Báo cáo khoa học: Roles of conserved arginines in ATP-binding domains of AAA+ chaperone ClpB from Thermus thermophilus pptx

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:
(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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