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 Walker
Trang 1of AAA+ chaperone ClpB from Thermus thermophilus
Takashi Yamasaki1, Yosuke Nakazaki1, Masasuke Yoshida2and Yo-hei Watanabe1
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.
Trang 2most 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.
Trang 3Disaggregation 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 arginexam-ines, Arg322 (P2)
and Arg323 (P1) of AAA-1, and Arg747 (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 Kd values were
calculated (Table 1) Increased fluorescence was
observed in all double mutants, and the Kdvalues 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 Kd
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 ADP or
Mg-ATP, and calculated the apparent Kd values of ADP
and ATP for the TClpB mutants (Table 1) The Kd
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 mgÆmL)1 j-casein At 55C, 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
Kdfor Mant-ADP (l M )
Kdfor ADP (l M )
Kdfor ATP (l M ) Wild type 1.00 ± 0.09 8.08 ± 0.17 31.1 ± 1.7a 1KT ⁄ AA 0.30 ± 0.15 4.36 ± 0.07 30.7 ± 0.4 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.
Trang 4(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 55C, 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 72C 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 55C 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 55C 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 WalWal-kerA and arginine mutants.
The measurements were performed at 1.5 l M TClpB monomer.
The error bars represent the standard deviation.
Trang 5the 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 T ClpB mutants (A) FITC-labeled
a-casein (3 l M ) was incubated with 0.05 l M T BAP (thick line),
T BAP–1R ⁄ A(P1) (thin line), T BAP–1R ⁄ A(P2) (dashed line) and
T BAP–2R ⁄ A (dotted line) as hexamer in the presence of 0.5 l M
T ClpP and 3 m M 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 T DnaK (0.6 l M ), T DnaJ (0.2 l M ), TGrpE (0.1 l M ) 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.
Trang 6300 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 m M (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 m M ADP, or 3 m M 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.
Trang 7that 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
recombinant plasmid pMCB1 [13], which contains the
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 TClpB 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
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 hexamono-mers, 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
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
lactate dehydrogenase and 3 mm ATP at 55C 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)
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,
TClpP, and 3 lm FITC-labeled casein] was preincubated at
55C 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
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
Trang 8absorbance at 340 nm in the assay solution [100 mm
3 mm glucose 6-phosphate] Similarly, a-glucosidase activity
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 55C, 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
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 (e384 nm) of 7800 m)1Æcm)1 [37]
the presence or absence of nucleotide were incubated for
more than 2 min at 55C 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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