Ultrafast pore-loop dynamics in a AAA+ machine point to a Brownian-ratchet mechanism for protein translocation

AAA+ ring-shaped machines, such as the disaggregation machines ClpB and Hsp104, mediate ATP-driven substrate translocation through their central channel by a set of pore loops. Recent structural studies have suggested a universal hand-over-hand translocation mechanism, in which pore loops are moving rigidly in tandem with their corresponding subunits. However, functional and biophysical studies are in discord with this model. Here, we directly measure the real-time dynamics of the pore loops of ClpB as they function in substrate threading, using single-molecule FRET spectroscopy. All pore loops undergo large-amplitude fluctuations on the microsecond timescale and change their conformation upon interaction with substrate proteins. Conformational dynamics of two of the pore loops strongly correlate with disaggregation activity, suggesting that they are the main contributors to substrate pulling through the central channel. Finally, the pore loops present differential dependence on ATP hydrolysis along the axial channel. This set of findings is rationalized in terms of an ultrafast Brownian-ratchet translocation mechanism, which likely acts in parallel to the much slower hand-over-hand process in ClpB and other AAA+ machines.