Translocating Loop‐Substrate Interactions Mediate Subunit Coordination and Regulate the Mechanochemical Coupling and Power Production in a AAA+ Protease Machine

ATP‐dependent proteases are crucial to maintain cellular protein homeostasis. Although previous single‐molecule studies of ClpXP have shown that this motor displays alternating dwells and bursts phases during translocation, the processes underlying them remain unknown. Here, we perform a series of experiments with optical tweezers and ATPase assays to show in unprecedented detail where and when each chemical transition of the ATP‐hydrolysis cycle occurs within the dwell/burst cycle of ClpXP, and identify the mechanisms that govern each phase of its mechanochemical cycle. Specifically, we show that ATP hydrolysis and phosphate release occur during the burst while ADP release/ATP binding during the dwell. In addition, we show that residues in the highly conserved translocating pore‐loops protruding the central pore of ClpX are optimized for efficient protein translocation and unfolding: their size and nature control ClpXP power generation ‐i.e. work produced per unit time‐ and the coupling efficiency between chemical and mechanical parts of the cycle. Interestingly, we observe that the conformational resetting of the pore loops between consecutive power strokes appears to time both the dwell duration and the release of ADP. Together, our results present important new insights into how evolution has optimized AAA+ proteases for efficient protein unfolding and translocation, and show that ClpXP's mechanism deviates from other well studied molecular motors (such as the Phi29 DNA packaging motor or the F1‐ATPase). Support or Funding Information This research was supported in part by the Searle Scholars Program (A.M.), NIH grant R01‐GM094497 (A.M.), NIH grant #2R01GM03243 (C.B), Howard Hughes Medical Institute (C.B.) and by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering Nanomachine Program under Contract No. DE‐AC02‐05CH11231 (single molecule force measurement (C.B.)(A) Cartoon of the mechanism of protein degradation by ClpXP.(B) Left: Top view of the ClpX ring showing the GYVG loops in purple. Right : Translocating GYVG loops of prokaryotic and eukaryotic AAA+ proteases. (C) Top : Experimental geometry of the optical tweezers assays. The ssrA‐tagged substrate has four titin I 27 domains permanently unfolded (Ti CM ) fused to a green fluorescent protein (GFP). Bottom: Single‐molecule trajectory of substrate translocation and unfolding by ClpXP.Translocating Loops Regulate the Mechanochemical Coupling and Power Production of ClpXP(A) Scheme that shows how upon the power stroke the GYVG loops experience a conformational change that resets its position, concomitantly inducing the release of ADP.(B) Dependence of the mechanochemical coupling and the Power efficiency on the bulkiness inside the crowded ClpX pore.