The ClpXP Protease Employs a Novel Mechanism of Translocation Using a Constant Frequency of Pulling but Different Gears

ATP-dependent proteases play important roles in the maintenance of cellular protein homeostasis, including the removal of misfolded, aggregated, or damaged proteins. The operating principles by which these proteases use ATP hydrolysis to power the mechanical tasks of protein unfolding and translocation remain unclear. Single-molecule optical tweezers experiments allow us to study the mechanism of force generation and inter-subunit coordination of ClpXP, a protease from E. coli. Our analysis reveals that phosphate release is the force-generating step in the ATP hydrolysis cycle, and ClpXP translocates substrates in cycles of dwells and bursts. During the dwell phase, the motor remains stationary on the polypeptide track, whereas during the burst phase, ClpX translocates polypeptide lengths of two to four nanometers. These translocation bursts result from highly coordinated conformational changes between two to four ATPase subunits. In order to successfully unfold stable substrates such as GFP, ClpXP must successively undergo four highly coordinated power strokes. Surprisingly, unlike many other molecular motors in which the duration of their operation cycles depends on the concentration of ATP while the step size remains constant, ClpXP does just the opposite: the duration of dwells between translocation bursts is constant regardless of the ATP concentration and number of hydrolyzing subunits, while the size of the translocation burst decreases as ATP concentration decreases from saturating to near-Km regimes. Our findings uncover a unifying mechanism of operation for inter-subunit coordination during both protein unfolding and polypeptide translocation and define a new archetype of motor operation (1).(1) Sen, et al. The ClpXP protease unfolds substrates using a constant rate of pulling but different gears. Accepted in Cell (2013).