AbstractAAA+ ring-shaped machines, such as ClpB and Hsp104, mediate substrate translocation through their central channel by a set of pore loops. Recent structural studies 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 and their response to substrate binding, 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. Pore-loop conformational dynamics are modulated by nucleotides and strongly correlate with disaggregation activity. The differential behavior of the pore loops along the axial channel points to a fast Brownian-ratchet translocation mechanism, which likely acts in parallel to the much slower hand-over-hand process.
Linking Enzyme Conformational Dynamics To Catalytic Function With Single-molecule FRET