ClpA, ClpB, and Hsp104 share similarities that have formed the basis for their classification. They are members of the AAA+ superfamily that are further classified as Hsp100 proteins for their roles in coupling ATPase activity to changes in the folding and/or assembly of substrate clients (Schirmer et al., 1996; Neuwald et al., 1999). Hsp100 members are partitioned into two classes based on the number of nucleotide binding domains (NBDs) contained per monomeric unit. Class I proteins, such as ClpA, ClpB, and Hsp104, contain two NBDs while Class II proteins, such as ClpX, contain a single NBD per monomer. In the presence of ATP, these proteins assemble into homohexameric ring-like structures that perform their chaperone activity. ATP binding and hydrolysis occur at canonical Walker A and B motifs contained within each nucleotide binding domain (Walker et al., 1982).

The protomer structures of ClpA, ClpB, and Hsp104 have been reported from various organisms in various nucleotide-bound states. In the case of ClpA, the monomer structure has been reported from Escherichia coli ClpA in the ADP-bound state (Guo et al., 2002b). For the disaggregases, atomic resolution crystal structures have been reported for Thermus thermophilus ClpB in the AMPPNP-bound state (Lee et al., 2003) and for Chaetomium thermophilum Hsp104 in complex with ADP (Heuck et al., 2016). Comparison of the available protomer structures (Figure 1A) as well as the primary sequences of the three motors, highlights their shared structural features. In general, each monomer is made up of an N-domain, nucleotide binding domains 1 (NBD1) and 2 (NBD2) joined by a linker region, and a C-terminal domain. The residues that separate the Walker A and Walker B motifs in each NBD have been modeled to form a loop that extends into the axial channel of the hexameric ring structures. Evidence from multiple studies has implicated conformational changes of these residues with ATP hydrolysis at each NBD in the mechanism of polypeptide substrate translocation by ClpA, ClpB/Hsp104, as discussed below in Sections Mechanisms of Polypeptide Translocation by ClpA and ClpAP and Mechanism of Translocation by ClpB/Hsp104, as well as other AAA+ motors (Yamada-Inagawa et al., 2003; Schlieker et al., 2004; Weibezahn et al., 2004; Hinnerwisch et al., 2005; Martin et al., 2008; Biter et al., 2012; Zeymer et al., 2014). As shown in Figure 1B, single particle reconstructions of the hexamer structures for the three motors predict similar arrangements of each domain within the quaternary structure. This structural similarity, in part, formed the basis for the hypothesis that ClpA, ClpB, and Hsp104 operate on substrate proteins through a shared mechanism.

ClpB and Hsp104 share an important feature that ClpA lacks. There is a middle domain (MD) located within the C-terminal end of NBD1 (Figure 1A). In the tertiary structure, this region adopts a coiled-coil fold, made up of four α-helices, that extends ~85 Å from NBD1 (Figure 1B). This domain is flexible and restriction of this flexibility has been shown to decrease disaggregation activity (Lee et al., 2003). MD flexibility has made its position and orientation within the hexamer difficult to assign in the multiple ClpB/Hsp104 structures available. The variable MD orientations in hexameric models have led to the hypothesis that nucleotide driven conformational switching of the MD may be an important part of the ClpB/Hsp104 disaggregation mechanism (Oguchi et al., 2012; Seyffer et al., 2012; Rosenzweig et al., 2013). Various studies have also shown the MD to be the binding target of ClpB/Hsp104 co-chaperones, DnaK/Hsp70 (Sielaff and Tsai, 2010; Miot et al., 2011; DeSantis et al., 2012; Seyffer et al., 2012; Lee et al., 2013; Rosenzweig et al., 2013; DeSantis et al., 2014; Doyle et al., 2015).

One other structural element distinguishes the protein translocase, ClpA, from the protein disaggregases ClpB/Hsp104: the presence or absence of a tripeptide motif requisite for the assembly with ClpP. ClpA hexamers interact with the protease ClpP through a conserved IGL/F motif nestled in a helix-loop-helix region near the C-terminal end of NBD2 (Kim et al., 2001). ClpB and Hsp104 lack that IGL/F motif, and accordingly, do not naturally associate with ClpP or any known protease.

In the presence of nucleotide, ClpA, ClpB, and Hsp104 oligomerize to form homo-hexamers that interact with client substrates and partner proteins. Structural models of the hexameric state have been reported for all three motors, in various nucleotide-bound states (Guo et al., 2002b; Lee et al., 2003, 2010; Wendler et al., 2007, 2009; Effantin et al., 2010; Carroni et al., 2014; Heuck et al., 2016; Yokom et al., 2016). In most cases, the hexameric state is reported to be a planar, ring-like structure with a central axial channel as shown in Figure 1C. In these models and single-particle reconstructions, the NBDs from each protomer align side-by-side around the hexamer, forming a NBD1 tier and a NBD2 tier. Hexamer models that capture the orientation of the flexible N-domain, have a third N-domain tier above NBD1, as seen for the hexameric single particle reconstructions in Figure 1C. ClpB and Hsp104 hexamers additionally have the MD protruding from the NBD1 tier.

Recently, an alternative asymmetric spiral structure has been reported for the Hsp104 hexamer in the AMPPNP-bound state, and in the ATPγS bound state with casein bound as a substrate (Yokom et al., 2016; Gates et al., 2017) spurring interest and speculation about its structural implications for the disaggregation mechanism. Similarly, Ripstein et al. recently reported images of another AAA+ protein, VAT, which threads protein substrates through its axial channel into the proteasome for degradation, in transient, asymmetric conformations (Ripstein et al., 2017). These asymmetric hexameric structures observed by cryo-EM are similar to the extended spirals reported previously in crystallographic studies (Guo et al., 2002b; Lee et al., 2003; Heuck et al., 2016). These provocative asymmetric structures invite further investigation. Biochemical assays will be key in determining how the asymmetric Hsp104 spiral structure fits into the disaggregation mechanism. This and other efforts to discern the mechanistic details of substrate processing by ClpA, ClpB, and Hsp104, will require the ability to precisely quantify the concentration of hexamers competent for polypeptide substrate binding.

Many studies have established that ClpA and ClpB reside in a distribution of oligomers in the absence of nucleotide (Maurizi et al., 1998; Zolkiewski et al., 1999; Akoev et al., 2004; Veronese et al., 2009; del Castillo et al., 2011). Hydrodynamic studies from Maurizi and co-workers concluded that ClpA resides in a distribution of monomers and dimers in the absence of nucleotide and that ATP is required for assembly into hexamers (Maurizi et al., 1998). In later work, Kress et al. report that ClpA hexamerization occurs through a transient tetramer intermediate (Kress et al., 2007). Using hydrodynamic and thermodynamic techniques, it was later shown that ClpA resides in a distribution of monomers, dimers, and tetramers in the absence of nucleotide (Veronese and Lucius, 2010; Veronese et al., 2011) thereby showing that the tetramer was not a transient intermediate on the pathway to assembly but was significantly populated at thermodynamic equilibrium independent of path. Notably, in the presence of excess nucleotide, ClpA hexamers as well as lower order oligomers remain in solution (Veronese et al., 2011; Li and Lucius, 2013). However, a complete quantification of the nucleotide linked assembly reaction is still needed.

On the other hand, the energetics of ClpB self-assembly in the absence and presence of nucleotide has been quantified (Lin and Lucius, 2015b, 2016). ClpB, like ClpA, resides in a distribution of monomers, dimers, tetramers, and hexamers. An important distinction between the two motors is the observation that ClpB, unlike ClpA, forms hexamers in the absence of nucleotide. A rigorous, in-depth investigation of the self-assembly of Hsp104 is currently lacking in the field, however recent results suggest that, similar to ClpB, Hsp104 populates hexamers and lower order oligomers in both the absence and presence of nucleotide (Weaver et al., 2017). Taken together, these quantitative investigations of ClpA and ClpB self-assembly reveal that macromolecular assembly is thermodynamically linked to nucleotide binding. This has fundamental implications for the driving forces that tune the population of each oligomer in solution.

Specifically, two thermodynamic driving forces govern the self-assembly of these enzymes into hexamers: the free monomer concentration and the free nucleotide concentration. As a result, assays performed on these enzymes in which the concentrations of protein or nucleotide change throughout the experiment, must account for the changing distribution of oligomers. Failure to do so can lead to conclusions about nucleotide processing at each NBD and NBD1-NBD2 interdependence that could otherwise be explained by changes in the macromolecular state.

In much of the published work on ClpA, ClpB, and Hsp104 it has been generally assumed that in the presence of 1–2 mM nucleotide concentrations, all of the protein is in the hexameric state. This assumption is generally supported with size exclusion chromatography (SEC). However, SEC is a non-equilibrium technique, meaning that the equilibrium is perturbed by running the sample through the column. That is to say, the chemical potential of both the protein and nucleotide are changing throughout the experiment and therefore the distribution of oligomeric states is changing throughout the experiment. Moreover, the observation of hexamers in SEC does not rule out the presence of smaller oligomers. Further, it does not rule out the possibility that the self-association equilibrium has been perturbed upon introduction of a mutation in the protein.

It is clear from the self-association and polypeptide binding properties of ClpA and ClpB that smaller order oligomers do persist at saturating concentrations of nucleotide (Veronese et al., 2011; Li and Lucius, 2013; Li et al., 2015a; Lin and Lucius, 2016). For example, Figure 2 shows the fraction of ClpB oligomers populated in the presence of 100 μM and 2 mM nucleotide as a function of total [ClpB], simulated from the reported energetic parameters for ClpB assembly (Lin and Lucius, 2015b, 2016). In the presence of 100 μM nucleotide (Figure 2A), a 1 μM ClpB sample would be made up of ~6% hexamers, while 94% of the population would reside in a mixture of monomers, dimers, and tetramers. In the presence of 2 mM nucleotide (Figure 2B), the same sample would reside in a distribution made up of 74% hexamers and 26% lower order oligomers. In fact, under these conditions, even at 10 μM ClpB, the hexameric state is not fully populated, with hexamers making up ~89% of the total [ClpB]. This fact severely limits the ability to draw conclusions about the ATPase activity of the hexamer from steady state kinetic measurements where a different distribution of oligomers is present at each substrate concentration.

The simplest explanation for why the assumption that all motor protein is in the hexameric state is problematic, is that the Michaelis-Menten equation is scaled linearly by the total enzyme concentration, i.e., V_max = k_cat × E₀. Recall, E₀ is the total amount of enzyme in the experiment, which is controlled by the experimentalist, whereas, E is the free (unbound) enzyme concentration at any given time and its concentration is unknown by the experimentalist. Thus, the maximum velocity is measured at saturating substrate concentration and divided by the known total enzyme concentration, E₀, and the k_cat is reported.

It is important to recall that V_max = k_cat × E₀ emerges from two assumptions in the derivation of the Michaelis-Menten equation. The first is that the substrate is in large excess over the enzyme. The total substrate concentration relative to the total enzyme concentration is controlled by the experimentalist but, mathematically, it results in being able to assume [S]k₁ is a constant in the first differential equation given by Equation (1) for Scheme 1.

Horwich and coworkers showed that ClpAP could catalyze global unfolding of an SsrA tagged GFP construct (Weber-Ban et al., 1999). This was done by incorporating the 11 amino acid SsrA tag, which is a known binding sequence for ClpA and ClpX, at the carboxy-terminus of GFP (Levchenko et al., 1997). When the GFP-SsrA construct was presented to ClpA in the presence of ATP, a slight decrease in fluorescence was observed. However, when the construct was presented to ClpAP in the presence of ATP, a near complete loss of fluorescence was observed. This was interpreted to mean that when ClpA unfolded the GFP in the absence of a protease, GFP was allowed to spontaneously refold. However, in the presence of the proteolytic component, GFP was degraded and thus complete loss of fluorescence was observed.

To examine directional translocation catalyzed by ClpA, Horwich and coworkers developed a FRET based assay (Reid et al., 2001). In this design, a donor fluorophore was placed in the central cavity of ClpP and an acceptor at various positions on model substrates all containing the SsrA sequence at the carboxy-terminus. If ClpA translocates the polypetide chain into the ClpP cavity from the SsrA sequence at the carboxy-terminus directionally to the amino-terminus, then FRET time courses would reveal this. FRET time courses were consistent with processive translocation from the carboxy-terminus to the amino-terminus. The results clearly showed that ClpA drives translocation of a polypeptide chain into the proteolytic chamber of ClpP.

Until recently, the elementary kinetic parameters governing this translocation reaction had not been reported. Moreover, most of the mechanistic investigations available were performed in the presence of ClpP. Thus, the critically important elementary kinetic mechanism for polypeptide translocation catalyzed by ClpA was missing from the field. Determining this mechanism required the development of techniques that would be sensitive to the elementary steps in polypeptide translocation in the absence of proteolytic degradation. Such approaches could then be broadly applied to a variety of enzymes that do not associate with proteases (see examples in the Introduction). This kinetic mechanism would include the elementary rate constants governing the reaction, kinetic step-size (amino-acids translocated between two rate-limiting steps), processivity (probability the enzyme will translocate vs. dissociate), and directionality (C to N vs. N to C).

A single-turnover fluorescent stopped flow assay was developed to elucidate these kinetic parameters (Rajendar and Lucius, 2010; Lucius et al., 2011). Figure 3 shows a generalized schematic representation of this rapid mixing assay. Synthetic polypeptide substrates containing the 11 amino acid SsrA binding sequence at the carboxy-terminus and a single cysteine at the amino-terminus were constructed. The sequence of the polypeptide was based on the Titin I27 domain because the long term goal was to move to full length tandem repeats of I27 as had been done for ClpX (Kenniston et al., 2003, 2005). The cysteine was labeled with fluorescein-5-maleimide. ClpA was bound to the SsrA sequence in the presence of the slowly hydrolysable ATP analog, ATPγS. Upon ClpA binding, fluorescence quenching was observed. Fluorescence quenching has since been observed for binding by both ClpB and Hsp104 to their respective substrates (Li et al., 2015b; Weaver et al., 2017). This sample was then loaded into one syringe of the stopped-flow fluorometer (see Figure 3). In the other syringe was loaded a large excess of ATP and unlabeled SsrA peptide to serve as a trap for ClpA, i.e., any free ClpA would rapidly bind to SsrA and not the fluorescently modified polypeptide (Rajendar and Lucius, 2010). The large excess of trap ensures single-turnover conditions with respect to the complex of ClpA bound to fluorescently labeled peptide.

In the single-turnover fluorescence assay, the two solutions are rapidly mixed within 2 ms in a stopped-flow fluorometer and fluorescence is observed as a function of time. Fluorescence was observed to increase with time indicating that ClpA dissociated from the polypeptide chain. The question is; do the kinetic time courses yield information on translocation before ClpA dissociates? In principle, if ClpA is taking multiple steps before dissociating then the observed kinetic time courses should reflect the number of steps the enzyme takes before dissociation. Thus, if the length of the peptide is increased, the number of steps the enzyme takes before reaching the end should also increase. That is to say, if the time courses are sensitive to processive translocation, then the time courses should depend upon substrate length.

To test the substrate length dependence of the kinetic time courses, time courses were collected as a function of polypeptide substrate length ranging from 30 to 50 amino acids (Rajendar and Lucius, 2010). Observed was a lag (constant fluorescence) followed by an increase in fluorescence. This lag was observed to increase in duration with increasing substrate length indicating that ClpA remained on the polypeptide for an increasing amount of time with increasing substrate length. This observation is interpreted to indicate that ClpA is taking more steps with each increase in substrate length. Therefore, the single-turnover fluorescence stopped-flow assay is sensitive to processive translocation.

To elucidate the elementary rate constants using transient state kinetics one needs to perturb the system. Variables like temperature, salt concentration and type, pH, etc. can be used for this perturbation. For a molecular motor that couples ATP binding and hydrolysis to repeated rounds of translocation, the simplest perturbation is to vary the ATP concentration. The initial experiments are usually carried out at excess ATP so that it can be assumed that ATP binding is not rate-limiting. As the [ATP] is reduced, the observed rate constant will reflect ATP binding, or a step coupled to ATP binding. Importantly, because the motor-peptide complex is preassembled prior to rapidly mixing with ATP (Figure 3), the signal is insensitive to the changing population of ClpA hexamers throughout the ATP range assayed. The kinetic time courses were collected as a function of ATP from ~125 μM to 5 mM. As the [ATP] was reduced, the observed kinetic rate constant decreased. This is further evidence that the time courses are reporting on translocation since simple dissociation would not be predicted to be ATP concentration dependent.

The kinetic time courses were subjected to global non-linear-least-squares (NLLS) analysis (Lucius et al., 2003, 2011). For ClpA, the enzyme translocated with a repeating rate constant, k_t = (1.39 ± 0.06) s⁻¹ and an overall rate of (19 ± 1) AA s⁻¹ at saturating ATP with a kinetic step-size of (14 ± 2) AA step⁻¹. It is important to note that the kinetic step-size represents the average number of amino acids translocated between two rate-limiting steps and may or may not represent physical stepping. While similar strategies have been successfully used to examine helicase catalyzed DNA unwinding and single strand DNA translocation (Fischer and Lohman, 2004; Fischer et al., 2004; Lucius et al., 2004; Lucius and Lohman, 2004), this was the first step-size reported for a polypeptide translocase (Rajendar and Lucius, 2010; Lucius et al., 2011).

The processivity is quantitatively defined as the rate constant for translocation divided by the summation of the rate constants for translocation and dissociation. For example, a translocating enzyme following the mechanism shown in Figure 4, where E·P represents the enzyme pre-bound to a peptide of length L, the enzyme can proceed forward with rate constant k_t or dissociate with rate constant k_d. I_(L−m) represents the first intermediate that has been translocated by some distance m (step-size).

The processivity is the probability given by Equation (10) (Lucius et al., 2003, 2011).

When k_d = 0, then P = 1 and every enzyme that binds will translocate to the end without dissociation. On the other hand, as k_d increases, P approaches zero, which would describe an enzyme with low processivity (an enzyme that has a higher propensity to dissociate than reach the end of the polypeptide chain). The processivity described as a probability, P, can be related to processivity expressed in terms of the average number of amino acids translocated per binding event, N, given by Equation (11) (for a complete derivation of Equation (11) see Appendix B of Lucius et al., 2003).

It is tempting to assume that a hexameric ring motor that encircles the linear lattice on which it translocates would be highly processive. However, this is not always true. For example, the hexameric ring helicase, DnaB exhibits a processivity of P ~0.89 (Galletto et al., 2004). The proposed model is that the ring opens and substrate can “escape” thereby resulting in a dissociation event. However, this primary replicative helicase likely exhibits much higher processivity in the context of the fast moving replication fork, likely due to interactions with other proteins. With respect to ClpB and Hsp104, both enzymes have been proposed to be in “rapid subunit exchange” (Werbeck et al., 2008; DeSantis et al., 2012). Thus, loss of a subunit in a hexameric ring could also result in a dissociation event. Moreover, like DnaB, partner proteins are likely to influence the processivity. Regardless of the mechanism, there is a dearth of quantitative measurements of processivity for polypeptide translocases.

In the initial examination of ClpA catalyzed polypeptide translocation with synthetic peptides, a measureable dissociation rate constant, k_d, was not detected above 500 μM ATP. However, at 300 μM ATP and below, a measureable dissociation rate constant was observed, allowing for the calculation of processivity. The processivity was determined to be P = (0.876 ± 0.006) at low [ATP]. Using Equation (11) a processivity of ~100 amino acids per binding event is predicted, which is 2-fold larger than the longest polypeptide used in this study. Thus, this is a preliminary estimate of the processivity at limiting [ATP] and methods allowing the examination of longer polypeptides are needed to rigorously test the processivity for this and related enzymes. Qualitatively, the findings support the idea that ClpA is highly processive, confirming that reported by Maurizi and coworkers (Thompson et al., 1994).

With a method in hand that is sensitive to polypeptide translocation in the absence of proteolytic degradation the question that could be addressed is, does ClpAP translocate using the same mechanism as ClpA alone? A qualitative assessment of stopped-flow time courses had been reported previously that concluded ClpAP translocated faster than ClpA alone but rate constants were not reported (Kolygo et al., 2009).

The single turnover stopped-flow method described above was employed to examine polypeptide translocation catalyzed by ClpAP. However, upon building a complex of polypeptide bound by ClpAP, a number of questions emerge. Hexameric ClpA can bind to either apical surface of ClpP forming a 1:1 complex, or to both apical surfaces of ClpP forming a 2:1 complex (see Figure 5). Should the experimental design conditions examine 1:1 or 2:1 hexameric ClpA to tetradecameric ClpP? Similarly, if the 2:1 complex is examined, should both sides of the enzyme be bound with peptide?

Based on activity measurements, Maurizi and coworkers reported an affinity for ClpA hexamer binding to ClpP tetradecamer to be ~4 nM (Maurizi et al., 1998). However, the fact that ClpA resides in a distribution of oligomers was not taken into account. ClpA resides in a distribution of monomers, dimers, and tetramers in the absence of nucleotide (Veronese et al., 2009; Veronese and Lucius, 2010). However, even at concentration of nucleotide above 1 mM there remains a distribution of oligomeric states (Veronese et al., 2011; Li and Lucius, 2013). Thus, it cannot be assumed that all of the ClpA present in solution is in the hexameric state.

For a macromolecule with two binding sites, one can be certain to ligate only one of the binding sites if the two-site macromolecule is maintained in large excess over the ligand. Thus, whether 1:1, 2:1 or a mixture of the ClpAP complexes are present in solution, by maintaining the complex in excess over the polypeptide only one peptide can be bound to any given ClpAP complex in the ensemble.

To build a peptide pre-bound complex, 86 nM tetradecameric ClpP and 1 μM monomer of ClpA were used in the presence of 150 μM ATPγS. Note that, unlike ClpA, ClpP forms stable tetradecamers (Maurizi et al., 1998) (E. Duran unpublished data). However, the question is; how much hexameric ClpA is present at 1 μM monomer? To address this question, sedimentation velocity experiments measured the concentration of hexameric ClpA in the presence of 150 μM ATPγS at 1 μM total ClpA monomer concentration. Under these conditions, the hexameric concentration was determined to be 130 nM. It is important to note that if the 1 μM total monomer concentration is divided by six, i.e., assume only hexamers are in solution, then one would predict 170 nM hexamers, an over estimate by 30% of the hexameric ClpA population. Under these conditions, a mixture of 1:1 and 2:1 complexes is predicted. With that in mind, binding the complex to 20 nM peptide maintains ClpAP (whether 1:1 or 2:1 complex) in large excess over the peptide. Keeping the ClpAP complex in excess over the peptide concentration ensures that peptide is only bound to one ClpA hexamer in a given ClpAP molecule. That is to say, it would be thermodynamically unfavorable to have a doubly peptide ligated 2:1 ClpAP complex.

Subjecting ClpAP to the same analysis as performed on ClpA alone revealed that, indeed, ClpAP does translocate with a faster overall rate of ~35 AA s⁻¹ (Miller et al., 2013). This is ~1.5 times faster than the ~20 AA s⁻¹ observed for ClpA alone. The overall rate is the product of the step size and the elementary rate constant governing that step. One of the strengths of the transient state kinetic approaches used is that it is sensitive to these two additional parameters. Interestingly, the kinetic step size for ClpAP was observed to be ~5 AA step⁻¹ in stark contrast to the ~14 AA step⁻¹ measured for ClpA alone (Rajendar and Lucius, 2010). Further, the rate constant governing translocation was found to be ~7 s⁻¹, which is ~5-fold faster than the ~1.4 s⁻¹ measured for ClpA (Miller et al., 2013).

As stated above, the kinetic step-size does not necessarily represent physical movement. However, a recent single-molecule examination of ClpAP translocation reports steps of ~1 nm (Olivares et al., 2014), which was reported to be consistent with the 5 AA step⁻¹ reported from the single turnover experiments described above (Miller et al., 2013). A single molecule experiment that would be sensitive to mechanical movement has not been performed on ClpA alone. Such an experiment would either confirm or refute the measured ~14 AA step⁻¹. Additional testing is necessary to determine whether or not this kinetic step-size represents mechanical movement.

All in all, it is clear that ClpP exerts an allosteric influence on ClpA catalyzed polypeptide translocation. Thus, ClpA and ClpAP should be considered to be two different enzymes that translocate with two different mechanisms. Moreover, questions remain regarding the activities of the 2:1 and 1:1 complexes.

The Walker A and Walker B motifs that form the ATP binding pocket are separated by a loop that extends into the axial channel of ClpA (Guo et al., 2002b). It has been proposed that the loop cycles up and down as the ATP binding site cycles through bound ATP to bound ADP + P_i and then release of ADP and P_i. This up and down motion is thought to drive translocation. Hinnerwisch and coworkers showed through crosslinking studies that polypeptide substrate crosslinked with the NBD2 loop in the central channel of ClpA (Hinnerwisch et al., 2005). From these observations, Hinnerwisch and coworkers proposed that the NBD2 loop was responsible for mechanical pulling on the substrate polypeptide being translocated. They proposed a cycle of translocation to consist of ATP binding at NBD2 with the NBD2 loop in the up conformation, followed by ATP hydrolysis that drives movement of the NBD2 loop to the down conformation and concurrent movement of the polypeptide substrate that is bound to the NBD2 loop. Consistently, synchrotron footprinting data showed that the NBD2 loop proceeds through a nucleotide-dependent conformational change (Bohon et al., 2008).

From examination of the ATP concentration dependence of the kinetic step-size and rate constant for ClpAP, the observed step immediately follows ATP binding (Miller et al., 2013). Coupling this observation with the Hinnerwisch model, the step detected in the single-turnover experiments could be either ATP hydrolysis or a conformational change; a conformational change that may represent movement of the NBD2 loop. Since a single repeating step was detected in each cycle of translocation, loop movement may represent movement by ~5 amino acids.

If the measured kinetic step-size for ClpAP truly represents mechanical movement by ~5 amino acids then why does ClpA alone exhibit a different kinetic step-size of ~14 AA step⁻¹? A potential answer to this question lies in the dependence of the overall translocation rate on [ATP] for ClpA and ClpAP. The translocation rate constant for ClpA alone exhibited a sigmoidal dependence on ATP. The isotherm could not be described by a simple rectangular hyperbola. Rather, it required analysis using a Hill model with a hill coefficient of ~2.5. In contrast, the translocation rate constant for ClpAP did not exhibit a sigmoidal dependence. Since ClpA contains two ATP binding sites per monomer and the single-turnover kinetic time courses are sensitive only to bound hexamer, the observation of a sigmoidal dependence suggests that there is cooperativity between multiple ATP binding sites that are involved in polypeptide translocation. On the other hand, since ClpAP did not exhibit any cooperativity, this indicates that the presence of ClpP relieves the cooperative interactions.

With these observations in mind, Figure 6 illustrates a working model for both ClpA and ClpAP polypeptide translocation, incorporating known structural information and various biochemical/biophysical studies. Figure 6A illustrates ClpA, in the absence of ClpP, with both the NBD1 and NBD2 loops in the up conformation and ATP bound to both domains. The polypeptide substrate is shown in black and is making contact with both the NBD1 and NBD2 loops. Crosslinking studies have shown that contacts between polypeptide substrate and ClpA were only observed with the NBD2 loop, but various single site mutations throughout the NBD1 loop abolished translocation activity (Hinnerwisch et al., 2005). Moreover, recent work indicates that both ATPase sites are involved in translocation catalyzed by ClpA in the absence of ClpP (Rajendar and Lucius, 2010). These two observations implicate the NBD1 loop in translocation. The next step would be for NBD1 to hydrolyze ATP and cause the NBD1 loop to move down and translocate (push) the substrate by up to 14 amino acids creating a polypeptide loop inside the axial channel of ClpA. The loop in the substrate can be accommodated in ClpA since it has been shown that ClpA forms a cavity between the NBD1 and NBD2 loops (Beuron et al., 1998; Guo et al., 2002a). NBD1 would contain ADP and P_i in the ATP binding site and therefore the NBD1 loop would have a reduced affinity for the polypeptide, which would allow for rebinding by another NBD1 loop loaded with ATP in a neighboring subunit in the hexamer (Farbman et al., 2007; Veronese et al., 2011). The NBD2 loop would cycle through multiple rounds of ATP hydrolysis coupled to translocation of the substrate by 2–5 amino acids per cycle with a rate constant of ~4 s⁻¹. This will occur several times thereby shortening the loop inside the cavity of ClpA before NBD1 translocates another ~14 amino acids of the polypeptide into the cavity with a rate constant of 1.4 s⁻¹.

Figure 6B illustrates the working model for how ClpA translocates when associated with ClpP. Since the ATP concentration dependence of the rate of ClpAP catalyzed polypeptide translocation suggests reduced cooperativity between ATP binding sites, it is hypothesized that NBD2 drives translocation in the ClpAP complex. Repeating cycles of ATP binding and hydrolysis could occur at NBD1, but they do not limit the observed translocation. Therefore, this model predicts repeating cycles of ATP binding and hydrolysis at NBD2 would lead to translocation of the substrate by distances of 2–5 aa step⁻¹.

The working model predicts that in the absence of ClpP, NBD1 should hydrolyze ATP with a rate constant of (1.39 ± 0.06) s⁻¹ and NBD2 should hydrolyze ATP with a rate constant of (7.9 ± 0.2) s⁻¹ in the presence of polypeptide substrate. Kress et al. examined the steady state rate of ATP hydrolysis catalyzed by ClpA both in the presence and absence of ClpP (Kress et al., 2009). Further, they made two variants of ClpA that are deficient in ATP hydrolysis at either NBD1 or NBD2, which allow for the examination of ATP hydrolysis at each domain in the absence of hydrolysis at the other domain, and in the presence or absence of ClpP and SsrA substrate. Interestingly, in the absence of ClpP and the presence of GFP-SsrA, NBD1 hydrolyzes ATP with a rate constant of (0.8 ± 0.2) s⁻¹, which is comparable to the rate constant determined for translocation of (1.39 ± 0.06) s⁻¹ determined using the single-turnover stopped flow experiments. Similarly, in the presence of ClpP and GFP-SsrA, NBD2 hydrolyzes ATP with a rate constant of (6.3 ± 0.5) s⁻¹, which is similar to the estimate of (7.9 ± 0.2) s⁻¹ (Miller et al., 2013).

ClpB and Hsp104 were initially observed to disaggregate clients only in the presence of co-chaperones. These disaggregating motors are far more potent in collaboration with co-chaperones, although conditions have since been found in which ClpB and Hsp104 have innate disaggregation abilities. The co-chaperone system for E. coli ClpB is made up of DnaK, DnaJ, and the nucleotide exchange factor GrpE (termed the KJE system). Yeast Hsp104 collaborates with the co-chaperones Hsp70 (analogous to DnaK) and Hsp40 (analogous to DnaJ). Like ClpB/Hsp104, DnaK/Hsp70 is an ATPase and a disaggregase that can function independently of co-chaperones. The full systems, ClpB/KJE and Hsp104/70/40, have ATPase and disaggregase activity greater than the sum of the components' activities. There are three proposed possibilities that could explain this enhanced activity: (1) DnaK modifies the aggregate making a better binding site for ClpB, (2) DnaK accepts substrate from ClpB after the substrate has been completely translocated, or (3) the ClpB-DnaK complex has greatly amplified disaggregation activity relative to ClpB alone, possibly through a fundamentally different mechanism.

Early attempts to identify which component of the system acted upon an aggregate or client first resulted in divergent findings. The Liberek group identified DnaK as the first actor. They found that DnaK, with DnaJ and ATP, remodeled aggregates to facilitate ClpB-catalyzed disaggregation. Neither a transient tertiary complex with ClpB or additional roles for DnaK downstream of ClpB's action were ruled out (Zietkiewicz et al., 2004, 2006). On the other hand, early work from the Bukau group concluded that ClpB acted first. Specifically, ClpB was observed to expose a substrate's hydrophobic regions, which could then be recognized by the KJE system (Goloubinoff et al., 1999). The development of a ClpB trap mutant (double Walker B variant, able to bind but not hydrolyze ATP) also revealed that ClpB_trap outcompeted DnaK for binding to a model substrate and inhibited DnaK activity (Weibezahn et al., 2003).

Over time, the idea of a ClpB-DnaK (Hsp104-Hsp70) complex has come into favor. One compelling observation in support of this finding is that the activity of the co-chaperones is species specific. ClpB works with DnaK but not Hsp70. Hsp104 works with Hsp70 but not DnaK. This suggests a direct interaction between the chaperones. Furthermore, both the Wickner and Tsai groups engineered sets of chimeras in which domains from ClpB were replaced by the analogous domain from Hsp104 and vice versa. Both groups found that the M domain dictates which species formed a productive cochaperone partnership. For example, Hsp104 with the M domain from ClpB partnered effectively with the KJE system, not the Hsp70 system. This finding is consistent with the identification of the M domain as the binding site for DnaK (Miot et al., 2011; Rosenzweig et al., 2013; Doyle et al., 2015).

Binding affinities of 17 and 25 μM have been reported for T. thermophilus ClpB and DnaK (Schlee et al., 2004; Rosenzweig et al., 2013). For E. coli ClpB and DnaK, the K_d has been estimated in the range of 7–30 μM (Kedzierska et al., 2005). Notably, while the ClpB-DnaK complex has been observed by co-elution assays (Schlee et al., 2004; Barnett et al., 2005) and NMR (Rosenzweig et al., 2013), the ternary complex of ClpB-DnaK-client has not been observed. Furthermore, despite the K_d measurements and estimates in the range of ~20 μM, biochemical assays are often carried out with nanomolar to low micromolar concentrations of DnaK, conditions in which a significant population of the ClpB-DnaK complex is not expected (Weibezahn et al., 2004; Haslberger et al., 2008; DeSantis et al., 2012; Seyffer et al., 2012; Rosenzweig et al., 2013; Aguado et al., 2015; Doyle et al., 2015). Nevertheless, in these cases observations are attributed to the interplay between ClpB and DnaK. Though the existence of a DnaK-ClpB or Hsp70-Hsp104 complex has become widely accepted, the role of co-chaperones upstream and/or downstream of that complex remains under investigation. The convergence of evidence suggests that DnaK acts on the aggregate first, possibly targeting the client to ClpB, and then DnaK binds ClpB unleashing the disaggregating power of ClpB (Weibezahn et al., 2004; Sielaff and Tsai, 2010; Miot et al., 2011; Seyffer et al., 2012). DnaK may also have additional roles in the proper refolding of the client after release from ClpB.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.