Under stressful conditions, such as high temperatures, many proteins lose their proper structure and clump together to form large irregular aggregates. To combat this effect, living organisms exposed to stress produce specialized proteins called chaperones, which can rescue the damaged proteins from aggregates.

Studies into this “disaggregation” process often use budding yeast as a model organism. The protein-recovery machinery in this yeast is composed of a ring-shaped enzyme called Hsp104, together with a chaperone called Hsp70 and its partner Hsp40. The Hsp104 enzyme converts molecules of ATP into ADP and uses the energy released from the reaction to move, or “translocate”, damaged proteins through its central channel and release them from the aggregates.

Previous studies had reported that ADP negatively affects Hsp104. Now, Kłosowska et al show that Hsp104 is almost inactive in a test-tube if the concentration of ADP is as high as that found inside a cell. This raises a question: how can Hsp104 efficiently remove proteins from aggregates in cells if the conditions are so unfavorable?

Using purified proteins, Kłosowska et al. go on to show that Hsp104 is able to tolerate the level of ADP found inside cells thanks to the Hsp70 chaperone. The experiments show that ADP weakens Hsp104’s ability to bind proteins while Hsp70 supports this ability and counteracts the negative effect of ADP. Further experiments demonstrate that Hsp104 is less affected by ADP, and binds more readily to ATP, when it is translocating proteins. These findings explain how the yeast disaggregating machinery can work even at relatively high concentrations of ADP, and reveal a new control mechanism in the disaggregation process.

Many important proteins have poorly organized fragments that can be recognized by Hsp104, and if Hsp104 was to bind to and translocate these proteins it could harm the cell. The findings of Kłosowska et al. suggest that Hsp70 helps Hsp104 to specifically bind to and act upon proteins in aggregates, while binding to partly unstructured proteins is limited by the high ADP concentration. Further studies are now needed to understand how the protein-recovery machinery can discriminate between aggregated and non-aggregated proteins.

Molecular chaperones maintain protein homeostasis by supporting protein folding and preventing aggregation. However, during severe stress the capacity of this protective system becomes exhausted due to appearance of excess amounts of misfolded proteins and their aggregation. Once physiological conditions are restored, the aggregation process is reversed by specialized chaperones, disaggregases, capable of reactivation of proteins trapped in aggregates (Aguado et al., 2015; Doyle et al., 2013; Kim et al., 2013; Mogk et al., 2015). In yeast Saccharomyces cerevisiae this role is played by the Hsp104 chaperone, essential for development of tolerance to stress (Sanchez and Lindquist, 1990) and for propagation of prions (Chernoff et al., 1995).

Hsp104 is an AAA+ superfamily member (ATPase Associated with various cellular Activities) and its homologs, Hsp104/ClpB proteins, are present in fungi, protozoans, plants and bacteria (Ammelburg and Frickey, 2006; Malinovska et al., 2015; Mishra and Grover, 2015). Hsp104 consists of four domains: the N-terminal domain, two Nucleotide Binding Domains (NBD1 and NBD2) and a coiled coil region protruding from the NBD1, called the M-domain (Lee et al., 2003). Hsp104 functions as a hexamer in which the Nucleotide Binding Domains form a double ring (Carroni et al., 2014; Lee et al., 2010; Parsell et al., 1994). Disaggregation is powered by ATP hydrolysis in NBD1 and NBD2 (Schaupp et al., 2007; Wendler et al., 2009). During disaggregation a polypeptide is disentangled from the aggregate, translocated through the central channel, and enabled to fold into the native structure (Lum et al., 2004; Tessarz et al., 2008; Weibezahn et al., 2004).

Hsp104 alone is insufficient for protein recovery from aggregates. A functional disaggregation machinery is composed of Hsp104 cooperating with the Hsp70 chaperone (Ssa1 in S. cerevisiae) and its cochaperone Hsp40 (Ydj1 and Sis1 in S. cerevisiae) (Glover and Lindquist, 1998; Krzewska et al., 2001). Hsp70 interacts with the M-domain of Hsp104 and facilitates Hsp104-mediated protein renaturation at several stages of the process (Miot et al., 2011; Rosenzweig et al., 2013; Schlee et al., 2004). Firstly, Hsp70 targets Hsp104 to aggregates and stabilizes the Hsp104-aggregate complexes (Acebrón et al., 2009; Okuda et al., 2015; Winkler et al., 2012). Secondly, Hsp70 activates Hsp104 by affecting the position of the M-domain against the surface of NBD1 (Haslberger et al., 2007; Lee et al., 2013; Lipińska et al., 2013; Oguchi et al., 2012; Seyffer et al., 2012; Sielaff and Tsai, 2010). Finally, Hsp70 might participate in folding events downstream of Hsp104.

The M-domain regulates the activity of the disaggregase. M-domains are assembled around the NBD1 ring in the hexamer. Each M-domain interacts with respective NBD1 via a network of ionic bonds and keeps Hsp104/ClpB proteins in a repressed, disaggregation-compromised state. Hsp70 binding to the M-domain causes its reposition, which transits Hsp104/ClpB into a derepressed state, manifested by highly stimulated ATPase and disaggregation activities (Oguchi et al., 2012). The latter state is permanent in the so-called derepressed mutants, which are partially independent from Hsp70 in protein disaggregation and display highly elevated ATPase activity (Carroni et al., 2014; Jackrel et al., 2014; Lee et al., 2013; Lipińska et al., 2013; Oguchi et al., 2012; Seyffer et al., 2012; Sielaff and Tsai, 2010). The M-domain mediated control of Hsp104 protein is critical for the cell, as the derepressed mutants are toxic (Lipińska et al., 2013; Schirmer et al., 2004). The mechanism coupling the M-domain with the increased Hsp104 activity remains not fully understood.

Both Nucleotide Binding Domains of Hsp104 possess typical motifs for an AAA+ protein: Walker A, responsible for ATP binding, Walker B, essential for ATP hydrolysis and arginine fingers, necessary for the intersubunit communication (Biter et al., 2012; Schaupp et al., 2007; Schirmer et al., 2001; Zeymer et al., 2014). ATP hydrolysis and translocation of protein substrates through the central channel are in a strict allosteric interdependence: polypeptide processing stimulates the ATPase activity of Hsp104 (Tessarz et al., 2008; Woo et al., 1992; Yamasaki et al., 2015), while the efficiency of polypeptide binding to Hsp104 is determined by the presence of ATP in NBD1 (Franzmann et al., 2011; Schaupp et al., 2007). Changes of the nucleotide states of NBDs are associated with domain rearrangements and movements of peptide binding loops located in the central channel. These conformational changes couple the energy generated during ATP hydrolysis with the protein-threading force (Lum et al., 2004; Wendler et al., 2009).

Remarkably, Hsp104 has low affinity towards ATP and relatively high towards ADP, therefore ADP strongly inhibits ATP hydrolysis (Franzmann et al., 2011; Glover and Lindquist, 1998; Grimminger et al., 2004). For this reason, studies of Hsp104 activity are usually performed under the conditions optimal for its ATPase activity, including saturating (5–10 mM) concentration of ATP and an ATP regeneration system to avoid ADP accumulation. Under such experimental conditions Hsp104 is a potent ATP-hydrolyzing and protein unfolding machine (Biter et al., 2012; Desantis et al., 2014; Franzmann et al., 2011; Tessarz et al., 2008). Yet, these nucleotide concentrations are very different from those observed in the cytosol, with ATP at 2.1–3.4 mM concentration and ADP at 0.5–1.5 mM, depending on the growth conditions (Canelas et al., 2008; Teusink et al., 2000; Wu et al., 2006). So far, little has been known about how Hsp104 disaggregase functions under conditions similar to the ones observed in the living cell, where ADP constitutes a significant proportion of adenine nucleotides. Therefore, we addressed the question: how does ADP affect Hsp104 activity?

Here, we report that ADP compromises ATP hydrolysis, substrate binding and translocation by Hsp104 in the absence of the Hsp70 chaperone. We also demonstrate that protein disaggregation at physiological concentrations of ATP and ADP is only effective due to the Hsp70 assistance. Hsp70 promotes Hsp104 binding to the aggregated protein substrate but does not support processing of non-aggregated proteins. Furthermore, we show that the process of polypeptide threading by Hsp104 facilitates binding of ATP, strongly stimulating the ATPase activity of the disaggregase in the presence of ADP. Based on these findings we propose a new level of regulation of the disaggregase by its chaperone partner Hsp70.

In this work we investigated in vitro Hsp104 functioning at physiological ATP and ADP concentrations. Under such conditions the activities displayed by Hsp104 alone, including ATP hydrolysis as well as binding and translocation of substrate proteins, are highly limited (Figure 7). Protein translocation through the central channel is initiated only when Hsp70 specifically recruits Hsp104 for disaggregation by facilitating its interaction with the aggregate. Translocation of the polypeptide substrate leads to the release of ADP inhibition. The release is due to a several-fold increase in Hsp104 affinity towards ATP. Consequently, the process of polypeptide threading highly stimulates the ATPase activity of Hsp104 at non-saturating concentration of ATP and in the presence of the competitive inhibitor, ADP (Figure 7). The presented results point to a novel, ADP and Hsp70 dependent, level of regulation of the disaggregase.

Figure 7

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The strong inhibition by ADP of Hsp104 in the absence of a protein substrate results from the discrepancy between ATP and ADP binding affinities (Grimminger et al., 2004). According to the previous studies (Canelas et al., 2008; Teusink et al., 2000; Wu et al., 2006), the cellular level of ATP (2.1–3.4 mM) is below saturation for Hsp104 (~10 mM). Furthermore, depending on growth conditions, residual ADP can constitute a significant fraction of adenine nucleotides in the cytosol (up to 1.5 mM) (Canelas et al., 2008; Larsson et al., 1997; Osorio et al., 2003; Ozalp et al., 2010). The conditions in yeast cytoplasm are therefore far from optimal for the biochemical activity of the disaggregase. In this work we measured the ATPase and the protein-processing activity of Hsp104 under a broad range ADP concentrations, establishing the relation between the degree of inhibition and ATP:ADP ratio. The highest ATP:ADP ratio reported for yeast cells is 7:1 (Theobald et al., 1997). Even at slightly higher ATP:ADP ratio 10:1 the activity displayed by Hsp104 alone was highly reduced (by over 50%) and under the intermediate physiological ATP:ADP ratio 2.6:1 (Canelas et al., 2008; Ozalp et al., 2010) it was almost completely abolished (inhibited by 90% or more). In contrast, the disaggregation activity of Hsp104 in the presence of Hsp70 at the ATP:ADP ratio 10:1 was reduced by less than 10% and at 2.6:1 - to 50%. 90% degree of inhibition occured below the ATP:ADP ratio 1.3:1, while the lowest proportion of ATP to ADP reported in yeast is 1.4:1 (Hynne et al., 2001). Therefore, inhibition by ADP and the counteracting effect of Hsp70 regulate Hsp104 activity across a whole range of physiological proportions between ATP and ADP.

The mechanism of ADP inhibition of Hsp104 can be explained in the light of the previous studies, which show that ADP predominantly occupies the NBD2 domain. It was proposed that ADP release from NBD2 could be a rate-limiting step in the ATPase cycle (Franzmann et al., 2011). Agreeably, we observed that ADP strongly inhibits the ATPase activity in NBD2 in the Hsp104 E285Q variant and exerts much milder effect on ATP hydrolysis by Hsp104 E687Q variant, in which NBD1 is the only active ATPase domain. This shows that ADP inhibits Hsp104 by affecting its NBD2.

Interestingly, WT Hsp104, in which both NBDs are functional, responds to ADP similarly as the Hsp104 E285Q variant, in which only the NBD2 domain is hydrolytically active. This suggests that in the WT protein the difficulties in ADP release from NBD2 prevent ATP hydrolysis also in NBD1. It has been established before that when ADP occupies NBD2, NBD1 is mostly in the nucleotide-free state (Franzmann et al., 2011). It has also been reported that the absence of nucleotide in NBD1 affects the ATPase activity of the neighbouring NBDs and interrupts protein binding to Hsp104 (Schaupp et al., 2007). This could explain how the inhibitory effect of ADP on NBD2 is allosterically transmitted between all the ATPase domains within the hexamer.

Results presented in this work imply an existence of two Hsp104 functional modes, depending on substrate translocation through the central channel: ADP-inhibited and active. In the ADP-inhibited mode both the ATPase activity and polypeptide binding are severely impaired and the protein substrate cannot undergo translocation through Hsp104. In the active mode the presence of the polypeptide in the central channel facilitates ATP binding, and most likely accelerates the rate-limiting step in the ATPase cycle: ADP release. These findings shed new light on how unfolded substrates stimulate ATP hydrolysis by Hsp104, however to fully understand the underlying mechanism detailed structural data collected in the presence of a substrate would be required.

Our data suggest that transition from ADP-inhibited into the active mode occurs upon a successful interaction with a substrate. Although substrate binding is allowed in the presence of ADP, the probability of this event decreases with the increasing proportion of ADP to ATP. It requires high level of protein substrate to compensate for this effect. This underlines the importance of the upstream role of Hsp70 in disaggregation, which involves initial aggregate remodeling and promoting of interaction between Hsp104 and the aggregate. Due to these activities of Hsp70, Hsp104 can bind aggregated substrates, which are at low concentration.

Curiously, the upstream role of Hsp70 is limited to aggregates. Hsp70 does not support binding of non-aggregated protein substrates, allowing Hsp104 activation only at the face of an aggregate. Thus, by specifically targeting Hsp104 towards aggregates, Hsp70 provides another level of control of the disaggregase.

The proposed regulatory mechanism works in addition to the activation of Hsp104 associated with Hsp70-dependent reposition of the M-domain, since the latter effect is not enough to overcome ADP inhibition. This is suggested by the observations that both the wild type Hsp104 and the derepressed D484K mutant of the M-domain are strongly affected by ADP and, in both cases, Hsp70 enables the Hsp104 variants to efficiently disaggregate proteins in the presence of the inhibitor. It needs to be kept in mind, however, that the two roles of Hsp70 are probably interdependent, as stimulation of the ATPase activity via the M-domain might favor formation of the complex between Hsp104 and an aggregate.

Interestingly, in the absence of Hsp70 addition of an efficient ATP regeneration system is enough to unleash the disaggregating potential of the derepressed Hsp104 D484K. Thus, when Hsp104 is not subjected to the repression by the M-domain, it depends on the Hsp70 chaperone partner exclusively in the context of the release of ADP inhibition.

The restrictive effect of ADP may play an important role in proper and efficient Hsp104 functioning in the cell. Firstly, inhibition of ATP hydrolysis decreases energy dispersing by Hsp104. Secondly, Hsp104 inhibition by ADP prevents a potentially harmful activity of the disaggregase. Such is manifested upon expression of derepressed Hsp104 variants in yeast, which causes disruption of cytoskeleton and inhibits cell division (Lipińska et al., 2013; Schirmer et al., 2004). This shows that the protein unfolding activity of Hsp104, although highly advantageous under stress conditions, needs to be under strict control, as instead of aggregates it can turn against other protein targets.

Notably, in our in vitro experiments, Hsp70 chaperone supports Hsp104 in processing of aggregates, but not of the model disordered protein substrates. The observation that Hsp70 inhibits both fRCMLa and α-casein proteolysis by the HAP variant of Hsp104 (and ClpP), implies that competition between the chaperones for their binding sites on the unfolded substrates may occur. This points to a plausible shielding role of Hsp70, protecting non-aggregated, intrinsically disordered proteins against the protein-threading activity of Hsp104.

Translocation of the disordered substrates by HAP WT was at very low level at the physiological concentrations of adenine nucleotides in the presence of Hsp70 (Figure 5). In comparison, the derepressed HAP D484K variant was still highly active under the same conditions, even upon inhibition by ADP (Figure 5—figure supplement 3). The high disaggregation activity of the derepressed Hsp104 comes at the price of high and unspecific protein unfolding activity, which might be responsible for the toxic effects of Hsp104 D484K in the cell (Lipińska et al., 2013). Meanwhile, the M-domain mediated repression, inhibition by ADP and, supposedly, competition by Hsp70 for binding sites on the partially disordered proteins ensures that the Hsp104 WT is tuned down to the level that is safe for the cell.

Due to its remarkable ability to dissolve protein aggregates related to stress, aging, as well as to neurodegenerative diseases (Erjavec et al., 2007; Jackrel et al., 2014; Torrente et al., 2016), Hsp104 and especially its derepressed variants have been the subject of intense search for their potential therapeutic application (Castellano et al., 2015). Our studies show that any plans for application of these protein remodeling machines in the novel environment should involve evaluation of ATP and ADP concentrations, which determine Hsp104 activity.

The knowledge of ADP influence on Hsp104 is also important for interpretation of in vitro experiments. As observed during fRCMLa proteolysis by HAP or GFP disaggregation by Hsp104 D484K, accumulating ADP inhibited the reaction before the total pool of the protein substrate has been processed. This affected the renaturation yield, a parameter that should not therefore be used to evaluate Hsp104 activity. Furthermore, the kinetics of the reaction is strongly influenced by the efficiency of different ATP regeneration systems.

In the light of Hsp104 dependence on adenine nucleotides, we integrate the established facts and reveal novel aspects of the complex interplay between the processes driving Hsp104-dependent disaggregation: the ATPase cycle, protein translocation trough the central channel, Hsp104 derepression trough the M-domain and the upstream action of Hsp70. The mechanism which emerges from our work implies that, in combination with the restrictive cellular ATP and ADP concentrations, Hsp70 provides an additional level of Hsp104 control, effectively limiting the toxic unspecific protein unfolding activity of Hsp104, at the same time supporting efficient recovery of aggregated proteins, the process crucial for cell survival after stress.

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Author details

1. Agnieszka Kłosowska

   Department of Molecular and Cellular Biology, Intercollegiate Faculty of Biotechnology, University of Gdańsk and the Medical University of Gdańsk, Gdańsk, Poland

   Contribution

   AK, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   agnieszka.jurczyk@biotech.ug.edu.pl

   Competing interests

   The authors declare that no competing interests exist.

2. Tomasz Chamera

   Department of Molecular and Cellular Biology, Intercollegiate Faculty of Biotechnology, University of Gdańsk and the Medical University of Gdańsk, Gdańsk, Poland

   Contribution

   TC, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

3. Krzysztof Liberek

   Department of Molecular and Cellular Biology, Intercollegiate Faculty of Biotechnology, University of Gdańsk and the Medical University of Gdańsk, Gdańsk, Poland

   Contribution

   KL, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   liberek@biotech.ug.edu.pl

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-7532-9279

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