PT  - JOURNAL ARTICLE
AU  - Hisham Mazal
AU  - Marija Iljina
AU  - Inbal Riven
AU  - Gilad Haran
TI  - Ultrafast Brownian-ratchet mechanism for protein translocation by a AAA+ machine
AID  - 10.1101/2020.11.19.384313
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.11.19.384313
4099  - http://biorxiv.org/content/early/2020/11/19/2020.11.19.384313.short
4100  - http://biorxiv.org/content/early/2020/11/19/2020.11.19.384313.full
AB  - AAA+ 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.Competing Interest StatementThe authors have declared no competing interest.