Iljina, M. | Search bioRxiv

bioRxiv2020

Ultrafast Brownian-ratchet mechanism for protein translocation by a AAA+ machine

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.

biophysics↗

bioRxiv2020

Single-molecule spectroscopy reveals dynamic allostery mediated by the substrate-binding domain of a AAA+ machine

ClpB is an ATP-dependent protein disaggregation machine that is activated on demand by co-chaperones and by aggregates caused by heat shock or mutations. The regulation of ClpBs function is critical, since its persistent activation is toxic in vivo. Each ClpB molecule is composed of an auxiliary N-terminal domain (NTD), an essential regulatory middle domain (MD) that activates the machine by tilting, and two nucleotide-binding domains that are responsible for ATP-fuelled substrate threading. The NTD is generally thought to serve as a substrate-binding domain, which is commonly considered to be dispensable for ClpBs activity, and is not well-characterized structurally due to its high mobility. Here we use single-molecule FRET spectroscopy to directly monitor the real-time dynamics of ClpBs NTD and reveal its involvement in novel allosteric interactions. We find that the NTD fluctuates on a microsecond timescale and, unexpectedly, shows little change in conformational dynamics upon binding of a substrate protein. During its fast motion, the NTD makes crucial contacts with the regulatory MD, directly affecting its conformational state and thereby influencing the overall ATPase and unfolding activity of this machine. Moreover, we also show that the NTD mediates signal transduction to the nucleotide-binding domains through conserved residues. The two regulatory pathways revealed here enable the NTD to suppress the MD in the absence of protein substrate, and to limit ATPase and disaggregation activities of ClpB. The use of multiple parallel allosteric pathways involving ultrafast domain motions might be common to AAA+ molecular machines to ensure their fast and reversible activation.

biophysics↗