Roles of conserved arginines in ATP-binding domains of AAA+ chaperone ClpB from Thermus thermophilus Takashi Yamasaki 1 , Yosuke Nakazaki 1 , Masasuke Yoshida 2 and Yo-hei Watanabe 1 1 Department of Biology, Faculty of Science and Engineering, Konan University, Okamoto, Kobe, Japan 2 Department of Molecular Biosciences, Kyoto Sangyo University, Motoyama-Kamigamo, Japan Introduction The expanded superfamily of ATPases associated with diverse cellular activities (AAA+) are involved in a variety of cellular activities, including membrane fusion, DNA replication, protein degradation, and dis- aggregation. Members of the AAA+ family contain one or more highly conserved AAA+ modules, and Keywords AAA+; aggregation; arginine finger; chaperone; ClpB Correspondence Y-h. Watanabe, Department of Biology, Faculty of Science and Engineering, Konan University, Okamoto 8-9-1, Kobe 658-8501, Japan Fax ⁄ Tel: +81 78 435 2514 E-mail: ywatanab@center.konan-u.ac.jp (Received 7 March 2011, revised 30 April 2011, accepted 6 May 2011) doi:10.1111/j.1742-4658.2011.08167.x ClpB, a member of the expanded superfamily of ATPases associated with diverse cellular activities (AAA+), forms a ring-shaped hexamer and coop- erates with the DnaK chaperone system to reactivate aggregated proteins in an ATP-dependent manner. The ClpB protomer consists of an N-termi- nal domain, an AAA+ module (AAA-1), a middle domain, and a second AAA+ module (AAA-2). Each AAA+ module contains highly conserved WalkerA and WalkerB motifs, and two arginines (AAA-1) or one arginine (AAA-2). Here, we investigated the roles of these arginines (Arg322, Arg323, and Arg747) of ClpB from Thermus thermophilus in the ATPase cycle and chaperone function by alanine substitution. These mutations did not affect nucleotide binding, but did inhibit the hydrolysis of the bound ATP and slow the threading of the denatured protein through the central pore of the T. thermophilus ClpB ring, which severely impaired the chaper- one functions. Previously, it was demonstrated that ATP binding to the AAA-1 module induced motion of the middle domain and stabilized the ClpB hexamer. However, the arginine mutations of the AAA-1 module destabilized the ClpB hexamer, even though ATP-induced motion of the middle domain was not affected. These results indicated that the three argi- nines are crucial for ATP hydrolysis and chaperone activity, but not for ATP binding. In addition, the two arginines in AAA-1 and the ATP- induced motion of the middle domain independently contribute to the sta- bilization of the hexamer. Structured digital abstract l TClpB binds to TClpB by molecular sieving (View Interaction 1, 2) Abbreviations AAA, ATPase associated with diverse cellular activities; AAA+, expanded superfamily of ATPases associated with diverse cellular activities; ABD-F, 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide; AMP-PNP, adenosine 5¢-(b,c-imido)triphosphate; ATPcS, adenosine 5¢-O-(thiotriphosphate); FITC, fluorescein isothiocyanate; G6PDH, glucose-6-phosphate dehydrogenase; Mant-ADP, 2¢(3¢)-O-N¢-methylaniloyl- aminoadenosine-5¢-diphosphate; P1, position 1; P2, position 2; T BAP, Thermus thermophilus BAP; TCEP, tris-(2-carboxyethyl)phosphine hydrochloride; TClpA, Thermus thermophilus ClpA; TClpB, Thermus thermophilus ClpB; TClpP, Thermus thermophilus ClpP; T DnaJ, Thermus thermophilus DnaJ; TDnaK, Thermus thermophilus DnaK; TGrpE, Thermus thermophilus GrpE. FEBS Journal 278 (2011) 2395–2403 ª 2011 The Authors Journal compilation ª 2011 FEBS 2395 most of them form ring-shaped oligomers [1,2]. The AAA+ module consists of a RecA-like nucleotide- binding domain and an a-helical domain. The RecA- like domain contains the WalkerA motif (GXXGXGKT, where X is any amino acid), the WalkerB motif (hhhhDE, where h is a hydrophobic residue), and conserved arginines called the arginine fingers. The roles of arginine fingers have been explored in several AAA+ proteins [3–8]. The arginine finger is typically located in the subunit interface, and interacts with the c-phosphate of ATP bound to the adjacent subunit; it is thought to participate in cataly- sis by stabilizing the transition state during ATP hydrolysis. Some AAA+ modules have two potential arginine fingers at position 2 (P2) and position 1 (P1) from the N-terminus [3]. Whereas the P1 arginine is highly conserved in most AAA+ proteins, the P2 argi- nine is conserved only in members of subfamilies of the AAA+ family, ATPases associated with diverse cellular activities (AAA) family, and several of the Clp ⁄ Hsp100 family members. However, little is known about the differences between the roles of P1 and P2 arginines. The molecular chaperone ClpB ⁄ Hsp104 is a member of the Clp ⁄ Hsp100 subfamily of the AAA+ family, and is essential for the survival of bacteria and yeast during severe thermal stress [9,10]. ClpB ⁄ Hsp104 coop- erates with the DnaK ⁄ Hsp70 chaperone system in the solubilization and reactivation of aggregated proteins by utilizing ATP hydrolysis [11–17]. The ClpB ⁄ Hsp104 monomer consists of an N-terminal domain, two AAA+ modules (AAA-1 and AAA-2 from the N-ter- minus), and a middle domain (Fig. 1A) [18]. The N-terminal domain is a highly mobile globular a-heli- cal domain. The middle domain is an 85-A ˚ coiled-coil structure tethered to the a-helical domain of the AAA-1 module, and is only found in ClpB ⁄ Hsp104. Like other AAA+ proteins, ClpB ⁄ Hsp104 forms a ring-shaped hexamer, and its stability is influenced by salt and pro- tein concentrations, temperature, and bound nucleotide [4,19–24]. The AAA-1 and AAA-2 of ClpB ⁄ Hsp104 contain two conserved arginines (P1 and P2) and one con- served arginine (P1), respectively (Fig. 1B,C). The crys- tal structure of ClpB from Thermus thermophilus (TClpB) shows that Arg322 (P2 of AAA-1) and Arg747 (P1 of AAA-2) are directed towards the c-phosphate of adenosine 5 ¢-(b,c-imido)triphosphate (AMP-PNP) that is bound to the neighboring subunit, whereas Arg323 (P1 of AAA-1) faces away from it (Fig. 1B) [18]. Previously, investigation of the effects of alanine substitutions in Escherichia coli ClpB showed that the alanine mutation of the P1 arginine of AAA- 1, R332A, decreased the chaperone activity and desta- bilized the hexamer, and that the alanine mutation of the P1 arginine of AAA-2, R756A, decreased the AT- Pase and chaperone activities [4]. Recently, the effects of alanine substitution of the P2 arginine of AAA-1, Arg322, of TClpB on nucleotide binding and ATPase were also investigated [25]. However, the exact roles of these arginines in the individual AAA+ modules are unclear. Fig. 1. Positions of the conserved arginines of TClpB. (A) Structure of TClpB. The N-terminal domain (green), the first AAA+ module (AAA-1) (blue), the second AAA+ module (AAA-2) (red) and the middle domain (yellow) are shown. (B) Close-up views of the subunit interface of AAA-1. AMP-PNP is bound to the left subunit (blue), and conserved arginines, Arg322 (P2) (yellow) and Arg323 (P1) (magenta), of the right subunit (cyan) are shown as sticks. (C) The subunit interface of AAA-2. AMP-PNP is bound to the left subunit (red), and conserved arginines, Arg747 (green), of the right subunit (pink) are shown as sticks. Roles of conserved arginine of ClpB chaperone T. Yamasaki et al. 2396 FEBS Journal 278 (2011) 2395–2403 ª 2011 The Authors Journal compilation ª 2011 FEBS Disaggregation of proteins by ClpB requires stable hexamer formation and threading of aggregated sub- strate proteins through the central pore of ClpB [26]. The ATP-induced motion of the middle domain stabi- lizes the ClpB hexamer, and is important for the disag- gregation process [18,27]. However, the relationships between these processes and the conserved arginines have not been clarified. Here, we used TClpB to exam- ine the roles of the conserved arginines, Arg322 (P2) and Arg323 (P1) of AAA-1, and Ar g747 (P1) of AAA- 2, and determined that the arginines of TClpB are not involved in nucleotide binding but are crucial for ATP hydrolysis, substrate threading, and disaggregation. In addition, the arginines of AAA-1 are important for stabilization of the hexameric form, but not for the ATP-induced motion of the middle domain. Results The conserved arginines are not involved in nucleotide binding To examine the roles of conserved arginines, we gener- ated three mutants of TClpB: R322A [1R ⁄ A(P2)], R323A [1R ⁄ A(P1)], and R747A (2R⁄ A). In addition, we made double mutants by combining these arginine mutations in AAA-1 or AAA-2 and the WalkerA mutations, replacement of Lys-Thr with Ala-Ala, in AAA-2 (2KT ⁄ AA) or AAA-1 (1KT ⁄ AA), respectively. Previously, we reported that the 1KT ⁄ AA and 2KT ⁄ AA mutants could not bind nucleotide to the AAA-1 and AAA-2 modules, respectively [23]. Nucleo- tide binding to the domain with the arginine mutation was estimated by measuring nucleotide binding to the double mutants 1R ⁄ A(P1)–2KT ⁄ AA, 1R ⁄ A(P2)– 2KT ⁄ AA, and 1KT ⁄ AA–2R ⁄ A. The fluorescence inten- sity increased when 2¢(3¢)-O-N¢-methylaniloyl-aminoad- enosine-5¢-diphosphate (Mant-ADP) bound TClpB. The extent of the changes in fluorescence intensity at 440 nm that was induced by wild-type and mutant TClpB were plotted against the concentrations of Mant-ADP (Fig. 2), and the apparent K d values were calculated (Table 1). Increased fluorescence was observed in all double mutants, and the K d values were 14.5 lm [1R ⁄ A(P1)–2KT ⁄ AA], 25.8 lm [1R⁄ A(P2)– 2KT ⁄ AA], and 0.71 lm [1KT ⁄ AA–2R ⁄ A]. These K d values were similar to those of corresponding single WalkerA mutants: 2KT ⁄ AA (11.0 lm) and 1KT ⁄ AA (0.30 lm) (Table 1). We also measured the decreases in Mant-ADP fluorescence by adding Mg-ADP or Mg- ATP, and calculated the apparent K d values of ADP and ATP for the TClpB mutants (Table 1). The K d values of ADP and ATP for these double mutants were similar to those for the corresponding single Wal- kerA mutants. These results indicated that the three arginines are not involved in nucleotide binding to the corresponding AAA+ module. The conserved arginines are indispensable for ATP hydrolysis in the corresponding AAA+ module We next measured the ATPase activities of the argi- nine mutants of TClpB with or without 0.1 mg Æ mL )1 j-casein. At 55 °C, wild-type TClpB hydrolyzed ATP at a rate of approximately 60 min )1 , and the addition of j-casein stimulated the rate approximately 1.7-fold (Fig. 3A). The ATPase activities of 1R ⁄ A(P1) and 1R ⁄ A(P2) were approximately six-fold lower, and that of 2R ⁄ A was approximately 40-fold lower, than that of the wild type (Fig. 3A). However, for all mutants, j-casein significantly stimulated ATPase activity Fig. 2. Mant-ADP binding to the TClpB mutants. The increases in fluorescence after mixing the indicated concentrations of Mant- ADP with wild-type (open circles), 1KT ⁄ AA (open squares), 2KT ⁄ AA (open triangles), 1R ⁄ A(P1)–2KT ⁄ AA (filled circles), 1R ⁄ A(P2)– 2KT ⁄ AA (filled squares) and 1KT ⁄ AA-2R ⁄ A (filled triangles) TClpB were plotted against the Mant-ADP concentrations. Theoretical curves are also shown. Table 1. Dissociation constants of nucleotides for T ClpB mutants. Standard deviations are shown. TClpB K d for Mant-ADP (l M) K d for ADP (l M) K d for ATP (l M) Wild type 1.00 ± 0.09 8.08 ± 0.17 31.1 ± 1.7 a 1KT ⁄ AA 0.30 ± 0.15 4.36 ± 0.07 30.7 ± 0.4 a 2KT ⁄ AA 11.0 ± 1.1 9.44 ± 0.55 56.4 ± 15.9 a 1R ⁄ A(P1)–2KT ⁄ AA 14.5 ± 0.4 13.5 ± 5.1 133 ± 73 1R ⁄ A(P2)–2KT ⁄ AA 25.8 ± 4.9 41.2 ± 24.5 96.4 ± 26.6 1KT ⁄ AA–2R ⁄ A 0.71 ± 0.13 8.34 ± 0.37 81.9 ± 29.6 a The values may only represent a lower limit, because of the intrin- sic ATPase activity of TClpB. T. Yamasaki et al. Roles of conserved arginine of ClpB chaperone FEBS Journal 278 (2011) 2395–2403 ª 2011 The Authors Journal compilation ª 2011 FEBS 2397 (Fig. 3A). To elucidate the effects of the arginine mutations on the ATPase activity in each AAA+ module, we measured the ATPase activities of 1R ⁄ A(P1)–2KT ⁄ AA, 1R ⁄ A(P2)–2KT ⁄ AA, and 1KT– AA-2R ⁄ A (Fig. 3B). Whereas the single WalkerA mutants, 1KT ⁄ AA and 2KT ⁄ AA, showed significant ATPase activities, all three double mutants showed no significant ATPase activity with or without j-casein. These results indicated that the three arginines play a crucial role in ATP hydrolysis in each AAA+ module. The threading activities of the arginine mutants T. thermophilus BAP (TBAP) is a TClpB mutant that has part of the T. thermophilus ClpA (TClpA) amino acid sequence that binds T. thermophilus ClpP (TClpP), YNVGPAIGFTSKEVDTESPLKA, instead of the Leu714–Val735 sequence. ClpP is a barrel- shaped protease that degrades the substrate proteins that are translocated by bound ClpA. By the use of TBAP, the threading activity of TClpB could be esti- mated by the degradation of a-casein in the presence of TClpP [28]. We combined TBAP with the arginine mutations and tested their threading activities. Degradation of fluorescein isothiocyanate (FITC)- labeled a-casein was monitored by increased fluores- cence intensities, and the initial rates of degradation were calculated (Fig. 4A,B). At 55 °C, TBAP and TClpP degraded casein at a rate of 0.14 s )1 . All three combined mutants could degrade casein in cooperation with TClpP, but the rates were low: 0.07 s )1 [TBAP– 1R ⁄ A(P1)], 0.04 s )1 [TBAP–1R ⁄ A(P2)], and 0.05 s )1 (TBAP–2R ⁄ A). The chaperone activities of the arginine mutants By using glucose-6-phosphate dehydrogenase (G6PDH) and a-glucosidase as substrate proteins, we tested the chaperone activities of the arginine mutants of TClpB. G6PDH and a-glucosidase were aggregated by incuba- tion at 72 °C for 8 min and 73 °C for 10 min, respec- tively, in the presence of 3 mm ATP and 1 mm dithiothreitol. Subsequently, T. thermophilus DnaK (TDnaK), T. thermophilus DnaJ (TDnaJ), T. thermo- philus GrpE (TGrpE) and wild-type or mutant TClpB was added, and the reaction mixtures were incubated at 55 °C for 90 min. The recovered activities of G6PDH and a-glucosidase were measured, and expressed as percentages of the activities of these enzymes before heat treatment. Whereas wild-type TClpB reactivated approximately 64% of heat-aggre- gated G6PDH, the yields of G6PDH that were reacti- vated by 1R ⁄ A(P1), 1R ⁄ A(P2) and 2R ⁄ A were only about 10% (Fig. 5A). Although the reactivation yields were low, a similar tendency was observed in the case of a-glucosidase (Fig. 5B). The conserved arginines in AAA-1 are important for stabilizing the hexameric form We examined the hexamerization properties of 1R ⁄ A(P1), 1R ⁄ A(P2) and 2R ⁄ A by using gel filtration chromatography. Gel filtration analyses were per- formed at 55 °C in the presence of 2 mm ATP, because stable hexamerization of TClpB is dependent on high temperature and ATP. When the elution buffer con- tained 150 mm KCl, the elution times of 1R ⁄ A(P1) and 1R ⁄ A(P2) were slightly delayed as compared with Fig. 3. ATPase activities of the TClpB mutants. ATPase activities of wild-type and mutant TClpB with 3 m M ATP were measured at 55 °C in the absence (open bars) or presence (filled bars) of 0.1 mgÆmL )1 j-casein. ATPase activities are expressed as turn- over ⁄ monomer TClpB. (A) ATPase activities of the wild-type and the single arginine mutants of TClpB. The measurements were per- formed at 0.3 l M TClpB monomer. (B) ATPase activities of the Wal- kerA mutants and the combined WalkerA and arginine mutants. The measurements were performed at 1.5 l M TClpB monomer. The error bars represent the standard deviation. Roles of conserved arginine of ClpB chaperone T. Yamasaki et al. 2398 FEBS Journal 278 (2011) 2395–2403 ª 2011 The Authors Journal compilation ª 2011 FEBS the wild type (Fig. 6A). At a higher concentration of KCl (300 mm), these delays increased, particularly in the case of 1R ⁄ A(P2) (Fig. 6B). In both conditions, the elution profiles of 2R ⁄ A were same as that of the wild type. The conserved arginines in AAA-1 are not involved in the nucleotide-induced motion of the middle domain The middle domain of TClpB is an 85 A ˚ coiled-coil that extends to the outside of the hexamer [18,29]. Nucleotide binding to AAA-1 causes the middle domain to lean towards AAA-1. This motion stabilizes the hexameric form of TClpB. This motion can be detected by the change in fluorescence intensity of 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide (ABD-F), a fluorescent probe that is conjugated to the cysteine introduced at position 419, which is at the edge of the middle domain [27]. We prepared three TClpB mutants, A419C, 1KT ⁄ AA–A419C, and 1R ⁄ A(P2)– A419C, with ABD-F-labeled cysteines. The labeling yields of these mutants were 90–110%. Following the addition of 3 mm ATP, the fluorescence intensity of the ABD-F-labeled A419C mutant decreased to 46% in the presence of 150 mm KCl (Fig. 7A,D). However, in the case of ABD-F-labeled 1KT ⁄ AA–A419C, the decrease in fluorescence intensity was marginal (to 80%) (Fig. 7B,D). These results were consistent with a previous report [27]. The fluorescence intensity of ABD-F-labeled 1R ⁄ A(P2)–A419C decreased similarly to that of the wild type (to 49%) with the addition of ATP (Fig. 7C,D). Similar decreases in fluorescence intensity were observed when ADP and adenosine 5¢-O-(thiotriphosphate) (ATPcS) were added (Fig. 7D). Similar results were observed in the presence of Fig. 4. Threading activities of the TClpB mutants. (A) FITC-labeled a-casein (3 l M) was incubated with 0.05 lM TBAP (thick line), TBAP–1R ⁄ A(P1) (thin line), TBAP–1R ⁄ A(P2) (dashed line) and TBAP–2R ⁄ A (dotted line) as hexamer in the presence of 0.5 l M TClpP and 3 mM ATP at 55 °C, and changes in fluorescence inten- sity were monitored. The excitation and emission wavelengths were 490 and 520 nm, respectively. (B) The initial rates of degrada- tion of the FITC-labeled a-casein calculated from changes in fluores- cence intensity are shown as turnover ⁄ hexamer TClpB. The error bars represent the standard deviation. Fig. 5. Chaperone activities of the TClpB mutants. G6PDH (A) or a-glucosidase (B) (final concentration, 0.2 l M monomers) was incu- bated at 72 °C for 8 min (G6PDH) or at 73 °C for 10 min (a-glucosi- dase) in the presence of 3 m M ATP. The temperature was shifted to 55 °C, and TDnaK (0.6 l M), T DnaJ (0.2 lM), TGrpE (0.1 lM) and TClpB or its mutant (0.05 l M hexamer) was added immediately to the solution. After incubation for 90 min at 55 °C, the activity of the recovered enzyme was measured. The recovery is shown as the percentage of the activity before heat inactivation. The error bars represent the standard deviation. T. Yamasaki et al. Roles of conserved arginine of ClpB chaperone FEBS Journal 278 (2011) 2395–2403 ª 2011 The Authors Journal compilation ª 2011 FEBS 2399 300 mm KCl (Fig. 7E). Together with the results of the gel filtration analysis, these results indicated that the middle domain of 1R ⁄ A(P2) leans towards AAA-1 upon nucleotide binding to AAA-1, regardless of the stability of the hexameric form. Discussion Previously, the effects of mutations of the P1 arginines in AAA-1 (R332A) and in AAA-2 (R756A) on chaper- one activity, ATPase activity and hexamerization prop- erties were investigated in E. coli ClpB [4]. However, as ClpB possesses two AAA+ modules, the roles of the arginines in individual AAA+ modules were not elucidated. The role of the other conserved arginine (P2) in AAA-1 was also unclear. Here, we investigated in detail the roles of these three arginines in TClpB. By combining an arginine mutation with the WalkerA mutation in the other AAA+ module, we tested the roles of the arginines in ATP binding and hydrolysis in each module. The arginine mutations did not affect nucleotide binding to the mutated AAA+ module (Fig. 2; Table 1) but inhibited hydrolysis of the bound ATP (Fig. 3A,B). These results suggested that these ar- ginines act as arginine fingers, as observed in other AAA+ proteins, such as FtsH and p97 [5,7,30]. In all three arginine mutants, the rates of the thread- ing of a-casein, a model denatured protein, decreased to 25–50% (Fig. 4A,B), and the chaperone activities were severely impaired (Fig. 5A,B). These results sug- gested that the AAA+ modules independently contrib- ute to substrate threading, but effective disaggregation requires the combination of these two motors. Although the ATPase activity of 2R ⁄ A was signifi- cantly lower than those of 1R⁄ A(P1) and 1R ⁄ A(P2), the threading and chaperone activities of 2R ⁄ A were comparable to those of 1R ⁄ A(P1) and 1R ⁄ A(P2). These differential effects might be caused by the differ- ences in stability of the hexameric structures of these mutants (Fig. 6A,B). Consistent with a previous report on E. coli ClpB [4], the hexameric structure of 1R ⁄ A(P1) was slightly unstable, whereas that of 2R⁄ A was stable. In addi- tion, we found that the hexameric structure of 1R ⁄ A(P2) was more unstable than that of 1R ⁄ A(P1) (Fig. 6A,B). According to the crystal structure of TClpB, the P2 arginine of AAA-1 is located near the c-phosphate of AMP-PNP bound to the neighboring subunit, whereas the P1 arginine of AAA-1 faces away from it (Fig. 1B) [18]. This structural difference might cause a difference in the degree of contribution to the stabilization of the hexamer. Previously, it was shown that ATP binding to AAA-1 caused a leaning motion of the middle domain towards AAA-1, and that this motion stabilized the hexameric form of TClpB [27]. Although the ATP-induced motion of the middle domain was observed for 1R ⁄ A(P2) even in the pres- ence of 300 mm KCl (Fig. 7E), the hexameric structure of this mutant was not stable. These results suggested Fig. 6. Stabilities of hexameric structure of the TClpB mutants. The wild-type and mutant TClpB were analyzed by gel filtration chroma- tography in the presence of 2 m M ATP at 55 °C. The elution buffer contained 150 m M (A) or 300 mM (B) KCl. In both panels, the elu- tion profiles of the wild-type, 1R ⁄ A(P1), 1R ⁄ A(P2) and 2R ⁄ A mutants, from top to bottom, are shown. The arrows indicate the calculated retention time that corresponds to 577, 385, and 192 kDa (hexamer, tetramer and dimer of 96.2-kDa TClpB), respectively. Fig. 7. Nucleotide-induced fluorescence changes of ABD-F-labeled TClpB mutants. Fluorescence spectra of ABD-F-labeled TClpB- A419C (A), 1KT ⁄ AA-A419C (B) and 1R ⁄ A(P2)-A419C (C) in the absence (solid lines) or presence (dotted lines) of 3 m M ATP. The excitation wavelength was 390 nm. (D) Relative fluorescence inten- sities at 512 nm of ABD-F-labeled TClpB in the absence or pres- ence of 3 m M ATP, 3 mM ADP, or 3 mM ATPcS. The intensities in the absence of nucleotide were considered to be 100%. (A–D) The buffer contained 150 m M KCl. (E) The same experiment as in (D) was performed, with 300 m M KCl. The error bars represent the standard deviation. Roles of conserved arginine of ClpB chaperone T. Yamasaki et al. 2400 FEBS Journal 278 (2011) 2395–2403 ª 2011 The Authors Journal compilation ª 2011 FEBS that both ATP-induced motion of the middle domain and the conserved arginines, especially the P2 arginine, in AAA-1 independently contributed to the stabiliza- tion of the hexamer. This model also explained the previous observation that ADP binding to AAA-1 induced motion of the middle domain but did not sta- bilize the hexamer [23,27]. As arginine has an extended and flexible side chain with a positively charged guani- dine group, this positive charge can interact with nega- tively charged groups, especially phosphate groups. By interacting with the c-phosphate of ATP, the arginines in AAA-1 might discriminate ATP from ADP and sta- bilize the hexamer only when ATP is bound to the neighboring subunit. Experimental procedures Proteins G6PDH from Bacillus stearothermophilus was purchased from Unitika (Tokyo, Japan), a-glucosidase from B. stearo- thermophilus, a-casein, and j-casein from Sigma (St Louis, MO, USA), and rabbit pyruvate kinase and hog lactate dehydrogenase from Roche (Basel, Switzerland). The recombinant plasmid pMCB1 [13], which contains the T. thermophilus ClpB gene, was used for the mutagenesis template. Site-directed mutagenesis was performed by using the overlap extension PCR method with Ex Taq DNA polymerase (Takara, Otsu, Japan) [31,32]. The mutations were confirmed by DNA sequence analysis. T ClpB and its mutants were expressed in E. coli BL21(DE3), and purified as described previously [27]. TDnaK, TDnaJ, TGrpE, and TClpP were expressed in E. coli BL21(DE3) with pMDK6 (TDnaK), pMDJ10 (TDnaJ), pMGE3 (TGrpE) and pET23a–TClpP (TClpP) vectors, respectively, and purified as described previously [28,33–36]. The concentrations of substrate proteins were expressed as monomers, and those of T. thermophilus chaperones were expressed as monomers for TDnaK and TDnaJ, dimer for TGrpE, and 14-mer for TClpP. TClpB and its mutants were expressed as mono- mers or hexamers, as indicated. Measurement of nucleotide binding Mant-ADP, a fluorescent nucleotide analog, was purchased from Invitrogen (Carlsbad, CA, USA). Nucleotide binding was detected as described previously [23], by monitoring the increase in fluorescence of Mant-ADP after incubation with TClpB mutants. The displacement of Mant-ADP by ADP or ATP was detected as described previously [23], by monitoring the decrease in fluorescence after addition of Mg-ADP or Mg-ATP. All measurements were performed at 55 °C. Fluorescence measurements were performed with a FP-6500 fluorometer (Jasco, Tokyo, Japan). The excita- tion and the emission wavelengths were 360 and 440 nm, respectively. The apparent dissociation constants of Mant- ADP, ADP and ATP for TClpB (monomer) were calcu- lated by fitting the data as described previously [23]. The data were analyzed with kaleidagraph 4.1 (Synergy Soft- ware, Reading, PA, USA). Measurement of ATPase activity The ATPase activities of wild-type or mutant TClpB were measured spectrophotometrically with an ATP-regenerating system that contained 2.5 mm phosphoenolpyruvate, 0.2 mm NADH, 50 lgÆmL )1 pyruvate kinase, 50 lgÆmL )1 lactate dehydrogenase and 3 mm ATP at 55 °C as described previously [28]. j-Casein (0.1 mgÆmL )1 ) was also added to the reaction mixture, if needed. The changes in absorbance at 340 nm were monitored in a V-650 spectrophotometer (Jasco). Measurement of threading activity FITC was purchased from Dojindo (Kumamoto, Japan). a-Casein (100 lm) was incubated in 50 mm Mops ⁄ NaOH (pH 7.5), 150 mm KCl, 5 mm MgCl 2 and 6 mm FITC for 1 h at 25 °C. Unreacted FITC was removed with a PD10 gel filtration column (GE Healthcare, Little Chalfont, UK). The labeling yield was 186%. Fluorescence measurements were performed with an FP-6500 fluorometer. The excita- tion and emission wavelengths were 490 and 520 nm, respectively. The assay mixture [50 mm Mops ⁄ NaOH (pH 7.5), 150 mm KCl, 5 mm MgCl 2 ,3mm ATP, 0.5 lm TClpP, and 3 lm FITC-labeled casein] was preincubated at 55 °C for 2 min. Subsequently, monitoring of the fluores- cence intensity of this mixture was commenced, and the TClpB mutant was then added (the final concentration was 0.05 lm hexamer). The time when the TClpB mutant was added was set as time zero. After incubation for 30 min, the proteins in the reaction mixture were precipitated by using 10% trichloroacetic acid and analyzed by SDS ⁄ PAGE (14%). The bands of any remaining a-casein were visualized by staining with Coomassie Brilliant Blue, and quantified by molecular imager fx pro plus (BioRad, Tokyo, Japan). There was a linear correlation between the fluorescence intensity and the amount of degraded FITC- labeled casein, and a calibration curve was constructed. The initial rates of casein degradation were calculated by using the calibration curve. Reactivation of heat-aggregated proteins The chaperone activities of TClpB mutants were measured as previously described [28]. G6PDH and a-glucosidase from B. stearothermophilus were used as substrate proteins. G6PDH activity was assayed at 55 ° C by monitoring the T. Yamasaki et al. Roles of conserved arginine of ClpB chaperone FEBS Journal 278 (2011) 2395–2403 ª 2011 The Authors Journal compilation ª 2011 FEBS 2401 absorbance at 340 nm in the assay solution [100 mm Tris ⁄ HCl (pH 8.8), 40 mm MgCl 2 ,1mm NADP + , and 3mm glucose 6-phosphate]. Similarly, a-glucosidase activity was assayed at 55 °C by monitoring the absorbance at 405 nm in the assay solution [50 mm sodium phosphate (pH 6.8) and 2 mm p-nitrophenyl-a-d-glucopyranoside]. Gel filtration analysis Gel filtration analysis of TClpB mutants was performed as previously described [28], with an HPLC gel filtration col- umn (TSK G-3000SWXL; Tosoh, Tokyo, Japan). Wild- type or mutant TClpB (1.73 lm as hexamer) was eluted at a flow rate of 0.5 mLÆmin )1 at 55 °C, and monitored spec- trophotometrically at 290 nm. The elution buffer contained 150 mm or 300 mm KCl, as indicated. The molecular mass standards were thyroglobulin (669 kDa), ferritin (440 kDa), DnaKJ complex from T. thermophilus (319 kDa), catalase (232 kDa), G6PDH from B. stearothermophilus (212 kDa), and aldolase (158 kDa). Detection of the motion of the middle domain ABD-F was purchased from Invitrogen. Tris-(2-carboxyeth- yl)phosphine hydrochloride (TCEP) was purchased from Sigma. TClpB mutants (4 mgÆmL )1 ) were incubated with 1mm ABD-F in 50 mm Mops ⁄ NaOH (pH 7.5), 150 mm KCl, 5 mm MgCl 2 and 5 mm TCEP at 55 °C for 2 h. Unre- acted ABD-F and TCEP were removed with an HPLC gel filtration column (G-3000SWXL). The amount of Cys- ABD was determined spectrophotometrically by using an extinction coefficient (e 384 nm ) of 7800 m )1 Æcm )1 [37]. ABD- F-labeled ClpB mutants (0.1 mgÆmL )1 )in50mm Mops ⁄ NaOH (pH 7.5), 5 mm MgCl 2 and 150 or 300 mm KCl in the presence or absence of nucleotide were incubated for more than 2 min at 55 °C. Fluorescence was measured with an FP-6500 fluorometer (excitation, 390 nm; emission, 400– 600 nm). Acknowledgements This work was supported by the Naito Foundation, the Sumitomo Foundation, the Inamori Foundation, a Grant-in-Aid for Young Scientists (B) Number 21770151 (to Y. Watanabe) and a Grant-in-Aid for Scientific Research on Priority Area Number 19058004 (to M. Yoshida and Y. Watanabe) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. 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Roles of conserved arginine of ClpB chaperone FEBS Journal 278 (2011) 2395–2403 ª 2011 The Authors Journal compilation ª 2011 FEBS 2403 . Roles of conserved arginines in ATP-binding domains of AAA+ chaperone ClpB from Thermus thermophilus Takashi Yamasaki 1 ,. domain. Results The conserved arginines are not involved in nucleotide binding To examine the roles of conserved arginines, we gener- ated three mutants of TClpB: R322A