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

Abstract

The bacterial AAA+ ClpX unfolds substrates using the energy from ATP hydrolysis and delivers them to the associated protease ClpP. A loop with an aromatic-hydrophobic motif protrudes into the central pore of the ClpX hexamer and was known to be important for activity. Now mutational analysis using covalently linked subunits provides evidence that this loop actually grips the substrate and undergoes conformational changes to drive its translocation and unfolding. 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.