The design of our substrate allowed identification of specific ClpXP unfolding and translocation events. For example, the spacing of the dwell periods flanking FLN1-domain unfolding is larger than for other FLN domains, because the FLN1 domain is separated from the HaloTag domain by a ~50-residue linker. Similarly, ClpXP unfolding of the HaloTag domain results in a larger increase in substrate length than unfolding of FLN domains. For future studies, additional natural or unnatural sequences or native protein domains could be placed between FLN1 and the HaloTag domain, allowing single-molecule studies of ClpXP unfolding and/or translocation of these elements.
We found that the velocity of ClpXP translocation decreases as the resisting force increased, indicating that at least one reaction in the overall translocation process is force dependent. However, the A value determined from fitting to the single-barrier Boltzmann model was far below 1, suggesting that a mechanical step is not rate limiting in the absence of an applied force. RNA polymerase also has a very low A value, and force-independent pyrophosphate release is known to be rate limiting for this enzyme (Wang et al., 1998
). It remains to be determined what reaction or reactions limit the rate of translocation and ATP hydrolysis by ClpXP, but the force-dependent step probably occurs at a rate at least 20-fold faster based upon the A value of 0.05. Curiously, we did not observe a change in unfolding dwell times as the force was increased. Because the ATPase rate and translocation rate appear to be tightly coupled, as shown here and previously (Martin et al., 2008c
), it seems very likely that the ATPase rate also slows at high forces, and one might therefore expect a corresponding increase in the average time required for unfolding. However, we suspect that force-induced destabilization of the substrate makes denaturation by ClpXP easier, offsetting any force-induced decrease in ATP-hydrolysis rates.
Motor proteins operate by power-stroke mechanisms, by Brownian-ratchet capture of random thermal motions, or by a combination of these mechanisms (Hwang and Lang, 2009
). The smallest ClpXP and ClpX translocation steps are ~5–8 amino acids in length, a size small enough to be driven by plausible structural changes in the ClpX hexamer during a power stroke (Glynn et al., 2009
) or by thermal motion. Although we cannot rule out some Brownian contributions, we favor a mechanism that relies predominantly on a power stroke for several reasons. First, the polypeptide substrate is threaded through the ClpX and ClpXP rings during translocation, an architecture that results in close enzyme-substrate contacts and favors a direct-drive mechanism. Second, translocation under load was highly unidirectional; ~150 consecutive forward steps were observed in some trajectories and reverse steps were extremely rare, except at the highest loads or at reduced rates of ATP hydrolysis. Thus, a Brownian mechanism would need to be exceptionally efficient at capturing thermal motion in the correct direction and preventing motion in the opposite direction to account for translocation. Notably, however, ClpXP can translocate substrates from the C-terminus to the N-terminus or in the opposite direction and does not require recognition of specific spacings of side chains or peptide bonds (Barkow et al., 2009
). Thus, unidirectional capture of thermal motion by binding to repeating molecular features of the polypeptide track seems highly unlikely. Third, we observe pre-unfolding dwells that indicate that the folded domain is in intimate contact with the ClpX motor, without significant motions or slack that would allow a Brownian mechanism to operate. Finally, it is difficult to envision a strictly Brownian mechanism that could account for the ability of ClpXP to accelerate protein-unfolding reactions by factors as large as 106
-fold (Kim et al., 2000
), whereas a power-stroke mechanism has this potential. Consistently, ClpXP can perform significant mechanical work. Indeed, a ~1 nm step against 20 pN of applied load corresponds to ~5 k
T of mechanical work per step, which would increase to ~7.5 k
T at the extrapolated stall force.
How might the pulling activity of the ClpX motor lead to protein unfolding? Depending upon the stability and unfolding properties of a domain, ClpXP probably operates using three general denaturation mechanisms. (i
) For meta-stable domains, one power stoke may result in cooperative denaturation. Indeed, the shortest unfolding dwells observed in our assays are consistent with events associated with ClpXP hydrolysis of a single ATP. (ii
) For more stable domains, each independent power stroke appears to have some probability of success linked to transient destabilization of the domain resulting from fluctuations in thermal energy, although fluctuations in the enzyme could also contribute to the probabilistic nature of unfolding. Notably, the largest unfolding dwell times in our assays were longer than 100 s, a period in which ClpXP could hydrolyze ~200 ATP molecules. As expected for a model in which the stability of a domain followed a Boltzmann distribution, the unfolding dwell times for specific FLN domains or the HaloTag domain were roughly exponentially distributed. (iii
) In principle, some ClpXP unfolding events could also occur in discrete steps. For example, if enzymatic pulling disrupted a small region of protein structure and translocation of that segment prevented refolding, then the partially unfolded protein might be sufficiently stable to persist. Under these circumstances, the energy barrier for unfolding of the remaining structure would be lowered, allowing subsequent power strokes to complete unfolding (Lee et al., 2001
). Approximately 15% of the FLN-unfolding events did show an unfolding intermediate, but these events also occurred in less than 5 ms, which is far faster than the average time required for ATP hydrolysis (> 250 ms). We cannot eliminate models in which a few coordinated power strokes contribute to these non-cooperative unfolding events, but the most likely model is that they also result from hydrolysis of a single ATP with subsequent thermally driven denaturation of the remaining elements of native structure. Thus, the FLN and HaloTag unfolding events observed in our experiments appear to occur by one of the first two mechanisms discussed. Stepwise ClpXP unfolding, which requires multiple ATP-hydrolysis events, is more likely to be observed for proteins like GFP and ribonuclease H, in which unfolding intermediates have higher kinetic or thermodynamic stabilities than the domains studied here (Hollien and Marqusee, 1999
; Dietz and Rief, 2004
; Kenniston et al., 2004
; Martin et al., 2008c
Although protein unfolding and thus degradation can be very energetically costly (Kenniston et al., 2003
), it is important to note that ClpXP and other AAA+ proteases must denature and degrade a highly diverse assortment of cellular proteins with radically different structures and stabilities. Thus, evolution is unlikely to have optimized ClpXP activity for any single substrate. Repeated pulling on a peptide tag is a simple mechanism that allows relatively low-cost degradation of metastable substrates but eventual high-cost degradation of hyperstable proteins as well. Some AAA+ family proteases appear have limited unfoldase activity and to specialize in degrading poorly structured proteins (Herman et al., 2003
). Because the degradation of unfolded substrates requires energy consumption that is inversely proportional to translocation step size, it will be important to determine if these proteases employ larger translocation steps, which minimize the net cost of ATP-fueled degradation but also limit unfolding power.
ClpXP appears to use a relatively low gear (small step size), allowing it to work against substantial loads. By contrast, cytoplasmic dynein, a dimeric AAA+ machine, takes longer steps (typically 8 nm) as it moves along the microtubule cytoskeleton but has a lower stall force (~7 pN; Gennerich et al., 2007
). Several molecular motors that track along nucleic acids also take relatively small steps and can work against substantial forces, including bacterial RNA polymerase (~25 pN; 1 base or ~0.4 nm steps; Wang et al., 1998
; Abbondanzieri et al., 2005
), the AAA+ 29 DNA-packaging motor (~50 pN; 2.5 base-pair or ~0.85 nm steps; Smith et al., 2001
; Moffitt et al., 2009
), and the ribosome (>20 pN; 3 base or ~1.3 nm steps; Wen et al., 2008
). Moreover, each of these machines encircle the single or double-stranded nucleic acid. Likewise, the polypeptide substrates of ClpXP are threaded through the enzyme, allowing intimate contacts that maintain processivity even when working against a substantial resisting force. We anticipate that other AAA+ machines with relatively small step sizes will also be able to generate high forces, allowing them to carry out mechanical work suited to their specific biological tasks.