The ClpXP Protease Unfolds Substrates using a Constant Rate of Pulling but Different Gears

ATP-dependent proteases(link is external) are vital to maintain cellular protein homeostasis(link is external). Here, we study the mechanisms of force generation and intersubunit coordination in the ClpXP protease from E. coli to understand how these machines couple ATP hydrolysis(link is external) to mechanical protein unfolding. Single-molecule analyses reveal that phosphate release is the force-generating step in the ATP-hydrolysis cycle and that ClpXP translocates substrate polypeptides(link is external) in bursts resulting from highly coordinated conformational changes(link is external) in two to four ATPase(link is external) subunits. ClpXP must use its maximum successive firing capacity of four subunits to unfold stable substrates like GFP. The average dwell duration between individual bursts of translocation is constant, regardless of the number of translocating subunits, implying that ClpXP operates with constant “rpm” but uses different “gears.”