Stochastic but highly coordinated protein unfolding and translocation by the CIpXP proteolytic machine

• Published in: Cell Vol. 158; no. 3; pp. 647 - 658
• Main Authors: Cordova, Juan Carlos, Olivares, Adrian O., Shin, Yongdae, Stinson, Benjamin M., Calmat, Stephane, Schmitz, Karl R., Aubin-Tam, Marie-Eve, Baker, Tania A., Lang, Matthew J., Sauer, Robert T.
• Format: Journal Article
• Language: English
• Published: 31.07.2014
• ISSN: 0092-8674; 1097-4172
• DOI: 10.1016/j.cell.2014.05.043

CIpXP and other AAA+ proteases recognize, mechanically unfold, and translocate target proteins into a chamber for proteolysis. It is not known if 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 CIpXP 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 multi-domain substrates and CIpXP 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 CIpX ring.