Cross-linking of Two
Subunits in the Closed Conformation in
F1-ATPase*
Satoshi P.
Tsunoda,
Eiro
Muneyuki,
Toyoki
Amano,
Masasuke
Yoshida
, and
Hiroyuki
Noji
From the Research Laboratory of Resources Utilization, Tokyo
Institute of Technology, 4259 Nagatsuta, Yokohama 226, Japan
 |
ABSTRACT |
In the crystal structure of mitochondrial
F1-ATPase, two
subunits with a bound
Mg-nucleotide are in "closed" conformations, whereas the third
subunit without bound nucleotide is in an "open" conformation. In
this "CCO" (
-closed
-closed
-open) conformational state, Ile-390s of the two closed
subunits, even though they are separated by an intervening
subunit, have a direct contact. We replaced the equivalent Ile of the
3
3
subcomplex of thermophilic
F1-ATPase with Cys and observed the formation of the
-
cross-link through a disulfide bond. The analysis of conditions
required for the cross-link formation indicates that: (i)
F1-ATPase takes the CCO conformation when two catalytic
sites are filled with Mg-nucleotide, (ii) intermediate(s) with the CCO conformation are generated during catalytic cycle, (iii) the Mg-ADP inhibited form is in the CCO conformation, and (iv)
F1-ATPase dwells in conformational state(s) other than CCO
when only one (or none) of catalytic sites is filled by Mg-nucleotide
or when catalytic sites are filled by Mg2+-free nucleotide.
The
3
3
subcomplex containing the
-
cross-link retained the activity of uni-site catalysis but lost
that of multiple catalytic turnover, suggesting that open-closed
transition of
subunits is required for the rotation of
subunit
but not for hydrolysis of a single ATP.
 |
INTRODUCTION |
F1, together with the membrane-embedded F0
part, constitutes ATP synthase which couples a transmembrane proton
flow to ATP synthesis/hydrolysis. F1 is easily and
reversibly separated from F0 part as a water soluble ATPase
and is called F1-ATPase. The F1-ATPase is
composed of five subunits,
,
,
,
, and
subunits, in a
molar ratio of 3:3:1:1:1 (1-3). In the crystal structure of bovine
mitochondrial F1-ATPase
(MF1)1 (4), three
and three
subunits are arranged alternately like the segments
of an orange around the central coiled-coil structure of the
subunit. F1-ATPase contains six nucleotide binding sites;
three of them are catalytic sites and mainly located on each
subunit, whereas the other three do not bear a catalytic role and are
mainly located on each
subunit (4). The
3
3
subcomplex of F1-ATPase
has been recognized as a minimum ATPase-active complex with stability
and characteristics similar to native F1-ATPase (5-7).
F1-ATPase shows a complicated kinetic behavior in ATP
hydrolysis. Three catalytic sites in F1-ATPase are not
independent but are related to one another in a cooperative manner.
Binding of ATP to the catalytic sites is governed by negative
cooperativity, and inversely, hydrolysis of bound ATP at each catalytic
site is governed by positive cooperativity. When substoichiometric amounts of ATP relative to the enzyme are added, ATP is hydrolyzed very
slowly (uni-site catalysis) (8-10). This reaction is accelerated by
chase-addition of an excess amount of ATP (chase-promotion) (8-10). As
a whole, ATP hydrolysis reaction by F1-ATPase usually exhibits negative cooperativity as a function of ATP concentration (11-16). These and other features had been unified into the binding change mechanism by Boyer (3). According to the mechanism, three
catalytic sites interchange their roles alternately and sequentially
during catalytic turnover accompanying the rotation of
subunit in
the center of the enzyme. Consistent with this rotary mechanism, the
3
3
subcomplex of thermophilic
Bacillus PS3 F1-ATPase (TF1)
containing two intact and one incompetent catalytic sites lost the
ability to mediate catalytic turnover while it showed uni-site ATP
hydrolysis and chase-promotion (17). The rotation of the
subunit
was supported by various methods (18-20) and was directly proved by
single molecule observation of the
3
3
subcomplex of TF1 (21).
Another complication of F1-ATPase kinetics is caused by the
"Mg-ADP inhibited form" (22-25). In general,
F1-ATPases from mitochondria, chloroplasts, and bacteria
are prone to develop turnover-dependent inactivation;
Mg-ADP trapped transiently in a catalytic site causes the slow
transition from an active form to an inhibited form called as Mg-ADP
inhibited form (26-28). The slow transition during catalysis is
accelerated by the simultaneous occupation of two catalytic sites by
Mg-nucleotides (25, 29). Mg-ATP bound to the noncatalytic nucleotide
binding site promotes dissociation of inhibitory Mg-ADP from the
affected catalytic site (30). Further, when the enzyme is preincubated
with stoichiometric Mg-ADP, the Mg-ADP inhibited form is generated. An
inhibitor of F1-ATPase, azide stabilizes the Mg-ADP
inhibited form (23-25), and an activator of F1-ATPase, N,N-dimethyldodecylamine-N-oxide
(LDAO) destabilizes it (31).
The crystal structure of MF1 (4) revealed that three
subunits in the MF1 molecule are in different states; one
(
TP) has an ATP analog, Mg-AMP-PNP, at its catalytic
site; another
(
DP) has Mg-ADP; the third
(
E) has none. The structures of
TP and
DP are very similar to each other, and they are in the
"closed" conformation, in which the carboxyl-terminal domain is
lifted close to the nucleotide binding domain. In contrast,
E adopts the "open" conformation, in which the
crevice for substrate binding is open. This crystal structure,
characterized by two closed and one open
subunits ("CCO"
conformational state) has been assumed to be a "snapshot" of
MF1 cycling the catalytic turnover along the pathway
predicted by the binding change mechanism. Whether this assumption is
really the case has not been established by experiments. To address
this question, we need a specific probe to detect the CCO
conformational state of F1-ATPase in solution. Looking at
the structure of MF1 carefully, we noticed that the two
closed
subunits, even though separated by an intervening
subunit, have a direct contact at the position of Ile-390 of each
subunit (see Fig. 1, A and B). If this Ile is
replaced with Cys, two closed
subunits in F1-ATPase in
the CCO conformation would be cross-linked by a disulfide bond, and it
would fix two
subunits in the closed conformation. Indeed, when we
examined this experiment using
3
3
subcomplex of TF1, the cross-link was formed in an
Mg-nucleotide-dependent, azide-facilitated manner with
concomitant loss of the activity of catalytic turnover.
 |
EXPERIMENTAL PROCEDURES |
Strains, Plasmids, and Preparation of
Subcomplexes--
Escherichia coli strains used were JM109
(32) for preparation of plasmids, CJ236 (33) for generating
uracil-containing single-stranded plasmid for site-directed
mutagenesis, and JM103D (uncB-uncD) (34) for expression of
the wild-type and mutant
3
3
subcomplexes of TF1. Plasmid pTABG3 (6), which carried genes for the
,
, and
subunit of TF1 was used for
mutagenesis and expression. A helper phage M13KO7 was obtained from
Amersham Pharmacia Biotech, Tokyo. The expression plasmid for the
mutant subcomplex was constructed as follows. The mutation (Ile
Cys at
-386) was introduced into pTABG3 (6) by using a synthetic oligonucleotide:
5'-TTGTCTTCATCCGACAGTTCATCCATCCCCAAGCATGCGATGATGTCTTGCAATTCTTTATAACGTTCGA-3' (changed bases are underlined) (33). The
EcoRI-BglII fragment from the resultant plasmid
was ligated into the EcoRI-BglII site of pKABG3
to produce the expression plasmid, pKAG3-
-Ile386Cys. Recombinant DNA
procedures were performed as described in the manual (35). The
wild-type and the mutant
3
3
subcomplexes were purified and stored as described previously except
that 2 mM dithiothreitol (DTT) was added in all buffers (6,
17). Just before use, they were subjected to gel-filtration HPLC with a
TSK-G3000SWXL column (Tosoh, Japan) equilibrated with 50 mM Tris-HCl (pH 7.0), 200 mM NaCl (Tris-NaCl buffer).
Gel-filtrated preparation of the mutant subcomplex did not contain a
detectable amount of endogenously bound adenine nucleotide (<0.1
mol/mol of subcomplex). In the presence of LDAO, the mutant subcomplex showed the steady-state ATPase activity of 15.2 µmol of ATP
hydrolyzed/min/mg at 25 °C, which is ~70% of that of the
wild-type subcomplex (21.3 µmol of ATP hydrolyzed/min/mg). In the
absence of LDAO, the activity was 4.6 (mutant subcomplex) and 7.7 (wild-type subcomplex) µmol of ATP hydrolyzed/min/mg.
Disulfide Cross-link Formation of the Mutant Subcomplex--
The
subcomplexes were incubated at 25 °C in Tris-NaCl buffer containing
indicated components. At the indicated time, an aliquot was taken out
and formation of the cross-link was analyzed with 10.5% polyacrylamide
gel electrophoresis in the presence of sodium dodecyl sulfate without
exposure to a reducing reagent (non-reducing SDS-PAGE) (36). To prevent
further oxidation during electrophoresis, all sample solutions were
treated with 10 mM N-ethylmaleimide (NEM) for 5 min prior to electrophoresis. The gels were stained with Coomassie
Brilliant Blue R-250. The yields of the cross-linked product were
determined from the staining intensity of bands with a
photodensitometer (model SM3, Howtec Inc.) using the
subunit band
as an internal standard.
Analysis of Bound Adenine Nucleotide--
The wild-type and
mutant subcomplexes were incubated in 20 µM ATP and 2 mM MgCl2 for 2.5 h at 25 °C. The yield
of cross-linked
dimer of the mutant subcomplex estimated from
non-reducing SDS-PAGE was nearly 100% after this incubation. As a
control, the samples treated with the same procedures, except that 10 mM DTT was included in the solution, were also analyzed. To
remove free adenine nucleotide, the samples were passed through an HPLC
column TSK-G3000SWXL. The protein fractions were recovered, and
perchloric acid was added to precipitate proteins. The amounts of
adenine nucleotides contained in the supernatant were measured with
reversed-phase HPLC on a Cosmosil 5C18-AR-II (Nacarai Tesque, Japan),
equilibrated, and eluted with 0.1 M sodium phosphate buffer
(pH 6.9).
Other Assays--
ATPase activity was measured at 25 °C in
the presence of an ATP-regenerating system (37). Unless stated, a
neutral detergent, LDAO, was included in the assay mixture to eliminate
the effect of the initial lag phase caused by the Mg-ADP inhibition
(31). The assay mixture contained 50 mM Tris-HCl (pH 8.0),
100 mM KCl, 2.5 mM phosphoenolpyruvate, 2.5 mM ATP, 2 mM MgCl2, 0.2 mM NADH, 50 µg/ml pyruvate kinase, 50 µg/ml lactate
dehydrogenase, and 0.1% LDAO. Uni-site ATPase activity and chase
promotion were measured at 26 and 8 °C using TNP-ATP as a substrate
as described previously (38). A reaction mixture (50 µl) containing
50 mM Tris-HCl (pH 8.0), 4 mM
MgCl2, 200 mM KCl, and 0.3 µM
TNP-ATP was incubated at 26 °C. The reaction was initiated by the
addition of an equal volume of 1 µM subcomplexes in 50 mM Tris-HCl (pH 8.0). The reaction was quenched by the
addition of 7.5 µl of ice-cold 24% perchloric acid. In
chase-promotion experiments, 10 µl of 30 mM ATP-Mg was added instead of perchloric acid. After 5 s, the reaction was quenched by the addition of 7.5 µl of ice-cold 24% perchloric acid.
The amounts of TNP-ATP and TNP-ADP were measured by HPLC (38). For
uni-site and chase-promotion experiments, the mutant subcomplex with
the cross-link lacking bound nucleotide was prepared. The mutant
subcomplex was treated with 100 µM CuCl2 for
24 h at 25 °C, and the subcomplex was separated from
CuCl2 with gel filtration (Sephadex G-25 column, 2 cm × 5 cm). Formation of the cross-link at 100% yield was confirmed with
SDS-PAGE before use. Protein concentration of
3
3
subcomplex was determined by UV
absorbance using the factor of 0.45 at 280 nm as 1.0 mg/ml.
 |
RESULTS |
Two
Subunits in the Closed Form Have a Contact Site--
In
the crystal structure of MF1, two
subunits with a bound
nucleotide,
TP and
DP, take the closed
conformation and contact each other at the carboxyl-terminal region
near the central axis (Fig. 1,
A and B, shown by an arrow). The
residues in the contact position are Ile-390s of each of
subunits
(Fig. 1, A and B, shown by red). The
distance between
carbons of the two Ile-390s is 0.79 nm. The
distance between the nearest carbon atoms (C
of
TP-Ile-390 and C
2 of
DP-Ile-390) is 0.42 nm, and the two residues actually
appear to have hydrophobic interaction. On the contrary, the distance
between
carbons of
TP-Ile-390 and
E-Ile-390 and that between
DP-Ile-390 and
E-Ile-390 are 2.40 nm and 2.58 nm, respectively. This
residue is located in a region just preceding the "DELSEED
sequence" (39) and is highly conserved in F1-ATPases from
various sources.

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Fig. 1.
A contact point between two
subunits in a closed conformation in
F1-ATPase. Illustrations are based on the
structure of bovine MF1 (4). Subunits TP,
DP, and E represent the subunits with
their nucleotide binding sites occupied by Mg-AMP-PNP, Mg-ADP, and
none, respectively. The contact point is indicated by an
arrow. The subunits are removed from the structure and
only a part of the subunit (A, residues 1-45, 73-90,
and 209-272; B, residues 1-45 and 215-272) is shown to
make the contact point visible. Ile-390s are shown by red
space-filling atoms. A, a side view; B, a
view from the membrane (F0) side. C,
two subunits are in the open conformation and one subunit is in
the closed conformation. D, three subunits are all in
the open conformation. C and D were generated by
replacing one ( DP) or two ( DP and
TP) subunits in the crystal structure with the subunit(s) in the open conformation ( E). Replacement was
carried out using Insight II computer program. The open subunit was
overlaid with the closed subunit with maximum overlapping at the
region of residues 9-150 and then the closed subunit was
erased.
|
|
The
3
3
Subcomplex with Cys at the
Contact Sites Formed
-
Cross-link in the Presence of Both
Mg2+ and AT(D)P--
We generated a mutant
3
3
subcomplex of TF1 in
which
-Ile-386, an equivalent residue to MF1
-Ile-390, was replaced with Cys. ATPase activity of the mutant
subcomplex was about 70% of that of the wild-type subcomplex (see
"Experimental Procedures"). The cross-linked
dimer in the
mutant subcomplex was detected by non-reducing SDS-PAGE after
incubation at 25 °C for 2 h in the Tris-NaCl buffer containing
0.25 µM CuCl2 and indicated compounds (Fig.
2). No new band other than three subunit
bands,
,
, and
was found in a reference sample, incubated
alone or with DTT (Fig. 2, lanes 1 and 2). When
the solution contained Mg-ATP (lane 8), Mg-ADP (lane
9), or Mg-ADP + azide (lane 10), a 100-kDa protein band
appeared. The exposure of the samples to a reducing reagent prior to
electrophoresis eliminated the band (lane 12). It was identified to be a cross-linked
dimer because two-dimensional electrophoresis showed that the 100-kDa band in the first non-reducing SDS-PAGE was developed into a
subunit band in the second reducing SDS-PAGE (data not shown). Consistently, the staining intensities of the
subunit band in lanes 8-10 decreased in parallel
with the increase of those of the
dimer band. Because the
cross-link was formed in Mg-ADP, catalytic turnover of ATP hydrolysis
is not absolutely required for the cross-linking. No cross-link was observed in AT(D)P + EDTA (lanes 5 and 6), ADP + azide + EDTA (lane 7), and Mg2+ alone
(lane 11). Therefore, both AT(D)P and Mg2+ are
necessary to form the cross-link. The cross-linked
dimer was formed
even under air oxygen without CuCl2 and essentially the
same dependence of the cross-linking on Mg-nucleotide were observed.
However, the time courses of the cross-linking under air oxygen varied
in the experiments in different days although they were reproducible in
one sequence of experiments in the same day. By this reason, unless
otherwise stated, we included 0.25 µM CuCl2
in the reaction mixtures in which the cross-linking occurred at
reproducible rates of the order of 10 min as described later. At high
concentration of CuCl2 (100 µM), the
cross-link was formed after a 2-h incubation in the absence of
Mg-AT(D)P, but the yield of the cross-linked
dimer was partial
(lane 3). At 100 µM CuCl2 in the
presence of Mg-AT(D)P, the cross-link was formed instantaneously (see
next paragraph). Prior treatment of the subcomplex with NEM prevented
the cross-link (lane 4).

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Fig. 2.
The formation of the cross-linked
dimer through a disulfide bond in the mutant
3 3
subcomplex. Lanes 1-11. The mutant
subcomplex was incubated for 2 h at 25 °C with following
reagents and analyzed on non-reducing SDS-PAGE: none (lane
1); 10 mM DTT and 0.25 µM
CuCl2 (lane 2); 100 µM
CuCl2 (lane 3); 2 mM NEM and 100 µM CuCl2 (lane 4); 2 mM ATP, 1 mM EDTA, and 0.25 µM
CuCl2 (lane 5); 2 mM ADP, 1 mM EDTA, and 0.25 µM CuCl2
(lane 6); 2 mM ADP, 10 mM
NaN3, 1 mM EDTA, and 0.25 µM
CuCl2 (lane 7); 2 mM ATP, 4 mM MgCl2, and 0.25 µM
CuCl2 (lane 8); 2 mM ADP, 4 mM MgCl2, and 0.25 µM
CuCl2 (lane 9); 2 mM ADP, 4 mM MgCl2, 10 mM NaN3,
and 0.25 µM CuCl2 (lane 10); and 4 mM MgCl2 and 0.25 µM
CuCl2 (lane 11). Lane 12, the sample
treated as lane 9 was then incubated for 1 min with 10 mM DTT prior to SDS-PAGE.
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|
The Enzyme Containing the Cross-link Was Inactive in Catalytic
Turnover--
As mentioned above, cross-linking occurs in Mg-ATP where
the enzyme is catalyzing hydrolysis of ATP. When we added 100 µM CuCl2 during continuous assay of ATPase,
ATP hydrolysis stopped almost immediately (<15 s), but the full
activity was recovered by addition of 150 mM DTT (Fig.
3A, trace a). It
should be noted that the solutions contained LDAO so that generation of
the Mg-ADP inhibited form was avoided. ATPase activity of the mutant
subcomplex pretreated with NEM (trace b) and that of the
wild-type subcomplex (trace c) were not affected either by
CuCl2 or DTT. This suggests that the cross-linking causes
the loss of the activity to cycle the catalysis. Indeed, the
time-course of the ATPase inactivation mirrored that of the yield of
the cross-linked
-
dimer (Fig. 3B), and the analysis
of their correlation showed that the subcomplex containing a
cross-linked
dimer completely lost the ability to mediate catalytic
turnover of ATP hydrolysis (Fig. 3C). The inactivated
subcomplex recovered a full activity by incubation with a reducing
reagent, for example 100 mM DTT (data not shown).

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Fig. 3.
The cross-linking inactivates ATPase
activity. A, traces of ATP hydrolysis catalyzed by the
subcomplexes. Hydrolysis was monitored by the oxidation of NADH with an
ATP-regenerating system as described under "Experimental
Procedures." The reaction mixture contained 0.1% LDAO to avoid the
influence of the Mg-ADP inhibited form. The reactions were initiated by
addition of the subcomplex at the time indicated by
arrowheads (a, 1.1 µg of the mutant subcomplex;
b, 1.1 µg of mutant subcomplex treated with 1 mM NEM for 20 min at 25 °C; c, 0.8 µg of
wild type subcomplex). Then 100 µM CuCl2 or
150 mM DTT were added at the time indicated by
arrowheads, respectively. These high concentrations of
CuCl2 and DTT were used to ensure the rapid reactions.
B, time courses of formation of the cross-linked -
dimer and inactivation of ATPase activity. The mutant subcomplex was
incubated at 25 °C with 2 mM ATP and 2 mM
MgCl2. At indicated times, aliquots were taken out, and
ATPase activity ( ) and the yield of the cross-linked - dimer
( ) were analyzed. C, correlation between the cross-linked
- dimer and residual ATPase activity.
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|
Cross-linking Occurred Most Rapidly in Mg-ADP + Azide--
Rates
of cross-linking were assessed by measuring the rates of ATPase
inactivation in the presence of nucleotide and azide (Fig.
4). To ensure that the residual ATPase
activities at each time point reflect correctly the fraction of the
native subcomplexes without the cross-link, they were measured in the
ATPase assay solutions containing LDAO in which subcomplexes showed
uninhibited linear activities without an initial lag. Inactivation of
ATPase activities proceeded with single exponential curves (solid
lines) most rapidly in Mg-ADP + azide, next in Mg-ADP, Mg-ATP + azide, and most slowly in Mg-ATP. The half-decay times were 3.6, 5.8, 10.2, and 21.0 min under the described conditions. Because the Mg-ADP
inhibited form is produced most efficiently when F1-ATPase is incubated with Mg-ADP and it is further facilitated by azide (23-25), the conformation of the Mg-ADP inhibited form appears to be
favorable for the cross-linking.

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Fig. 4.
Effect of Mg-AT(D)P and NaN3
on the rates of ATPase inactivation caused by cross-linking.
The reactions were initiated by addition of the mutant subcomplex into
the following solutions: (final concentrations) 2 mM ATP
and 2 mM MgCl2 ( ); 2 mM ATP, 2 mM MgCl2 and 10 mM NaN3
( ); 2 mM ADP and 2 mM MgCl2
( ); or 2 mM ADP, 2 mM MgCl2, and
10 mM NaN3 ( ). All the solutions contained
0.25 µM CuCl2. The mixtures were incubated at
25 °C. At every 5 min, aliquots were taken out, and residual ATPase
activities were measured in the ATPase assay solutions containing 0.1%
LDAO.
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|
Effect of Mg-Nucleotide Concentrations on the
Cross-linking--
To estimate how many nucleotides were required for
the formation of the cross-link at 0.25 µM
CuCl2, the mutant subcomplex was incubated with various
concentrations of Mg-ADP and Mg-TNP-ADP, and the final yield of the
cross-linked
dimer was measured. At low concentrations of
nucleotides, cross-linking proceeded slowly and it was safe to wait for
24 h to reach the final, maximum yield (Fig.
5). Formation of the cross-link was
saturated when the concentrations of Mg-nucleotide, expressed as a
molar ratio Mg-nucleotide:subcomplex, reached 3:1. It should be noted
that at molar ratio 1:1, cross-linked
dimer was formed in only a small fraction of the subcomplex (20%, Mg-ADP; 3%, Mg-TNP-ADP) but at
molar ratio 2:1, 50% or more populations of the subcomplexes had a
cross-linked
dimer. Similar results were obtained when the
subcomplex was incubated with Mg-ATP and Mg-TNP-ATP (not shown). It was
demonstrated previously from difference absorption spectrum titration
that, when added with 1:1 molar ratio at micromolar range, almost all
Mg-TNP-ADP bound to a catalytic site of the subcomplex (7). Also,
nearly all Mg-ADP added at 1:1 molar ratio was bound to the subcomplex
(22, 40). Therefore, for cross-linking to occur, occupation of a single
catalytic site by Mg-nucleotide is not sufficient but at least two
catalytic sites need to be filled by Mg-nucleotides.

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Fig. 5.
Effect of Mg-nucleotide concentrations on the
cross-linking. The 1 µM mutant subcomplex was
incubated with ADP or TNP-ADP ( ) in the presence of 2 mM MgCl2 at 25 °C for 24 h, and the
yield of the cross-linked - dimer was analyzed. The
concentrations of nucleotides are expressed as molar ratio to the
subcomplex.
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|
Subcomplex Containing the Cross-link Had Two Bound
Nucleotides--
The amount of bound nucleotide in the mutant
subcomplex was measured after the cross-link was formed in Mg-ATP
(Table I). Unbound nucleotide was removed
by gel filtration, and nucleotide bound to the subcomplex in a stable
manner was analyzed. For comparison, two samples, the wild-type
subcomplex incubated with Mg-ATP and the mutant subcomplex incubated
with Mg-ATP + DTT were also analyzed. The mutant subcomplex incubated
with Mg-ATP + DTT had only a trace amount of bound nucleotide. The
mutant subcomplex after formation of the cross-link retained about 2 mol/mol bound ADP (Mg2+ was not analyzed). This result
indicates that when the carboxyl-terminal domains of the two
subunits are fixed in the closed conformation by the cross-link, two
Mg-ADPs remain trapped in their catalytic sites. The wild-type
subcomplex had about 1 mol/mol bound ADP (Table I). It can bind as much
as 3 mol/mol Mg-ADP when analyzed with equilibrium dialysis (5), but 2 mol of Mg-ADP probably dissociated upon separation from unbound Mg-ADP
during gel filtration. Related to our observation, it was reported that
when
subunit and
(or
) subunit of E. coli
F1-ATPase were cross-linked, entrapped nucleotide (ATP or
ADP) cannot be released (41).
The Enzyme Containing the Cross-link Catalyzed Uni-site Catalysis
and Chase-promotion--
Hydrolysis of a substoichiometric amount of
substrate (uni-site catalysis) and its acceleration by chase-added ATP
(chase-promotion) of the subcomplex with the cross-link were measured.
To avoid confusion arising from bound Mg-ADP, nucleotide-free
subcomplex with the cross-link was prepared in 100 µM
CuCl2 in the absence of nucleotide. The subcomplex thus
prepared did not have the activity of steady-state ATP hydrolysis, but
it retained the ability to catalyze uni-site hydrolysis of TNP-ATP
(Fig. 6). The rates of uni-site catalysis
of the subcomplexes with and without the cross-link were almost the
same at 26 °C (Fig. 6, A and B, closed
circles), and chase-promotion was also observed for both
(open circles). This result indicates that a single high
affinity catalytic site and its communication with the second catalytic
site of the subcomplex are not lost by the cross-linking. However, the
efficiency of the communication was diminished because at low
temperature, 8 °C, chase-promotion observed for the subcomplex with
the cross-link was very poor compared with that of the subcomplex
without the cross-link (Fig. 6, A and B,
insets).

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Fig. 6.
Uni-site hydrolysis of substoichiometric
TNP-ATP and chase-promotion by the mutant subcomplex.
A, the mutant subcomplex without cross-link; B,
the mutant subcomplex with the cross-link. The reaction mixture
contained 0.5 µM mutant subcomplex and 0.13 µM TNP-ATP. Uni-site catalysis ( ) and chase promotion
( ) were assayed at 26 °C as described under "Experimental
Procedures." The experimental data of chase promotion are shown at
the time when the reactions were stopped. Inset, uni-site
catalysis and chase promotion measured at 8 °C.
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 |
DISCUSSION |
-
Cross-linking as a Specific Probe to Detect the CCO
Conformational State of F1-ATPase--
Intersubunit
cross-linking has been proved to be a useful method in the study of
F1-ATPase to know relative location and motion of subunits
(19, 41, 42). In the crystal structure of the
3
3 subcomplex of TF1, the
whole structure is arranged in an exact three-fold symmetry, and all
three
subunits are in the open conformation (43). The distance
between Ile-386s of the two
subunits in this subcomplex is 3.27 nm,
and a disulfide bond would not be possible even if
-Ile-386s are
replaced with Cys. By substituting the closed
subunit in the
crystal structure of MF1 with the open
E
subunit with the aid of a computer, we generated the structure of the
3
3
subcomplex with one closed and two
open
subunits (COO conformational state) (Fig. 1C) and that with three open
subunits (OOO conformational state) (Fig. 1D).
-Ile-390s in the COO conformational state are
distant from each other. The distances between Ile390 of the closed
and that of each of the two open
s are 2.42 and 3.65 nm, too far for
cross-linking. This is also the case for the OOO conformational state;
-Ile390s are far away from each other, just similar to
3
3 subcomplex of TF1 (43).
Only when two
subunits take the closed conformation (CCO
conformational state) do their lifted carboxyl-terminal domains bring
-Ile-390s into the contact position (Fig. 1, A and
B). One might think of a CCC conformational state, but when
we generated CCC conformational state based on the MF1
structure,
subunit could not be accommodated in the center of the
molecule without steric collision. Therefore, it is unlikely that all
three
subunits take closed conformations. Thus,
-
cross-linking between the introduced Cys at the position
-386 of
TF1 is a specific means to identify the CCO conformational
state of F1-ATPase in solution. This rationale should be
valid for F1-ATPases from other sources.
F1-ATPase Takes the CCO Conformation in Catalytic Cycle
and in the Mg-ADP Inhibited Form--
The cross-link was formed in the
solution containing Mg-ATP and ATP regeneration system where catalysis
was going on (Fig. 3A). Therefore, at least one of the
intermediates in the catalytic cycle of the subcomplex takes the CCO
conformation.2
Because azide stabilizes the Mg-ADP inhibited form (23-25),
accelerated formation of cross-linking in Mg-ATP by azide (Fig. 4)
indicates that the Mg-ADP inhibited form, which is probably derivatized
from the catalytic intermediate(s) with a CCO conformation as mentioned
above, also takes a CCO conformation. This contention is supported by
the fact that cross-linking occurs even more efficiently in Mg-ADP and
Mg-ADP + azide, conditions where the Mg-ADP inhibited form is produced
efficiently. Requirement for 2 mol of Mg-nucleotide per mol of the
subcomplex for the cross-linking (Fig. 5) is consistent with the
observation that the rate of development of azide inhibition during ATP
hydrolysis was saturated at an ATP concentration of about 10 µM, a concentration range of bi-site catalysis where the
enzyme operates with two catalytic sites being occupied by substrates
(22).
Existence of COO (or OOO) Conformational State of
F1-ATPase Is Suggested--
It has been known that Mg-free
AT(D)P can bind to catalytic sites of F1-ATPase but not to
noncatalytic nucleotide binding site (44, 45). The
3
3
subcomplex can bind 3 mol of
Mg-free ADP per mol (5). Nonetheless, the cross-linked
dimer was not formed in Mg-free AT(D)P (Fig. 2). This indicates that subcomplex with bound Mg-free AT(D)P at catalytic site(s) dwells in a
conformational state, probably either in a COO or OOO conformational
state, in which two or three
subunits are in open conformations. In
this respect, the liganding of Mg2+ to nucleotide at the
catalytic site has a great effect on the conformation of the
subunit; shifting the carboxyl-terminal domain from the open position
to the closed one. Probably related to this, the profound effect of
Mg2+ on the binding affinity of AT(D)P to the catalytic
sites of F1-ATPase was reported by Senior's group (44).
Also, we previously observed that chemical labeling of
-Glu-190, a
catalytic residue acting as a general base in ATP hydrolysis reaction
(17), by dicyclohexylcarbodiimide was completely blocked when
Mg2+ was liganded in a catalytic site (46).
Subunit Cannot Rotate without Open-Closed Transition of
Subunits--
The mutant subcomplex cannot catalyze multiple catalytic
turnover when the cross-link was formed (Fig. 3). Because multiple catalytic turnover of F1-ATPase couples with rotation of
subunit, indication of this result is that, when two
subunits
are fixed in the closed conformation, the rotation of the
subunit
is blocked. The open-closed conformational transition of each of the
subunits and exchange of the CC (
-closed,
-closed) pair among
three
subunits appear to be necessary for the rotation of the
subunit and mechanical prevention of this transition by the
cross-linking results in blocking the rotation and, thereby, turnover
of ATPase cycle. Probably by the same reason, E. coli
F1-ATPase in which the residue
-381, an equivalent
residue of TF1
-E391, is labeled by a bulky fluorescent
dye is inactive in ATPase catalytic turnover (42). In a sense, the
mechanism of inactivation caused by cross-linking may be similar to
that of the Mg-ADP inhibition, blocking sequential transition of the
intermediate states in catalysis of the enzyme. The difference is that
the former blocks physically and the latter does kinetically. Once the
cross-link is formed in the Mg-ADP inhibited form, the transition is
now physically blocked.
Based on the experiments of the
-
cross-linked E. coli
F1-ATPase, Capaldi's group has proposed that uni-site
catalysis does not accompany the rotation (47). Our result, that the
subcomplex with the
-
cross-link can catalyze uni-site TNP-ATP
hydrolysis (Fig. 6), again reinforces this conclusion. Mere hydrolysis
of a single ATP probably does not require the open-closed transition of
the
subunits. Furthermore, because the uni-site catalysis by the
subcomplex with the
-
cross-link was promoted by chase-added ATP
(Fig. 6), communication between two
subunits in the enzyme is still
possible without open-closed transition of the
subunits. Probably
the open-closed transition of the
subunits is needed to drive the
critical catalytic step, i.e. simultaneous exchange of the
role of each catalytic site.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Research Laboratory of
Resources Utilization, R-1, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226, Japan. Tel.: +81 45 924 5233; Fax: +81 45 924 5277; E-mail: myoshida{at}res.titech.ac.jp.
2
One can argue that the Mg-ADP inhibited form,
but not an active intermediate, is the molecular species in which
cross-link is formed under the conditions in Fig. 3A because
the Mg-ADP inhibited form, which also takes the CCO conformation, is
generated more or less in a dynamic equilibrium during catalysis.
However, this argument may not be the case. The solution contained
LDAO, an activator known to keep F1-ATPases from falling
into the Mg-ADP inhibited form during catalysis and ATP regenerating
system to convert the free ADP into ATP. Therefore, the fraction of the Mg-ADP inhibited form should be minimal in the solution. The
inactivation of ATPase activity, which was caused by cross-linking,
occurred immediately when 100 µM CuCl2 was
added. If the cross-link had been formed only from the Mg-ADP inhibited
form, the inactivation would have proceeded very slowly in the presence
of LDAO.
 |
ABBREVIATIONS |
The abbreviations used are:
MF1, F1-ATPase from mitochondria;
TF1 , F1-ATPase from thermophilic Bacillus strain PS3;
PAGE, polyacrylamide gel electrophoresis;
LDAO, N,N-dimethyldodecylamine-N-oxide;
DTT, dithiothreitol;
TNP-AT(D)P, 2',3'-O-(2,4,6-trinitrophenyl)
derivatives of AT(D)P;
HPLC, high performance liquid
chromatography;
NEM, N-ethylmaleimide;
AMP-PNP, adenosine
5'-(
,
-imino)triphosphate;
TNP-ATP, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine
5'-triphosphate.
 |
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