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

Abstract

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 orhow power strokes relate to the ATP-hydrolysis cycle. Single-molecule optical trapping allows ClpXP unfolding to be directly visualized and reveals translocation steps of ∼1-4nm 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.