Single-Molecule Protein Unfolding and Translocation by the AAA+ Protease, ClpXP

Abstract

All cells employ ATP-fueled AAA+ proteases for protein-quality control and regulation. In the ClpXP protease, the ring-shaped AAA+ molecular motor ClpX first recognizes and mechanically unfolds specific protein substrates, then translocates the denatured polypeptide through a central axial pore into the barrel-shaped peptidase ClpP for degradation. Although the mechanisms of ATP utilization and substrate degradation have been extensively studied in solution and recently at the single-molecule level[1, 2], the molecular details underlying mechanical substrate unfolding and stepping along the polypeptide track remain unexamined.Using a dual-laser optical trap, we probed how ClpXP unfolds and translocates a multi-domain protein substrate at the single-molecule level. In our assay, a ClpXP-substrate complex is tethered between two trapped polystyrene beads held in a dumbbell configuration [2]. Motility records of ClpXP along the polypeptide track show unique, fingerprint-like, substrate unfolding and translocation events. Following rapid and cooperative unfolding of individual domains, we find that ClpX translocates the polypeptide into ClpP, taking small steps of 5-8 amino acids. The nature of the polypeptide track affects ClpXP mechanochemical activity. ClpX step size does not depend on ClpP, though we observe substantial substrate refolding and slippage events when only ClpX is examined. Our results support a power-stroke model of denaturation in which successful unfolding requires mechanical pulling by the enzyme to coincide with transient stochastic protein destabilization.[1] Shin, Y., Davis, J.H., Brau, R.R., Martin, A., Kenniston, J.A., Baker T.A., Sauer, R.T., Lang, M.J. Single-molecule denaturation and degradation of proteins by the AAA+ ClpXP protease. PNAS, 106, 19340 (2009)[2] Aubin-Tam, M. E., Olivares, A. O., Sauer, R. T., Baker, T. A., Lang, M. J. Single-molecule protein unfolding and translocation by an ATP-fueled proteolytic machine. Cell, 145, 257-67 (2011)