Pore loops of the AAA+ ClpX machine grip substrates to drive translocation and unfolding

Abstract

Proteolytic AAA+ unfoldases use ATP hydrolysis to power conformational changes that mechanically denature protein substrates and then translocate the polypeptide through a narrow pore into a degradation chamber. We show that a tyrosine residue in a pore loop of the hexameric ClpX unfoldase links ATP hydrolysis to mechanical work by gripping substrates during unfolding and translocation. Removal of the aromatic ring in even a few ClpX subunits results in slippage, frequent failure to denature the substrate and an enormous increase in the energetic cost of substrate unfolding. The tyrosine residue is part of a conserved aromatic-hydrophobic motif, and the effects of mutations in both residues vary with the nucleotide state of the resident subunit. These results support a model in which nucleotide-dependent conformational changes in these pore loops drive substrate translocation and unfolding, with the aromatic ring transmitting force to the polypeptide substrate.

Figure 1. Substrate binding and degradation

(a) Native substrates are recognized by the ClpX unfoldase via exposed peptide tags and unfolded as they are translocated through a narrow axial pore and into the ClpP peptidase for degradation. (b) Ar-Φ pore-loop motifs in prokaryotic and eukaryotic AAA+ unfoldases. (c) Mutations in the Ar-Φ loop of ClpX weaken binding to ssrA-tagged substrates. K_M values for titin^CM-ssrA degradation by ClpP in complex with ClpX RWE/RWE or variants with the Y153A, V154F, or V154A mutations in different classes of subunits were determined by Michaelis-Menten analyses of initial degradation rates (Table 1). Errors in K_M were ±10% based on replicate measurements (n=3).

Figure 2. The Ar-Φ loop in the central pore of ClpX provides a “grip” on substrates during unfolding and translocation

(a) Thermodynamic efficiencies for titin^CM-ssrA translocation by ClpX RWE/RWE with mutations in the Ar-Φ loop. Maximal degradation rates are plotted against the ATP-hydrolysis rate at saturating titin^CM-ssrA concentrations for ClpX RWE/RWE (○) and variants with Y153A (red), V154F (blue), or V154A (cyan) mutations in empty-state R subunits (△), hydrolyzing W subunits (□), or ATP-state E subunits (▽). Variants that consume similar amounts of ATP for each substrate degraded cluster close to the lines shown. (b) The first residue in the Ar-Φ loop contacts translocating substrates. Titin-ssrA was unfolded by modification of its cysteines with DTNB, incubated with ATP and single-chain ClpX hexamers bearing Cys153 in a W or E subunit, and disulfide-crosslinked products were detected by western blotting after non-reducing SDS-PAGE. DTNB-modified titin with a C-terminal AA→DD mutation in the ssrA tag is not degraded by ClpX and serves as a negative control. (c) Thermodynamic efficiencies of native titin-ssrA unfolding and translocation by ClpX RWE/RWE with Ar-Φ-loop mutations. Symbols for ClpX variants are the same as in panel A.

Figure 3. Ar-Φ-loop motions propel substrate through the central pore of ClpX

(a) The cartoon depicts two neighboring ClpX subunits cycling through ATP-bound, hydrolyzing, and empty states. ATP-dependent conformational changes in the Ar-Φ loop of one subunit translocate the substrate, and an adjacent subunit binds the polypeptide and prevents slipping before the next power stroke. (b) Weakened grip during substrate translocation. Degradation of GFP-titin^CM-ssrA by ClpXP RWE/RWE stops at GFP, leaving a 38-residue titin tail. Y153A mutations in the R, W, or E subunits of ClpX RWE/RWE result in an additional product with a 45-residue tail. These mutations may reduce the enzyme’s ability to pull GFP tightly against the ClpX pore or allow the substrate to slip after it reaches the pore.