Single-molecule FRET probes the allosteric effect of ATP on the protein-translocating pore loops of a AAA+ machine

AAA+ proteins (ATPases associated with various cellular activities) comprise a family of powerful ring-shaped ATP-dependent translocases that carry out numerous vital substrate-remodeling functions ranging from protein unfolding and disaggregation to DNA melting and unwinding. ClpB is a AAA+ disaggregation machine that forms a two-tiered hexameric ring, with flexible pore loops which protrude to its center and bind to substrate-proteins, and two nucleotide-binding domains (NBD1-2), responsible for ATP binding and hydrolysis. It remains unknown whether and how the two nucleotide-binding sites interact with each other and affect the pore loops. Recently, we applied single-molecule FRET (smFRET) spectroscopy to directly measure the dynamics of substrate-binding pore loops in ClpB in aqueous solution. We have reported that the three pore loops of ClpB (PL1-3) undergo large-scale fluctuations on the timescale of microseconds and change their conformations in response to substrate-protein binding. Here, using smFRET, we study the allosteric coupling between the two ATP-binding sites and the pore loops in ClpB. Using Walker mutations that perturb either ATP hydrolysis or binding, we demonstrate that nucleotide states of the NBDs tune the pore loop dynamic equilibrium. This communication is remarkably long-range, and in particular, PL2 and PL3 are each affected by mutations in both NBD1 and NBD2. We characterize the allosteric paths connecting the NBDs to the pore loops by molecular dynamics simulations, and find that these paths can be altered by changing the ATPase state of ClpB. Further, a rigorous thermodynamic double-mutant cycle analysis reveals the coupling of the two ATP-binding sites in their effects on pore loop dynamics. Surprisingly, abolishing ATP hydrolysis in both NBDs results in an order of magnitude stronger substrate-protein binding to PL2, but not to the other pore loops. Importantly, PL3 which is highly conserved in AAA+ machines, is found to favor either an upward or a downward conformation depending on which of the NBDs remains active. These results explicitly demonstrate a significant long-range allosteric communication between the ATP binding sites and the pore loops, and shed new light on the Brownian ratchet substrate translocation mechanism of AAA+ machines. ### Competing Interest Statement The authors have declared no competing interest.