypeptide chain (A) or as a disulfide-bonded pair of polypeptides (B and C). The presence of the disulfide cross-link requires that two or three polypeptide chains must pass simultaneously through the axial pore of the ClpP tetradecamer.