Role of a conserved aspartic acid in nucleotide binding domain 1 (NBD1) of Hsp100 chaperones in their activities
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- Nowicki, Ł., Leźnicki, P., Morawiec, E. et al. Cell Stress and Chaperones (2012) 17: 361. doi:10.1007/s12192-011-0312-4
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Besides its beneficial role in thermotolerance, the chaperone protein Hsp104 is involved in the inheritance of yeast Saccharomyces cerevisiae prions. Guanidine hydrochloride was previously shown to interfere with Hsp104 chaperone activity in vivo, thus impairing thermotolerance and resulting in prion curing. It was also reported that guanidine inhibits Hsp104 ATPase and disaggregation activity. We show that in vitro guanidine significantly inhibits the disaggregation activity of ClpB, the bacterial orthologue of Hsp104. However, guanidine exerts opposite effects on the ATPase activities of Hsp104 and ClpB. While the ATPase activity of Hsp104 is inhibited, the analogous ClpB activity is stimulated several-fold. Mutation of the universally conserved aspartic acid residue in position 184 to serine (D184S) in HSP104 and the analogous mutation in clpB (D178S) resulted in chaperones with lower disaggregating and ATPase activities. The activities of such changed chaperones are not influenced by guanidine, which suggests the role of this residue in the interaction with guanidine.
KeywordsChaperone-dependent protein disaggregationProtein aggregationPrion curing agent—guanidine hydrochlorideHsp70Hsp100
Severe heat shock conditions cause aggregation of many intracellular proteins. Hsp104 from Saccharomyces cerevisiae and ClpB from Escherichia coli, members of the Hsp100 family of molecular chaperones, are crucial factors required for thermotolerance (Squires et al. 1991; Kitagawa et al. 1991; Sanchez and Lindquist 1990), as well as the disaggregation of protein aggregates which occurs once the cell is returned to the physiological temperature (Parsell et al. 1994a; Laskowska et al. 1996; Mogk et al. 1999). To mediate protein disaggregation, chaperones of the Hsp100 family require the assistance of the Hsp70 chaperone system (reviewed in Liberek et al. (2008)). Recent studies postulate that the Hsp70 system remodels aggregates at the initial step of disaggregation, enabling subsequent action by the Hsp100 chaperones (Ziętkiewicz et al. 2004, 2006; Weibezahn et al. 2004).
Hsp104 and ClpB, both Hsp100 proteins, belong to the AAA+ (ATPase associated with a variety of cellular activities) family whose members possess characteristic ATPase domain(s). AAA+ proteins self-assemble into oligomeric structures and use the energy derived from ATP hydrolysis to remodel their protein substrates (Vale 2000; Bösl et al. 2006). Hsp104 and ClpB have two nucleotide binding domains (NBD1 and NBD2; AAA+ domains) which are essential for hexamerization and chaperone function (Krzewska et al. 2001a; Weibezahn et al. 2003; Schirmer et al. 2001; Watanabe et al. 2002; Mogk et al. 2003). The first AAA+ domain of both Hsp104 and ClpB contains an additional coiled-coil region called the “middle domain,” located C-terminally to the ATP binding site (Lee et al. 2003). This middle domain is characteristic of Hsp100 disaggregating chaperones.
In addition to its role in thermotolerance, Hsp104 plays a role in the propagation and inheritance of yeast prions [PSI+], [URE3], and [RNQ+] (Wickner 1994; Sondheimer and Lindquist 2000). It was shown that the [PSI+] prion is lost in a yeast strain overexpressing Hsp104, as well as in an hsp104 deletion strain (Chernoff et al. 1995). Remarkably, the addition of millimolar concentrations of guanidine hydrochloride (GuHCl), a chaotropic salt, to the yeast medium leads to prion curing (Tuite et al. 1981; Ferreira et al. 2001; Jung and Masison 2001; Jung et al. 2002). Due to this property, GuHCl became a tool which allows studying the propagation of yeast prions. Moreover, GuHCl-treated strains show a reduced heat stress tolerance and, similar to the hsp104 deletion strain, display impaired intracellular reactivation of heat-inactivated luciferase (Ferreira et al. 2001; Jung and Masison 2001). It was also shown that various substitutions of the aspartic acid residue in position 184 in Hsp104 affect both prion curing by guanidine and thermotolerance. However, the thermotolerance of cells expressing these mutant proteins is diversely influenced by guanidine (depending on the mutation), and the degree of thermotolerance does not correlate with prion stability (Jung et al. 2002).
Recently, it has been shown that GuHCl reduces the ATPase activity of Hsp104 by approximately 50% (Grimminger et al. 2004). Guanidine selectively binds to the nucleotide-bound Hsp104 hexamer and increases the affinity of the chaperone for adenine nucleotides, thus promoting (or stabilizing) the oligomerization of the chaperone (Grimminger et al. 2004). However, it is not fully understood how the change in Hsp104 ATPase activity correlates with its ability to disaggregate protein aggregates. Wegrzyn et al. (2001) reported that prion curing in the presence of GuHCl was significantly slower than curing by removal or inactivation of Hsp104. Therefore, it has been suggested that GuHCl only partially inhibits various Hsp104 activities, as is the case for ATP hydrolysis (Grimminger et al. 2004).
The influence of GuHCl on the activity of ClpB, the E. coli orthologue of Hsp104, has not been investigated until now. We note that the Hsp104 NBD1 domain encompassing amino acid residue 184, critical for GuHCl-dependent prion curing and thermotolerance, is highly conserved among members of the Hsp100 family (Lee et al. 2003). Here, we show that GuHCl completely abolishes the disaggregation activity of Hsp104 and significantly inhibits the analogous activity of ClpB. However, GuHCl exerts opposite effects with respect to the ATPase activities of Hsp104 and ClpB. While the ATPase activity of Hsp104 is inhibited by GuHCl, the analogous ClpB activity is stimulated several-fold. Mutation in HSP104 changing the aspartic acid residue in position 184 to serine (D184S) as well as the analogous mutation in clpB (D178S) results in the formation of chaperones with lower disaggregating activities. However, the activities of such mutated chaperones are no longer influenced by GuHCl, indicating that this aspartic acid residue is important for interaction with guanidine.
Materials and methods
Published protocols were used for the purification of E. coli DnaK, DnaJ, GrpE (Zylicz et al. 1989), ClpB, and ClpB (D178S) (Woo et al. 1992). Yeast S. cerevisiae proteins Hsp104, Hsp104 (D184S), and Ydj1 were expressed in E. coli and purified as described in references Parsell et al. (1994b) and Cyr and Douglas (1994), respectively. Ssa1 protein was overexpressed in S. cerevisiae and purified according to the published protocol (Ziegelhoffer et al. 1995). GFP was expressed in E. coli and purified as described (Ziętkiewicz et al. 2004). Firefly luciferase (E 1701) was purchased from Promega. Protein concentrations were determined using the Bio-Rad Protein Assay system with BSA as a standard. Molar concentrations given are based on the monomeric structure for all chaperones.
Site-directed mutagenesis of pET15b-HSP104 and pET22b+clpB was performed using Pfu DNA polymerase and the oligonucleotide primers 5′-CGTCAAGGTAAACTTTCCCCTGTCATCGGCCGTG-3′ and 5′-GCCGAACAGGGCAAACTCTCTCCGGTGATTGGTCG-3′, respectively, as described in the QuickChange manual (Stratagene). All constructs were confirmed by sequencing.
Yeast cells were grown at 30°C in SC medium prepared as described (Sherman 1991) for W3031B, and in SC-His medium for other strains (Sherman 1991). Overnight cultures were diluted to an OD600 of 0.2. Heat shock was performed on exponentially growing cells adjusted to an OD600 of 0.4. Cells were pre-incubated at 37°C for 30 min, and then incubated with slight agitation at 50°C for 45 min. Aliquots were withdrawn at the indicated time points, cells were mixed by vortexing, serially diluted, and subsequently spotted on SC or SC-His solid media with or without 5 mM GuHCl.
The ATPase activity of Hsp104, Hsp104 (D184S), ClpB, and ClpB (D178S) was measured using a coupled enzymatic assay (Norby 1988). This assay enables ATP regeneration and thus eliminates ADP, a potent competitive inhibitor of the reaction. Assays were carried out as described previously with minor modifications (Grimminger et al. 2004). Assays were performed in thermostated 200-μl cuvettes at 25°C using a Beckman DU650 spectrophotometer. Reactions were assembled in buffer A (50 mM Hepes/KOH, pH 7.5; 150 mM KCl; 20 mM MgAc) supplemented with 5 mM ATP. Reactions were started by addition of the protein of interest (1 μM Hsp104 or 4 μM ClpB in GuHCl titration experiment). ATP turnover was estimated from the slope of dA340/dt curve as described previously (Grimminger et al. 2004).
Refolding of urea-denatured luciferase
Firefly luciferase (4 μM) was denatured for 3 h at 30°C in buffer B (50 mM Tris, pH 7.4; 150 mM KCl; 20 mM MgAc; 5 mM DTT) containing 6 M urea. Refolding reactions were performed by addition of denatured luciferase (to the final concentration of 50 nM) to buffer A containing indicated chaperone proteins (2 μM Hsp104, 3 μM Ssa1p, 1 μM Ydj1p for yeast chaperones; 0.5 μM ClpB, 1 μM DnaK, 0.2 μM DnaJ, 0.1 μM GrpE for bacterial chaperones), 5 mM ATP, the ATP regenerating system (10 mM phosphocreatine, 100 μg/ml phosphocreatine kinase), and 0.15 mg/ml bovine serum albumin. Reaction mixtures were incubated for the indicated time at 24°C, and the luciferase activity was measured in a Beckman LS 6000 TA scintillation counter using the Luciferase Assay System (Promega E1500).
In order to analyze the influence of GuHCl on the oligomeric state of both Hsp104 and Hsp104 (D184S), 20 μg of each protein diluted in buffer D (40 mM Hepes/KOH, pH 7.8; 150 mM NaCl; 10 mM MgCl2) were loaded onto a 3.5-ml 15–45% glycerol gradient in buffer C (40 mM Hepes/KOH, pH 7.8; 150 mM NaCl; 10 mM MgCl2; 5 mM 2-mercaptoethanol) supplemented with 5 mM ATP and (where indicated) with 5 mM GuHCl. The gradients were centrifuged at 4°C for 16 h in a Beckman SW 60 rotor at 46,000 rpm. Following centrifugation, 14 fractions (250 μl each) were collected from the top and analyzed by SDS-PAGE (12.5%) together with the remaining protein eluted from the bottom of the tube.
Partial trypsin digestion
Four micrograms of either ClpB or Hsp104 protein were incubated in 10 μl of buffer A (with 2 mM ATP) at 30°C for 10 min in the presence or absence of GuHCl (5 mM). Partial proteolysis was started by addition of 0.07 μg of trypsin (Sigma). At the indicated times, reactions were stopped by addition of SDS sample buffer. Proteolysis products were analyzed using SDS-PAGE (10%) and Coomassie Blue staining.
Isothermal titration calorimetry
Isothermal titration calorimetry (ITC) was performed using a MicroCal AUTO-ITC (MicroCal-LLC, Northampton) as described previously (Grimminger et al. 2004). All experiments were performed at 25°C using assay buffer (50 mM Hepes/KOH, pH 7.5, 150 mM KCl, and 10 mM MgCl2). For the binding of GuHCl to Hsp104- or Hsp104 (D184S)-nucleotide complexes, experiments were carried out using 13.82 mM GuHCl in the injection syringe and an injection volume of 5 μl. The protein concentration in the cell was 20 μM, and the ADP concentration was 2 mM. Data analysis was performed using the Origin software package (Origin Lab, Northampton, MA, USA).
GFP disaggregation experiments
Experiments were performed as described previously (Ziętkiewicz et al. 2004). Briefly, GFP at 5 mg/ml concentration was heat-aggregated by incubation at 85°C for 10 min. The reactivation was started by addition of heat-aggregated GFP (9 μg) to the reaction mixture containing chaperone proteins (2 μM Hsp104, 3 μM Ssa1p, 1 μM Ydj1p for yeast chaperones; 0.5 μM ClpB, 1 μM DnaK, 0.2 μM DnaJ, 0.1 μM GrpE for bacterial chaperones) in buffer A supplemented with 5 mM ATP and the ATP regenerating system (10 mM phosphocreatine, 100 μg/ml phosphocreatine kinase). Reactions were carried out in thermostated 120-μl cuvettes at 25°C, and GFP reactivation (observed as an increase in GFP fluorescence) was monitored in real time using a Perkin Elmer LS 50B spectrofluorometer with excitation at 395 nm and emission at 510 nm. The excitation slit was set to 4 nm and the emission slit to 12 nm; 1% attenuator was used.
Heat-aggregated GFP (9 μg) was incubated in buffer A containing the indicated chaperone proteins (2 μM Hsp104, 7.5 μM Ssa1p, 2.9 μM Ydj1p for yeast chaperones; 0.5 μM ClpB, 4.4 μM DnaK, 0.34 μM DnaJ, 0.45 μM GrpE for bacterial chaperones), 5 mM ATP, the ATP regenerating system and, where stated, 5 mM GuHCl. Disaggregation reactions were performed at 24°C for 15 min (when bacterial chaperones were used) or 45 min (for yeast chaperones). Reactions were stopped by addition of EDTA to the final concentration of 40 mM, and reaction mixtures (100 μl) were loaded onto a 3.5-ml 15–45% glycerol gradient in buffer C (40 mM Hepes/KOH, pH 7.8; 150 mM NaCl; 10 mM MgCl2; 5 mM 2-mercaptoethanol) supplemented with 1 mM ATP. The gradients were centrifuged for 2 h at 4°C in a Beckman SW 60 rotor at 40,000 rpm. Following centrifugation, 20 fractions (180 μl each) were collected from the top and, together with the remaining protein eluted from the bottom of the tube, analyzed by SDS-PAGE (12.5%) and subsequent western blotting using anti-GFP mouse monoclonal antibodies (Roche Applied Science).
Residue D184 of Hsp104 is important for interaction with guanidine
It was shown that the presence of GuHCl in the medium abolishes thermotolerance and cures the prion phenotypes in yeast cells (Tuite et al. 1981; Ferreira et al. 2001; Jung and Masison 2001; Jung et al. 2002). Both effects are dependent on the presence of functional Hsp104 chaperone (Ferreira et al. 2001; Jung and Masison 2001). The aspartic acid residue in position 184 was identified as crucial for both prion curing and thermotolerance development. The specific amino acid substitution D184S results in a mutant protein that retains nearly normal thermotolerance but lacks prion curing ability in the presence of GuHCl (Jung et al. 2002).
The influence of increasing concentrations of GuHCl on the efficiency of Hsp104-, Ssa1p-, and Ydj1p-dependent reactivation of urea-denatured firefly luciferase was also monitored. GuHCl efficiently inhibited the disaggregation of luciferase; 2 mM GuHCl nearly abolished luciferase disaggregation (Fig. 2c). Similar reactivation experiments performed with Hsp104 (D184S) showed that the mutant protein possesses a substantially lower disaggregation activity compared to wild type. However, in contrast to wild-type Hsp104, the activity of the mutant protein was hardly inhibited by 2 mM GuHCl (Fig. 2d). Only at high GuHCl concentration (10 mM), the efficiency of luciferase disaggregation was inhibited twofold (Fig. 2b). As a consequence, in the presence of 5 mM GuHCl, the ability of Hsp104 (D184S) to disaggregate and refold luciferase was substantially higher than that of wild-type Hsp104, which was efficiently inhibited under these conditions (Fig. 2e). Our in vitro analysis of Hsp104 and Hsp104 (D184S) activities is in good agreement with the in vivo thermotolerance assay showing that in the presence of GuHCl, the (D184S) mutant, but not the wild-type Hsp104, retains thermotolerance.
Partial trypsin digestion was used to further examine the protein conformation, since it was previously reported that changes in a protein’s digestion pattern occur in response to protein oligomerization (Rudyak et al. 2001). Accordingly, we observed that in the presence of ATP, the addition of GuHCl inhibits the proteolytic cleavage of Hsp104 (Fig. 4b). Not only was the disappearance of major protein bands restricted but also the patterns of digestion products were also different in the presence of GuHCl. This suggests that in the presence of GuHCl, the structure of Hsp104 changes in such a way that the protein becomes more resistant to trypsin digestion. Under the same experimental conditions, the presence of GuHCl had no effect on either the pattern or the rate of trypsin cleavage for Hsp104 (D184S) (Fig. 4b). It is worth noting that digestion of the Hsp104 (D184S) mutant by trypsin was substantially slower compared to wild-type Hsp104.
Taken together, this data suggests that the aspartic acid residue in position 184 in Hsp104 is involved in the interaction with guanidine, which presence severely inhibits both ATPase and disaggregation activities and promotes hexamerization of wild-type Hsp104. Mutation D184S results in a protein possessing lower ATPase and disaggregation activities. However, these activities are only weakly influenced by GuHCl. The oligomeric state of Hsp104 (D184S) is shifted towards hexamers, and the presence of GuHCl does not further influence its oligomeric status.
GuHCl efficiently stimulates ClpB’s ATPase activity but inhibits its disaggregating activity
Our in vitro experiments indicated that the presence of GuHCl influences the rate but not the total amount of ClpB-mediated disaggregation. To test possible in vivo effects, we monitored E. coli thermotolerance in the presence of GuHCl. No phenotype was observed. However, when we monitored the removal of aggregates formed in bacterial cells following heat shock, we did observe a mild in vivo effect on the disappearance of temperature-induced intracellular aggregates (Supplementary Fig. S3). This delay is consistent with our in vitro results showing an impaired rate of ClpB-dependent disaggregation.
Since the aspartic acid in position 184 in Hsp104 was identified as crucial for thermotolerance development and interaction with GuHCl, and is found in a highly conserved region, we decided to introduce an analogous mutation in ClpB. The resultant protein, ClpB (D178S), was purified and compared with the wild-type ClpB. In the absence of GuHCl, the ATPase activity of this mutant is similar to that of wild-type ClpB (Fig. 6d). Yet, in contrast to wild-type ClpB, no increase in the ATP hydrolysis rate was observed in the presence of GuHCl (Fig. 6d). Next, we analyzed the ability of ClpB (D178S) to disaggregate aggregated luciferase. Like the analogous Hsp104 (D184S) mutant, the disaggregating activity of ClpB (D178S) is lower than that of wild-type ClpB (compare Fig. 6b, e). Moreover, its disaggregating activity is not influenced by the presence of GuHCl (Fig. 6e). In our sedimentation experiments, both ClpB and its D178 mutant were found in the fractions characteristic for a monomer, and GuHCl did not influence their sedimentation profiles (Supplementary Fig. S4).
However, partial trypsin digestion showed that addition of GuHCl to the sample containing ClpB and ATP inhibited the proteolytic cleavage of ClpB (Supplementary Fig. S5). Under the same experimental conditions, the presence of GuHCl had no effect on the rate of trypsin cleavage for ClpB (D178S). Similarly to Hsp104 (D184S), the ClpB (D178S) mutant was digested substantially slower by trypsin as compared to wild-type protein (Supplementary Fig. S5). These experiments show that GuHCl does not influence an oligomeric status of ClpB as efficiently as it does in case of Hsp104. However, the partial trypsin digestion approach suggests that GuHCl does have some effect on wild-type ClpB but not the D178S mutant.
GuHCl inhibits the Hsp100-dependent step of disaggregation
The process of protein disaggregation is not solely dependent on Hsp100 chaperones in that it requires the specific cooperative action of Hsp100 and Hsp70 chaperones (Glover and Lindquist 1998; Krzewska et al. 2001b). Therefore, we made an effort to test more directly whether it is indeed only Hsp100, and not Hsp70, which is inhibited by GuHCl. We first analyzed the influence of GuHCl on Hsp104-dependent disaggregation of a substrate other than firefly luciferase, namely GFP protein. GFP has been successfully used to analyze the disaggregation activity of the bacterial ClpB-DnaK, DnaJ, and GrpE chaperone system (Ziętkiewicz et al. 2004). As expected, the addition of yeast chaperones Hsp104, Ssa1p, and Ydj1p to aggregated GFP resulted in its reactivation, monitored in real time as an increase in GFP fluorescence (Fig. 7A). The disaggregation process was efficiently blocked by the presence of 5 mM GuHCl in the reaction mixture (Fig. 7A). As stated above, inhibition of the GFP disaggregation rate by the bacterial chaperones was also observed in the presence of 5 mM GuHCl (Fig. 7B and Supplementary Fig. S2A). Because of these observations using two different substrates in two different systems, we continued to test whether GuHCl specifically influences the Hsp100 or the Hsp70 component of the bacterial and yeast chaperone disaggregating systems.
We took advantage of the fact that the Hsp70 system works upstream of Hsp100 (Ziętkiewicz et al. 2004; Weibezahn et al. 2004) and is able to disentangle single polypeptides from aggregates on its own (Ziętkiewicz et al. 2006). In the absence of Hsp100, the released polypeptides do not fold properly into their native structures and re-aggregate, forming a new class of aggregates characterized by a smaller size and a significantly lower sedimentation coefficient (Ziętkiewicz et al. 2006). These observations allowed us to investigate the influence of GuHCl on the specific disaggregation steps. Sedimentation conditions (40,000 rpm, SW 60 Beckman rotor; 2 h; 4°C) were chosen in such a way that following centrifugation, native GFP remained at the top of the gradient (fractions 1–3; Fig. 7C, subpanel c); the large aggregates of GFP formed a pellet at the bottom of the tube (Fig. 7C, subpanel c), but GFP aggregates remodeled by the KJE chaperone system sedimented to the middle of the gradient (fractions 5–9; Fig. 7C, subpanel a). The yeast Hsp70 chaperone system (Ssa1p/Ydj1p) was also capable of efficiently remodeling GFP aggregates, and the reaction produced analogous, small aggregates sedimenting in the middle of the gradient (fractions 4–12; Fig. 7C, subpanel b). When bacterial (KJE) or yeast (Ssa1p/Ydj1p) Hsp70 chaperone systems were supplemented with ClpB or Hsp104, respectively, GFP was found at the top of the gradients in the position characteristic for native GFP (Fig. 7C). These top fractions exhibited green fluorescence (result not shown), indicating that the action of the Hsp70-Hsp100 chaperones resulted in disaggregation and efficient reactivation of GFP. When the complete chaperone reactions, bacterial KJE-ClpB and yeast Ssa1p/Ydj1p-Hsp104, were supplemented with 5 mM GuHCl, the formation of fluorescent GFP refolded by the bacterial system was significantly inhibited (Fig. 7C, subpanel a) and essentially blocked in the case of the yeast system (Fig. 7C, subpanel b). However, it is important to note that under these conditions, the initial large aggregates of GFP, which typically form a pellet at the bottom of the tube, were efficiently converted by bacterial chaperones to small aggregates that sediment in the middle of the gradient (Fig. 7C, subpanel a). Similar, albeit slightly less efficient conversion, was also observed for yeast chaperones (Fig. 7C, subpanel b). These observations suggest that the activity of the bacterial (KJE) and yeast (Ssa1p/Ydj1p) Hsp70 chaperone systems was not affected by the presence of GuHCl. Since the hydrolysis of ATP was shown to be required for Hsp70-dependent remodeling of aggregates (Ziętkiewicz et al. 2004), this observation is also consistent with the lack of influence of GuHCl on the ATPase activity of both the KJE and Ssa1p/Ydj1p systems (Supplementary Table S1). Therefore, we conclude that GuHCl affects the Hsp100-dependent step in the disaggregation process of both bacterial and yeast chaperones.
In this study, we analyzed the influence of guanidine hydrochloride on the abilities of two homologous bi-chaperone systems, bacterial KJE-ClpB and yeast Ssa1/Ydj1-Hsp104, to disaggregate protein substrates. Our results clearly show that guanidine inhibits the disaggregating activity of both bi-chaperone systems, albeit to different degrees. Disaggregation in the yeast system is blocked completely, whereas the effect is less pronounced in the bacterial system. We observed a two- to three-fold inhibition in the rate of disaggregation mediated by bacterial chaperones, but the efficiency was unaffected. Our attempts to observe the effect of GuHCl on E. coli thermotolerance were unsuccessful (results not shown), although we did observe a somewhat minor in vivo effect on the disappearance of temperature-induced intracellular aggregates. This delay in removal of aggregated proteins is consistent with our in vitro results showing an impaired rate of disaggregation. The inhibitory effect of GuHCl on bacterial chaperones has not been described previously either in vivo or in vitro.
For both Hsp104 and ClpB, the change of the conserved aspartic acid (D184 and D178, respectively) to serine resulted in the formation of protein with lower disaggregating activity, yet in both cases, the activities of the respective mutant proteins did not change in response to GuHCl, unlike their wild-type counterparts. Analysis of the structural models of Hsp104 and ClpB (Lee et al. 2003, 2010; Ziętkiewicz et al. 2010) shows that D184 and D178, respectively, are located in the middle of NBD1 domain, in close proximity to the first ATP binding site and not at the interface between adjacent subunits. Therefore, it is unlikely that the observed effect is due to the direct interaction of GuHCl with the amino acids at the subunit interface, which in turn influences the oligomerization of the chaperones. Collective data suggest that D184 in Hsp104 is involved in the interaction with positively charged guanidine. Lack of negative charge at this position in the D184S mutant results in the lack of interaction between Hsp104 and guanidine. This mutant possesses substantially lower ATPase and disaggregating activities in vitro, yet in vivo, the observed phenotype is almost normal. This suggests that a certain robustness in the functioning of the Hsp104 protein in the chaperone network exists. It is worth mentioning that both Hsp104 and Hsp104 (D184S) were expressed in yeast cells at the same level (Supplementary Fig. S6), and three independent Hsp104 (D184S) purifications resulted in a similarly active chaperone protein. An analogous robustness in ClpB functions also exists in E. coli since the observed two- to three-fold guanidine-dependent inhibition of ClpB activity in the presence of GuHCl does not result in an observable phenotypic effect.
Our results clearly assign the guanidine-dependent inhibition of the Hsp70/Hsp100 bi-chaperone system to the Hsp100-dependent disaggregation step. This is based on the observations that in both bacterial and yeast systems, neither Hsp70’s ATPase activity nor its ability to remodel protein substrate aggregates was disturbed by the presence of GuHCl. Thus, at which point(s) in the Hsp100-dependent step does GuHCl influence disaggregation? And what is the difference(s) between the two studied systems? Guanidine affects the ATPase activities of the bacterial and yeast Hsp100 chaperones oppositely, stimulating ClpB and inhibiting Hsp104. Yet, in both cases, disaggregation activity is inhibited. These results suggest that there is no simple correlation between the rate of ATP hydrolysis by Hsp100 chaperones and their ability to disaggregate proteins. Recently, it was shown that restrained ATP hydrolysis promoted substrate remodeling activity of ClpB and Hsp104, most probably by inducing the balance between substrate holding and unfolding (Doyle et al. 2007).
In contrast to its opposite effect on ATPase activities, GuHCl seems to have a stimulatory effect on the oligomerization of both ClpB and Hsp104. The effect is much more pronounced for Hsp104 since stable hexameric structures were easily detected by us and others (Grimminger et al. 2004) in sedimentation analysis. The effect for ClpB was observed only using trypsin digestion approach suggesting the transient stabilization of the hexameric structure. Interestingly, inhibition of the disaggregation reaction correlated with the increased stability of the oligomeric chaperone structures. One can speculate that during the disaggregation process, Hsp100 oligomerization and deoligomerization events must take place, and thus, the stabilization of Hsp100 oligomers does not necessarily lead to an increase in the overall efficiency (or rate) of the disaggregation reaction. On the other hand, it has to be remembered that results obtained for ClpX show that a stable hexameric variant of this chaperone is fully active in a translocation assay (Martin et al. 2005).
We thank Dr. Dariusz Wyrzykowski for his help with ITC experiments. We thank Drs. Szymon Ziętkiewicz, Debbie Ang, and Wojciech Sawula for discussions and critical reading of the manuscript. This work was supported by the Polish Ministry of Science and Higher Education grant No. N301 507338. E.M. and N.L. were supported by TEAM/2009/-3/5 program.
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