Mechanism of Hsp104/Clpb Inhibition by Prion Curing Guanidinium Hydrochloride ⇑ ⇑ Eva Kummer, Yuki Oguchi 1, Fabian Seyffer 1, Bernd Bukau , Axel Mogk

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FEBS Letters 587 (2013) 810–817

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Mechanism of Hsp104/ClpB inhibition by prion curing Guanidinium hydrochloride ⇑ ⇑ Eva Kummer, Yuki Oguchi 1, Fabian Seyffer 1, Bernd Bukau , Axel Mogk

Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH) and Deutsches Krebsforschungszentrum (DKFZ), DKFZ–ZMBH Alliance, Im Neuenheimer Feld 282, Heidelberg D-69120, Germany

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Article history: The Saccharomyces cerevisiae AAA+ protein Hsp104 and its Escherichia coli counterpart ClpB cooper- Received 24 December 2012 ate with Hsp70 chaperones to refold aggregated proteins and fragment prion fibrils. Hsp104/ClpB Revised 23 January 2013 activity is regulated by interaction of the M-domain with the first ATPase domain (AAA-1), control- Accepted 5 February 2013 ling ATP turnover and Hsp70 cooperation. Guanidinium hydrochloride (GdnHCl) inhibits Hsp104/ Available online 14 February 2013 ClpB activity, leading to prion curing. We show that GdnHCl binding exerts dual effects on Edited by Jesus Avila Hsp104/ClpB. First, GdnHCl strengthens M-domain/AAA-1 interaction, stabilizing Hsp104/ClpB in a repressed conformation and abrogating Hsp70 cooperation. Second, GdnHCl inhibits continuous ATP turnover by AAA-1. These findings provide the mechanistic basis for prion curing by GdnHCl. Keywords: AAA protein Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Protein disaggregation Prion Hsp104 ClpB Hsp70

1. Introduction and a middle (M) domain that is inserted into AAA-1 [12,13]. ATP- ase and threading activities of Hsp104/ClpB are controlled by the Severe protein folding stress may exceed the refolding or controlled interaction of M domain with AAA-1. M-domain binding degrading capacity of the cellular protein quality control system to AAA-1 keeps Hsp104/ClpB in a repressed, inactive state, whereas and result in protein aggregation, which is a major threat for cellu- M-domain detachment causes derepression and allows for further lar homeostasis. Aggregated proteins can be reactivated in vivo activation by substrate [14,15]. Hsp70 stabilizes Hsp104/ClpB in a from bacteria to human in a process involving molecular chaper- derepressed state, leading to activation of the AAA+ partner at the ones [1]. In fungi and bacteria a bi-chaperone system consisting surface of protein aggregates [16]. Activation enables Hsp104/ClpB of Hsp104 (Saccharomyces cerevisiae) or ClpB (Escherichia coli) and to thread single aggregated polypeptides trough their central a cooperating Hsp70 chaperone system refolds proteins from an translocation channel in an ATP-driven reaction [8,17]. aggregated state [2–5]. This activity enables cellular survival of In addition to its role in solubilizing stress-induced protein stress conditions, which otherwise would be lethal, a phenomenon aggregates, Hsp104 is also required for the inheritance of several referred to as thermotolerance [6,7]. The disaggregation mecha- protein-based genetic elements (prions), including [PSI+], which nism involves an essential activity of the Hsp70 system during result from conversion of soluble Sup35 into ordered amyloid-like the initial phases of the process [8,9]. The binding of Hsp70 to aggregates [18]. Hsp104 fragments prion ﬁbrils, generating propa- aggregates is a prerequisite for Hsp104/ClpB interaction with gons, which act as templates to incorporate soluble Sup35 mono- aggregated proteins and allows for substrate delivery to ring- mers to ﬁbril ends [19,20]. shaped, hexameric Hsp104/ClpB [10,11]. Low concentrations (3–5 mM) of Guanidinium hydrochloride Hsp104/ClpB are members of the AAA+ protein family and con- (GdnHCl) represent a tool of particular importance in prion re- sist of two ATPase domains (AAA-1, AAA-2), an N-terminal domain search and are used to cure prions by reversibly inhibiting Hsp104 activity. The isolation of a GdnHCl-resistant Hsp104 variant demonstrates that Hsp104 is the main chaperone target of GdnHCl in vivo [21]. Despite its extensive use in prion research, lit- ⇑ Corresponding authors. Fax: +49 6221 545894. tle is known about the mechanism by which GdnHCl inhibits E-mail addresses: [email protected] (B. Bukau), a.mogk@zmbh. Hsp104. GdnHCl reduces Hsp104 ATPase activity at high ATP con- uni-heidelberg.de (A. Mogk). 1 These authors contributed equally to this work. centrations, indicating that it disturbs the Hsp104 ATPase cycle in,

0014-5793/$36.00 Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2013.02.011 E. Kummer et al. / FEBS Letters 587 (2013) 810–817 811 however, unknown manner [22,23]. GdnHCl also inhibits ClpB 3. Results and discussion chaperone activity but leads to increased ATPase activity [23]. These unexplained differences underline the need for further 3.1. GdnHCl keeps Hsp104 in a repressed state and abrogates Hsp70 mechanistic analysis of GdnHCl-mediated inhibition of Hsp104/ cooperation ClpB activity. Here, we set out to determine the conformational changes within Hsp104 and ClpB induced by GdnHCl binding and We analyzed the conformational changes in Hsp104 induced by the consequences on ATP turnover by the two ATPase domains. GdnHCl binding using time-resolved amide hydrogen exchange Our findings indicate that GdnHCl exerts the same inhibitory ef- (HX), which determines solvent accessibility and structural flexi- fects on Hsp104 and ClpB by abrogating Hsp70 cooperation and bility of amide hydrogens [25]. We recently applied HX to analyze altering ATPase cycles. the conformational dynamics of ClpB, demonstrating that this technique faithfully reports on ATP-driven structural changes 2. Materials and methods [14]. Hsp104 conformation was analyzed in presence of ATPcS, since GdnHCl binding is nucleotide-dependent [22]. HX analysis 2.1. Plasmids and proteins was performed in absence and presence of 20 mM GdnHCl, which caused complete inhibition of Hsp104-dependent reactivation of Mutant derivatives of hsp104/clpVH/clpB/clpVB were generated aggregated Luciferase (Suppl. Fig. 1A). GdnHCl binding resulted by PCR mutagenesis in pET24a and pDS56 and were verified by in multiple minor changes in HX protection throughout Hsp104. sequencing. Hsp104 and ClpB variants used in this study are sum- A pronounced difference in deuteron incorporation (DD > 10%) marized in Supplementary Table 1. Wild type and mutant was, however, only noticed for a few peptides (Fig. 1A). These com- Hsp104/ClpB, ClpV, DnaK, DnaJ, GrpE, Ssa1, Ydj1, Sse1, VipA/VipB prise the Walker A motif of AAA-1 (I211—E223), M-domain motif 2 and Luciferase were purified as previously described [16]. Protein (T477-E527) and a segment located next to the substrate process- concentrations were determined with the Bradford assay from ing pore2 loop (L646-G661), which were all protected from HX by Bio-Rad. GdnHCl. We next analyzed the structural changes induced in ClpB by 2.2. Biochemical assays 20 mM GdnHCl (Suppl. Fig. 1B), which reduced the ClpB-dependent reactivation of aggregated Luciferase (Suppl. Fig. 1C). GdnHCl HX-MS experiments were performed as described earlier [14]. caused minor deprotection of various peptides distributed Hsp104/ClpB peptides were analyzed on an electrospray ionization throughout both the protein. Similar to Hsp104, protection from quadrupole time-of-flight mass spectrometer (QSTAR Pulsar; Ap- HX was only determined for ClpB M-domain motif 2 and for a pep- plied Biosystems) as described [14]. ATPase and chaperone activi- tide encompassing the AAA-1 Walker A motif (I205-G218) (Suppl. ties were determined in Reaction buffer A (50 mM Hepes, pH 7.5, Fig. 1B). Thus, Hsp104 and ClpB share conserved structural changes

10 mM MgCl2, 50 mM KCl, 2 mM DTT) for Hsp104 and in Reaction induced by GdnHCl. buffer B (50 mM Tris pH 7.5, 150 mM KCl, 10 mM MgCl2,2mM GdnHCl-induced hyperprotection of the Hsp104/ClpB AAA-1 DTT) for ClpB as described [16]. Activities were calculated based Walker A segment and M-domain motif 2 from HX exchange is on at least two independent experiments. Single turnover ATPase reminiscent of structural changes observed for hyper-repressed measurements were performed using 50 lM Hsp104 and 25 lM ClpB M-domain variants, which exhibit a stabilized interaction be- ATP/[a32P]-ATP. Nucleotides were separated by thin layer chroma- tween M-domain motif 2 and AAA-1 [14]. The effect of GdnHCl on tography and ATPase rates were determined as described [24]. Pro- M-domain/AAA-1 interaction can be explained by the position of tein disaggregation activities of Hsp104/ClpB were determined in the GdnHCl binding site. The conserved aspartate residue 184 of presence of cognate Hsp70 chaperone systems and were calculated Hsp104 (D178 in ClpB) is crucial for the interaction with positively based on initial refolding rates of aggregated firefly Luciferase as charged GdnHCl, as a D184S variant is resistant towards GdnHCl described [14]. Chaperones were used at the following concentra- inhibition in vivo and does no longer bind GdnHCl in vitro tions: 1 lM ClpB/Hsp104 (wild type or derivatives); 1 lM DnaK, [21,23]. D184 is located close to M-domain motif 2 and the first 0.2 lM DnaJ, 0.1 lM GrpE and 2 lM Ssa1, 1 lM Ydj1, 0,1 lM Sse1. a-helix of the C-terminal AAA-1 subdomain (V345-H362), which Disassembly of VipA/VipB tubules (0.5 lM) by 0.25 lM ClpVH, makes crucial electrostatic interactions with motif 2 (Fig. 1B) ClpVB or ClpV and variants was analyzed by monitoring the de- [14,15]. GdnHCl binding might therefore either directly stabilize crease of turbidity as described [16]. Unfolding of Casein–YFP M-domain motif 2 attachment or indirectly, via strengthening (0.3 lM) by 1 lM Hsp104 was followed by monitoring YFP fluores- AAA-1/motif 2 interaction. cence as described [14]. The bacterial Hsp70, DnaK, interacts with a detached M-domain motif 2 of ClpB [16]. Repressed ClpB variants mutated in the M-do- 2.3. Fluorescence microscopy main are therefore affected in DnaK interaction and show a defect in DnaK-dependent targeting to heat stress-induced protein aggre- Yeast BY4741 cells coexpressing yEmCitrine–Luciferase and gates [16]. We therefore monitored the targeting of functional Hsp104-CFP were grown to mid exponential growth phase at Hsp104-CFP to heat-shock induced protein aggregates in S. cerevi- 30 °C, further incubated at 37 °C for 45 min and heat-shocked to siae cells in absence and presence of 5 mM GdnHCl (Fig. 1C). Pro- 45 °C for 20 min. Cells were resuspended in PBS buffer, immobi- tein aggregates were visualized by co-expressing thermolabile lized on 1% agarose pads, sealed with grease, covered with cover- mCitrine–Luciferase, which forms multiple foci upon temperature slips and imaged using PerkinElmer UltraView ERS Spinning Disk up-shift to 45 °C. Co-localization of Hsp104-CFP and mCitrine– Microscope equipped with a Plan-APOCHROMAT 100Â/1.4 Oil Luciferase aggregates was abrogated in presence of GdnHCl, which DIC objective. CFP and Citrine were excited at 440 nm and correlated with a substantial reduction in Luciferase reactivation 514 nm, respectively. 3D imaging was performed with 0.2 lm step in vivo (Fig. 1C, Suppl. Fig. 1D). Respective experiments could not size. Confocal settings were chosen to allow for clear spectral sep- be conducted in E. coli cells, since ClpB-dependent reactivation of aration of the simultaneously visualized fluorophore pair and cor- aggregated Luciferase was not affected at 30 mM GdnHCl in vivo, responding planes for both channels and the merged images are probably due to inefficient GdnHCl uptake (data not shown). To- shown. gether these findings indicate that GdnHCl inhibits Hsp104/ClpB 812 E. Kummer et al. / FEBS Letters 587 (2013) 810–817

A 10 5 0 -5 -10 -15 -20 211-223 211-223 211-243 611-623 508.527 166-174 174-190 244-248 249-261 262-271 278-282 283-299 300-310 321-330 332-352 351-372 401-414 415-423 424-431 431-447 477-493 508-520 537-549 546-553 551-569 579-586 594-610 624-631 632-638 647-661 663-670 671-683 675-683 684-696 700-717 722-729 726-733 733-748 745-753 794-806 813-831 832-837 843-856 877-898 878-902 446-455 646-655 769-775 773-785 857-875

R356 (R358) E480 (D484) ATP D178 (D184) E355 (E357) AAA-1

E358 (E360) K476

(K480) M domain

C Citrine- Citrine- Hsp104-CFP Luciferase merge Hsp104-CFP Luciferase merge

0 mM

5 mM

Fig. 1. GdnHCl prevents Hsp104-Hsp70 cooperation. (A) Changes in structural dynamics of Hsp104 upon GdnHCl binding. Difference in deuteron (D) incorporation after 60 s between ATPcS-bound Hsp104 in absence and presence of 20 mM GdnHCl at peptic peptide level. An Hsp104 domain organization is given. (B) Detail of E. coli ClpB AAA-1 and M-domain motif 2. Positions of D178 (D184), E355 (E357), R356 (K358), E358 (E360), K476 (K480) and E480 (D484), which form a saltbridge network, are indicated. Hsp104 numbering is given in brackets. Bound ATP molecule is shown in green. (C) Saccharomyces cerevisiae cells expressing mCitrine–Luciferase and Hsp104-CFP were grown at 30 °C in absence and presence of GdnHCl (5 mM), shifted to 37 °C for 45 min and heat-shocked at 45 °C for 20 min. Co-localization of aggregated mCitrine–Luciferase and Hsp104-CFP foci was monitored. Scale bar: 4 lm. by keeping the proteins in a repressed conformation, thereby tained higher activity in presence of 5 mM GdnHCl compared to inhibiting Hsp70 cooperation. Hsp104 wild type (wt), however, the variant was still inactivated at higher GdnHCl concentrations (Fig. 2A). Similarly, derepressed 3.2. GdnHCl can inhibit Hsp104 independent from M-domain ClpB-K476C remained GdnHCl-sensitive, although the variant re- conformation tained higher activity in presence of GdnHCl compared to Hsp104-K480C (Suppl. Fig. 2A). We analyzed whether binding of Derepressed ClpB variants with mutated M-domain (e.g. ClpB- GdnHCl could restore a stable interaction of AAA-1 with M-domain K476C) exhibit strong deprotection of M-domain motif 2 in HX motif 2 in Hsp104-K480C and determined the HX proﬁle of the experiments, caused by detachment of motif 2 from AAA-1 [14]. derepressed variant in absence and presence of 20 mM GdnHCl We speculated that such conformational changes might counteract (Fig. 2B and C). The derepressed conformational state of Hsp104- GdnHCl inhibition (which stabilize AAA-1/motif 2 interaction) and K480C was conﬁrmed by determining strong deprotection of the tested for sensitivity of the respective Hsp104-K480C variant to- M-domain in HX compared to Hsp104-wt (Fig. 2B). Surprisingly, wards GdnHCl during protein disaggregation. Hsp104-K480C re- the M-domain protection pattern remained almost unchanged in E. Kummer et al. / FEBS Letters 587 (2013) 810–817 813

Fig. 2. Derepressed Hsp104 remains GdnHCl-sensitive. (A) Luciferase disaggregation activities of Hsp104-wt and Hsp104-K480C in presence of Ssa1, Ydj1 and Sse1 were determined in absence and presence of increasing GdnHCl concentrations. The activity of Hsp104-wt in absence of GdnHCl was set as 100%. (B and C) Difference in deuteron incorporation (D) after 60 s at peptic peptide level between ATPcS-bound Hsp104-K480C and Hsp104-wt, (B) and Hsp104-K480C in absence and presence of 20 mM GdnHCl (C). presence of 20 mM GdnHCl (Fig. 2C). Similarly, the M-domain proof Hsp104/ClpB is replaced by the N-domain of the AAA + family tection pattern of ClpB-K476C remained unchanged in presence of member Vibrio cholerae ClpV. ClpVH/ClpVB bind in an Hsp70-inde- GdnHCl (Suppl. Fig. 2B). We instead noticed a variety of changes in pendent manner to tubular VipA/VipB polymers and disassemble Hsp104-K480C HX protection, which was most pronounced the tubules under ATP consumption [16]. VipA/VipB tubule disas- (DD > 10%) for a peptide encompassing F244-L248, which is lo- sembly by ClpV was not affected by GdnHCl, excluding any unspe- cated next to the substrate-processing pore1 loop (Suppl. ciﬁc inhibitory effects of GdnHCl in this experimental setup (Suppl. Fig. 2C). Hsp104-K480C sensitivity towards GdnHCl is therefore Fig. 3). linked to subtle structural changes in both AAA domains but not ClpVH and ClpVB exerted a limited VipA/VipB disassembly in the M-domain. These ﬁnding indicate that GdnHCl must have activity, which was sensitive towards GdnHCl, demonstrating that additional, M-domain independent consequences on Hsp104/ClpB GdnHCl inhibits Hsp104/ClpB also independent from Hsp70 coop- structure and activity. eration (Fig. 3A). To test whether GdnHCl also inhibits Hsp104/ To test whether GdnHCl can inhibit Hsp104/ClpB chaperone ClpB independent from M-domain conformation, we employed activity independent from M-domain conformation and the the ClpVB-Y520D variant, which is hyperactive in VipA/VipB disas- Hsp70 partner, we employed hybrid variants of Hsp104 and ClpB, sembly due to permanent detachment of M-domain motif 2 from ClpVH and ClpVB, respectively, in which the N-terminal domain AAA-1 and ClpVB-DM, which lacks the entire M-domain [14,16]. 814 E. Kummer et al. / FEBS Letters 587 (2013) 810–817

A ClpVH ClpVB 120 120 100 100 0 mM GdnHCl 80 80 60 60 5 mM GdnHCl 40 40

Turbidity (%) Turbidity 20 (%) Turbidity 20 30 mM GdnHCl 0 0 0 15 30 45 60 0 15 30 45 60 Time (min) Time (min)

B ClpVB-Y520D ClpVB- M 120 120 100 100 80 80 0 mM GdnHCl

60 60 5 mM GdnHCl 40 40 Turbidity (%) Turbidity 20 (%) Turbidity 20 30 mM GdnHCl 0 0 0 15 30 45 60 0 15 30 45 60 Time (min) Time (min)

Fig. 3. GdnHCl can inhibit Hsp104/ClpB activity independent from Hsp70 cooperation. (A) Disassembly of VipA/VipB complexes by ClpVH (left) and ClpVB (right) in absence and presence of GdnHCl. (B) VipA/VipB disintegration by hyperactive ClpVB-Y520D (left) and ClpVB-DM (right) in absence and presence of GdnHCl. Initial VipA/VipB turbidity was set as 100%.

The high disassembly activities of both, ClpVB-Y520D, and ClpVB- whether ATPase activities of either AAA-1 or AAA-2 are particularly DM, were strongly reduced by GdnHCl (Fig. 3B). ClpVB-Y520D re- sensitive towards GdnHCl, we included Hsp104 variants that are tained minor disassembly activity at 30 mM GdnHCl concentra- deﬁcient in ATP hydrolysis at AAA-1 (E285A) or AAA-2 (E687A) tions, resembling partial luciferase disaggregation activity of due to mutations of Walker B motifs (Fig. 4A). The basal ATPase derepressed Hsp104/ClpB in presence of GdnHCl (Fig. 2A, Suppl. activity of Hsp104 was not perturbed by GdnHCl, in agreement Fig. 2A). In conclusion, GdnHCl can inhibit Hsp104/ClpB activity with previous studies, which noticed a twofold reduction of ATPase independent from M-domain docking states, suggesting that activity only at higher (10–15 mM) ATP concentrations (Fig. 4A) GdnHCl additionally affects the Hsp104/ClpB ATPase cycle. [22,23]. GdnHCl reduced substrate-stimulated ATP hydrolysis by 2.1-fold at 30 mM GdnHCl. Hsp104-E285A (which allows ATP 3.3. GdnHCl inhibits continuous ATP turnover in AAA-1 hydrolysis only at AAA-2) exhibited a strongly (7.6-fold) increased basal ATPase activity compared to Hsp104-wt, in agreement with We investigated the consequences of GdnHCl on Hsp104 ATP- previous ﬁndings [26,27]. Both, basal and substrate stimulated ase activity in steady-state turnover experiments. We determined ATPase activity of Hsp104-E285A were slightly increased (1.4-fold the basal and substrate (casein)-stimulated ATPase activity of and 2-fold, respectively) in presence of GdnHCl. In sharp contrast, Hsp104 in presence of 2 mM ATP, representing saturating concen- GdnHCl strongly reduced the ATPase activity of Hsp104-E687A, trations in disaggregation assays (data not shown) [26]. To analyze which only hydrolyzes ATP at AAA-1, in absence (fourfold) or pres-

35 casein 30 + casein 25

5 Relative ATPase activty (fold) ATPase Relative

0 0 5 30 0 5 30 0 5 30 0 5 30 mM GdnHCl Hsp104-wt Hsp104-E285A Hsp104-E687A Hsp104-K480C

Fig. 4. GdnHCl inhibits continuous ATPase turnover in AAA-1. ATPase activities of Hsp104-wt, Walker B-mutants (E285A, E687A) and derepressed Hsp104-K480C were determined in absence and presence of casein and GdnHCl. The basal ATPase activity of Hsp104 was set as 1. E. Kummer et al. / FEBS Letters 587 (2013) 810–817 815

A ClpVH-E302A ClpVH-E704A 120 120 100 100 80 80 0 mM GdnHCl 60 60 5 mM GdnHCl 40 40 Turbidity (%) Turbidity Turbidity (%) Turbidity 20 20 30 mM GdnHCl 0 0 0 15 30 45 60 0 15 30 45 60 Time (min) Time (min)

B Hsp104-E285A Hsp104-K480C 1000 1000 800 800 600 600 MGdnHCl mM GdnHCl mM 400 0 mM 400 0 mM 5 mM 5 mM 200 200 20 mM 20 mM 0 0 YFP fluorescence (a.u.) YFP fluorescence (a.u.) YFP 0 20 40 60 0 15 30 45 60 Time (min) Time (min)

C 120

100

Refolding activity (%) 20

0 0 5 20 0 5 20 0 5 20 mM GdnHCl Hsp104-wt Hsp104-E285A Hsp104-E687A D 200 180 160 140 120 100 80 60

Refolding activity (%) 40 20 0 0 20 0 20 0 20 0 20 0 20 mM GdnHCl KJE ClpB-wt ClpB-K476C ClpB-E279A ClpB- K476C/ E279A

Fig. 5. GdnHCl-sensitivity of Hsp104-E285A depends on Hsp70-cooperation, (A) Disassembly of VipA/VipB complexes by ClpVH-E302A (left) and ClpVH-E704A (right) in absence and presence of GdnHCl. Initial VipA/VipB turbidity was set as 100%. (B) Unfolding of Casein–YFP by Hsp104-E285A (left) and Hsp104-K480C (right) in absence and presence of GdnHCl was followed by monitoring YFP fluorescence. (C and D) Luciferase disaggregation activities of Hsp104/ClpB-wt and variants were determined in presence of cognate Hsp70 chaperone systems in absence and presence of increasing GdnHCl concentrations. The activity of Hsp104/ClpB-wt was set as 100%. 816 E. Kummer et al. / FEBS Letters 587 (2013) 810–817 ence of casein (6.6-fold) (Fig. 4A). The same observation was made M-domain motif 2 interaction, thereby abrogating Hsp70 coopera- for the respective ClpB AAA-2 Walker B variant E678A (Suppl. tion. Second, it inhibits continuous ATP turnover at AAA-1 by Fig. 4). Thus irrespective of the different consequences of GdnHCl affecting a post-hydrolysis step. The AAA-1 Walker B mutant on the basal ATPase activities of Hsp104 and ClpB, the compound Hsp104-E285A variant is resistant towards GdnHCl in Hsp70-inde- inhibits continuous ATP turnover at AAA-1 in both proteins. The pendent activity assays, as ATP-loaded AAA-1 never exists in a impact of GdnHCl on ATP hydrolysis at AAA-1 can be explained (post hydrolysis) nucleotide state that is sensitive towards GdnHCl. by the location of its binding site (D178 in E. coli ClpB, D184 in S. To validate the dual inhibitory consequence of GdnHCl binding, we cerevisiae Hsp104), which is located close to ATP bound at AAA-1 determined the activity of AAA-1 mutant Hsp104-E285A in Hsp70- (Fig. 1B). dependent protein disaggregation and included AAA-2 mutant We also analyzed the consequences of GdnHCl binding on the Hsp104-E687A as control. Both Walker B variants exhibited partial ATPase activity of derepressed M-domain mutants Hsp104- protein disaggregation activity in presence of the Hsp70 system. K480C and ClpB-K476C (Fig. 4, Suppl. Fig. 4). Hyperstimulation of Reactivation of aggregated Luciferase by both Hsp104 mutants derepressed Hsp104-K480C/ClpB-K476C ATPase activities by case- was reduced in presence of 5 mM GdnHCl, demonstrating that in was abrogated by GdnHCl, explaining why the variants remain Hsp104-E285A remains GdnHCl-sensitive in Hsp70-dependent sensitive to GdnHCl. disaggregation (Fig. 5C). Similar results were obtained for respec- We next performed single ATP turnover experiments to analyze tive ClpB Walker B mutants (Suppl. Fig. 8). These findings also whether GdnHCl is directly inhibiting ATP hydrolysis at AAA-1 or is demonstrate that blocking ATP hydrolysis at AAA-1 does not pre- affecting a different step of the ATPase cycle. GdnHCl had only a vent GdnHCl binding, which we further confirmed by ITC measure- minor effect on single rounds of ATP hydrolysis in Hsp104- ments employing ClpB-E285A/E687A (Suppl. Fig. 9). The opposing E687A, suggesting that GdnHCl affects a post-hydrolysis step of sensitivities of Hsp104 and ClpB Walker B variants towards the AAA-1 ATPase cycle (Suppl. Fig. 5). This observation is consis- GdnHCl in Hsp70 dependent (protein disaggregation) and indepen- tent with the noticed 2.5-fold increase in ADP affinity for dent (VipA/VipB disassembly, unfolding of Casein–YFP) activity as- GdnHCl-bound Hsp104 [22], suggesting that changes in ADP bind- says indicate that GdnHCl binding has dual inhibitory ing and release leads to inhibition of ATP hydrolysis at AAA-1 dur- consequences on Hsp104/ClpB activity. Consistently, we show that ing steady-state conditions. the double mutant ClpB-E287A/K476C is insensitive to GdnHCl in Hsp70-dependent reactivation of aggregated Luciferase (Fig. 5D). 3.4. Different GdnHCl sensitivities of Hsp104-E285A in Hsp70- ClpB-E287A/K476C harbors a detached M-domain motif 2 and can- independent and dependent substrate processing not hydrolyze ATP at AAA-1, thereby overcoming both inhibitory effects of GdnHCl. The varying sensitivities of Hsp104/ClpB Walker B mutant’s In summary, we demonstrate that GdnHCl inhibits Hsp104 and ATPase activities (in AAA-1 or AAA-2) towards GdnHCl let us to ClpB activity via the same mechanism. GdnHCl binding at AAA-1 analyze whether their chaperone activities are also differently af- prevents M-domain motif 2 detachment thereby abrogating fected by the compound. We employed the Hsp70-independent Hsp70 cooperation and inhibits continuous ATP turnover at AAA- VipA/VipB tubule disassembly assay and analyzed the activities 1. Both inhibitory effects can be explained by local conformational of respective ClpVH Walker B variants (E302A in AAA-1, E704A in changes at the GdnHCl binding site, which is located close to the AAA-2). ClpVH-E704A exerted a slightly increased disassembly ATP binding site of AAA-1 and to the docking site of the M-domain. activity compared to ClpVH-wt and was inhibited by GdnHCl (Fig. 5A), matching ATPase inhibition of Hsp104-E687A in presence Note added in proof of GdnHCl (Fig. 4A). Disassembly activity of ClpVH-E302A was strongly increased compared to ClpVH-wt (see Fig. 3A) and resisted While this article was in revision, Reinstein and coworkers re- the presence of even 30 mM GdnHCl (Fig. 5A). Similar results were ported that GdnHCl binding to AAA-1 modulates nucleotide bind- obtained for respective ClpVB-variants (Suppl. Fig. 6), underscoring ing properties. This findings provides the mechanistic rationale that blocking ATP hydrolysis at AAA-1 increases unfolding power for the observed inhibition of continuous but not single ATP turn- and causes GdnHCl resistance. The high disassembly activity of over in presence of GdnHCl [29]. ClpVH-E302A is reminiscent of the unleashed unfolding activity of Hsp104-E285A towards the fusion protein LA-EYFP, which har- Acknowledgements bors loosely folded human lactalbumin for Hsp104 recognition [26]. To further substantiate our findings we employed Casein– This work was supported by Grants from the Deutsche Fors- YFP as alternative Hsp70-independent substrate. In contrast to chungsgemeinschaft (BU617/17-1) to B.B. and A.M.F.S. and E.K. Hsp104-wt, which did not process the folded YFP moiety, AAA-1 were supported by the Hartmut-Hoffmann-Berling International mutant Hsp104-E285A but not AAA-2 mutant Hsp104-E687A Graduate School of Molecular and Cellular Biology (HBIGS). Y.O. caused a rapid decrease of YFP fluorescence, reflecting YFP unfold- was supported by a Humboldt fellowship. ing (Fig. 5B, Suppl. Fig. 7). The subsequent regain of YFP fluorescent represents YFP refolding, consistent with previous findings for Appendix A. Supplementary data unfolding of GFP-SsrA by the Hsp100 member ClpA [28]. Unfolding of Casein–YFP by Hsp104-E285A was still observed in presence of Supplementary data associated with this article can be found, in GdnHCl and occurred at a slightly faster rate (Fig. 5B). For compar- the online version, at http://dx.doi.org/10.1016/j.febslet.2013. ison, we tested for GdnHCl-sensitivity of Casein–YFP processing by 02.011. derepressed M-domain mutant Hsp104-K480C. YFP unfolding by Hsp104-K480C was faster and more efficient compared to References Hsp104-E285A, however, unfolding was inhibited by 5 mM GdnHCl (Fig. 5B). The differences in GdnHCl-sensitivities indicate [1] Tyedmers, J., Mogk, A. and Bukau, B. (2010) Cellular strategies for controlling that the mechanistic bases for hyperactivity of derepressed and protein aggregation. Nat. Rev. Mol. Cell Biol. 11, 777–788. ATPase deficient AAA-1 Walker B mutants are different. [2] Glover, J.R. and Lindquist, S. (1998) Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins. Cell 94, 73–82. Our results presented so far show that GdnHCl binding has two [3] Goloubinoff, P., Mogk, A., Peres Ben Zvi, A., Tomoyasu, T. and Bukau, B. (1999) functional consequences on Hsp104/ClpB. First, it stabilizes AAA-1/ Sequential mechanism of solubilization and refolding of stable protein E. Kummer et al. / FEBS Letters 587 (2013) 810–817 817

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