Stochastic but Highly Coordinated Protein Unfolding and Translocation by the ClpXP Proteolytic Machine

ClpXP and other AAA+ proteases recognize, mechanically unfold, and translocate target proteins into a chamber for proteolysis. It is not known whether these remarkable molecular machines operate by a stochastic or sequential mechanism or how power strokes relate to the ATP-hydrolysis cycle. Single-molecule optical trapping allows ClpXP unfolding to be directly visualized and reveals translocation steps of ∼1–4 nm in length, but how these activities relate to solution degradation and the physical properties of substrate proteins remains unclear. By studying single-molecule degradation using different multidomain substrates and ClpXP variants, we answer many of these questions and provide evidence for stochastic unfolding and translocation. We also present a mechanochemical model that accounts for single-molecule, biochemical, and structural results for our observation of enzymatic memory in translocation stepping, for the kinetics of translocation steps of different sizes, and for probabilistic but highly coordinated subunit activity within the ClpX ring. [Display omitted] •ClpXP takes physical translocation steps of 1–4 nm in a stochastic pattern•The stability of degron-proximal structure determines substrate unfolding rates•One ATP-hydrolysis event can drive more than one power stroke•Commitment to unfolding is the slow step in solution degradation An optical trapping tool, which allows visualization of single-molecule degradation, reveals that ClpXP proteolytic machine operates by a stochastic rather than sequential mechanism. Depending upon which ATP-bound subunit in the hexameric ClpX ring hydrolyzes ATP first, kinetic bursts of 1–4 power strokes drive translocation steps of different sizes, requiring highly coordinated mechanisms of subunit-subunit communication.