From the
A set of mutants of the Escherichia coli F
The yield of
covalent linkage of
An H
Low resolution
cryoelectron microscopy studies show the F
We have been examining structure-function
relationships in ECF
In the site-directed mutagenesis step (Kunkel
et al., 1987) the oligonucleotides CTTCAGACAGACAATCCATACCC and
TTGTCTTCTTCACACAGTTCATCCAT were used for replacing a glutamate at
position
The mutations were inserted in an unc operon containing
plasmid in two steps: (i) the 5.8-kb XhoI/ NsiI
fragment from pRA100 and pRA102, respectively, was inserted in
pBluescript SK+ (Stratagene), which had been previously modified
by placing an NsiI linker (New England Biolabs) in the
EagI site. These plasmids, pRA13 and pRA14, contained the
unc C coding for wild-type
ATP synthase was reconstituted
into egg-lecithin vesicles by first adding 10% sodium deoxycholate to
ECF
Mutants in the DELSEED region were prepared in which a
cysteine was introduced in place of a glutamate at residue 381
(
Experiments similar to those shown in
Fig. 5
were conducted using
The studies presented here, using mutants in which Cys
residues have been incorporated into the DELSEED region of the
Importantly, the fact that both
When cross-linking on ECF
The recently published 2.8-Å resolution
structure of MF
The important role of the
The key
observation related to function is that covalent linkage of a
The DELSEED region of the
In addition to the cross-linked products between
F
-type ATPase has been generated by
site-directed mutagenesis as follows:
E381C,
S383C,
E381C/
S108C, and
S383C/
S108C. Treatment of
ECF
isolated from any of these mutants with CuCl
induces disulfide bond formation. For the single mutants,
E381C and
S383C, a disulfide bond is formed in essentially
100% yield between a
subunit and the
subunit, probably at
Cys
based on the recent structure determination of F
(Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E.
(1994) Nature 370, 621-628). In the double mutants, two
[Medline]
disulfide bonds are formed, again in essentially full yield, one
between
and
, the other between a
and the
subunit via Cys
. The same two cross-links are produced
with CuCl
treatment of ECF
F
isolated from either of the double mutants. These results show
that the parts of
around residue 87 (a short
-helix) and the
subunit interact with different
subunits.
to
is nucleotide dependent and highest
in ATP and much lower with ADP in catalytic sites. The yield of
covalent linkage of
to
is also nucleotide dependent but in
this case is highest in ADP and much lower in ATP. Disulfide bond
formation between either
and
, or
and
inhibits
the ATPase activity of the enzyme in proportion to the yield of the
cross-linked product. Chemical modification of the Cys at either
position 381 or 383 of the
subunit inhibits ATPase activity in a
manner that appears to be dependent on the size of the modifying
reagent. These results are as expected if movements of the catalytic
site-containing
subunits relative to the
and
subunits
are an essential part of the cooperativity of the enzyme.
-ATPase is found in the plasma membrane of
bacteria, the inner membrane of mitochondria, and the thylakoid
membrane of chloroplasts. This enzyme catalyzes both ATP synthesis
coupled to an electrochemical gradient and ATP hydrolysis-driven proton
translocation. The enzyme complex is made up of two parts: an F
part composed of five different subunits,
,
,
,
, and
in the molar ratio 3:3:1:1:1 and an F
sector which, in bacterial enzymes such as that from
Escherichia coli (ECF
F
),
(
)
contains subunits a, b, and c in the ratio 1:2:10-12
(reviewed in Cross, 1988; Senior, 1990; Boyer, 1993).
part separated
from the F
by a narrow stalk of around 45 Å in length
(Gogol et al., 1987; Lücken et al., 1990). This
stalk region includes parts of the
,
, and
subunits of
F
and b subunits of the F
part (Capaldi et
al., 1994; Wilkens et al., 1994). Catalytic site events
(ATP synthesis and ATP hydrolysis) appear to be linked to proton
translocation via conformational changes in these stalk-forming
subunits. Electron microscopy studies have also shown that the
and
subunits alternate in a hexagonal arrangement around a
central cavity containing the
subunit (Gogol et al.,
1989a, 1989b). The recent high resolution structure of MF
(mitochondrial F
) (Abrahams et al., 1994)
adds detail to this picture. The
and
subunits have a very
similar fold, each made up of three domains. At the top (farthest from
the F
part) is an amino-terminal six-stranded
barrel.
The more central domain contains the nucleotide-binding domain, which
in the
subunits includes the (three) catalytic sites, while the
subunits contain the (three) non-catalytic sites. The third and
bottom domain (closest to the F
moiety) consists of a
carboxyl-terminal bundle of seven
helices in the
subunit
and six
helices in the
subunit. In the COOH-terminal domain
of the
subunits is the so-called ``DELSEED'' region
which has been implicated in binding of the
subunit (Dallmann
et al., 1992) and which is seen from the x-ray data to
interact tightly with a segment of the
subunit (Abrahams et
al., 1994).
F
by introducing Cys
residues at various sites in the enzyme complex, which can be used to
incorporate reporter groups of the environment of the site, as well as
(conformational) changes that occur around the site during functioning
of the enzyme. In recent work, we have introduced Cys residues into the
and
subunits, and by using fluorescent probes (Turina and
Capaldi, 1994a, 1994b) as well as cross-linking reagents ( e.g. Aggeler et al., 1992, 1993), have provided evidence for
large conformational changes in these subunits during coupling of ATP
hydrolysis to proton translocation. Here we describe experiments in
which we have introduced Cys residues into the DELSEED region of the
subunit in conjunction with mutating a Cys residue into the
COOH-terminal region of the
subunit (
S108C). With these
mutants, we were able to reversibly generate disulfide bonds between
subunits and both the
and
subunits. The reactivity of
the Cys residues in the DELSEED region with maleimides has been
studied. In addition, the functional consequences of such
modifications, and of disulfide bond formation between
and
and between
and
subunits both together and independently,
have been examined.
Construction of Plasmids Containing Mutations in the
unc D and unc C genes
A 1.01-kb NcoI fragment encoding
the COOH-terminal part of the subunit was isolated from pRA100
(Aggeler et al., 1992) and inserted into M13mp18 (New England
Biolabs) in an NcoI site that was created by introduction of
an NcoI linker (Boehringer Mannheim, GmbH.) into the
SmaI site.
381 and a serine at position
383 each with cysteine.
The successful incorporation of the mutations was determined by
sequencing (Sequenase from United States Biochemical Corp., Cleveland,
OH ).
subunit and mutant
S108C, respectively; (ii) the NcoI fragments containing
mutations
E381C and
S383C were then inserted in pRA13 and
pRA14. Finally, the 5.8-kb XhoI/ NsiI fragments from
these four constructs were ligated to the 6.8-kb
XhoI/ NsiI portion of pRA100. The resulting plasmids
were pRA133, pRA134, pRA135, and pRA136, containing the mutations
E381C,
E381C/
S108C,
S383C, and
S383C/
S108C, respectively.
CuCl
ECFInduced Cross-linking of ECF
and ECF
F
-ATPase
was precipitated for 1 h in 70% ammonium sulfate at 4 °C and
collected by centrifugation at 10,000
g for 15 min.
The protein was dissolved in 50 m
M MOPS, pH 7.0, 0.5 m
M EDTA, and 10% glycerol and passed through two consecutive
centrifuge columns in Sephadex G-50 equilibrated in the same buffer at
a concentration between 2-5 mg/ml, as described in Aggeler et
al. (1992). Cross-linking was carried out in either of two ways:
(i) the enzyme was passed through two centrifuge columns, equilibrated
in 50 m
M MOPS, pH 7.0, 10% glycerol, and 20 µ
M CuCl
(Bragg and Hou, 1986; Tozer and Dunn, 1986); (ii)
the ATPase solution was diluted to 0.1 m
M EDTA, supplemented
with 2.5 m
M MgCl
and 2 m
M nucleotide,
followed by incubation with 20 or 50 µ
M CuCl
for 3 h at room temperature.
F
from the sucrose gradient at 1 mg/ml to a
final concentration of 0.5%. After 10 min on ice, 25 µl of 20 mg/ml
egg-lecithin (in 1.5% sodium deoxycholate) was added per ml, followed
by another 10-min incubation on ice. The mixture was then passed
through a Sephadex G-50 column (medium, 1
45 cm) (Brunner
et al., 1978), equilibrated in 50 m
M Tris-HCl, pH
7.5, 2 m
M MgCl
, 2 m
M DTT, and 10%
glycerol. Turbid fractions containing ATPase activity were pooled and
vesicles pelleted by centrifugation for 1 h at 45,000 revolutions/min
at 4 °C in a Beckman Ti60 rotor. The pellets were resuspended in
the same buffer and kept frozen in liquid nitrogen. For cross-linking
with CuCl
, 1 mg of ECF
F
was brought
to a volume of 2 ml with 50 m
M MOPS, pH 7.0, 2 m
M MgCl
, and 10% glycerol and centrifuged twice at 20
°C for 25 min at 70,000 revolutions/min in a Beckman TLA100.2 rotor
to remove reducing agent. The pellet was resuspended in the same buffer
at 0.7-1 mg/ml, samples were supplemented with 2 m
M nucleotide, and cross-linked with 20 µ
M CuCl
for 3 h at room temperature. ATPase activity was measured with or
without prior treatment with 10-20 m
M DTT for 2 to 3 h
at room temperature.
Maleimide Modifications of ECF
The
rate of incorporation of [C]NEM into Cys
of the
subunit of
E381C/
S108C was determined by
reacting ATPase at a concentration of 0.4 mg/ml in 50 m
M MOPS,
pH 7.0, 0.5 m
M EDTA, 5 m
M ATP, and 10% glycerol with
25 µ
M [
C]NEM (specific activity 40
mCi/mmol) at room temperature. Aliquots were withdrawn at time
intervals of 1, 10, 30, 60, and 150 min and quenched by the addition of
10 m
M
L-cysteine. For determination of stoichiometry
of incorporation (mol [
C]NEM/mol F
),
an additional aliquot denatured by 2% SDS was reacted with 200
µ
M [
C]NEM for 150 min. The aliquots
were then electrophoresed on a 10-18% SDS-polyacrylamide gel and
the radioactivity in individual subunits measured as described in
Aggeler et al. (1987). The change of ATPase activity, due to
the incorporation of the maleimides NEM or CM into
Cys
, was determined in a parallel experiment under
identical conditions. Aliquots of a reaction mixture containing 25
µ
M maleimide were quenched by the addition of 20 m
M DTT at the time intervals 1, 10, 30, 60, and 150 min and ATPase
activity determined.
Other Methods
E. coli strains used for
routine cloning procedures (Davis et al., 1986; Maniatis
et al., 1982) and site-directed mutagenesis according to
Kunkel et al. (1987) were XL1-Blue from Stratagene
and CJ236 from New England Biolabs. For expression of mutant
ATPase and synthase, the plasmids pRA133-pRA136 were used to transform
the uncstrain AN888 (Aggeler et al. 1992). ECF
and ECF
F
were
isolated as described by Wise et al. (1981), modified by Gogol
et al. (1989a) and Aggeler et al. (1987).
-Depleted ECF
was prepared by the use of the
monoclonal antibody
-4 as described by Dunn (1986). Protein
concentrations were determined using the BCA protein assay from Pierce.
ATPase activity was measured with a regenerating system described by
Lötscher et al. (1984). For the analysis of cross-link
products, SDS containing 10-18% polyacrylamide gels were run
(Laemmli, 1970), and polypeptides were blotted on nitrocellulose
membranes and identified with monoclonal antibodies (Aggeler et
al., 1990; Mendel-Hartvig and Capaldi, 1991a). Protein bands on
gels were visualized by staining with Coomassie Brilliant Blue R
according to Downer et al. (1976).
E381C) or a cysteine replacing a serine at residue 383
(
S383C). In addition, double mutants were generated with the same
mutations in the DELSEED region along with the mutation of a serine to
a cysteine at position 108 of the
subunit (
E381C/
S108C;
S383C/
S108C). The four different mutants grew well, and the
activities of the isolated F
of each and
F
F
from the two double mutants were similar to
wild-type enzyme, i.e. in the range 9-15 µmol
ATP/min/mg for the F
and 25-35 µmol/min/mg for
the F
F
preparations. All four mutants showed
normal LDAO activation, i.e. 8-fold for the F
and
3-4-fold for the F
F
.
The DELSEED Region Interacts with Both the
CuCl and
Subunits
was used to induce disulfide bond
formation using the isolated F
and F
F
from the various mutants with or without gel filtration
centrifugation. Fig. 1 shows data for the two double mutants. Passage
of ECF
through a centrifuge column in the presence of 20
µ
M of CuCl
generated four major cross-link
products, as revealed by SDS-polyacrylamide gel electrophoresis of
samples in the absence of reducing agents (Fig. 1), which Western
blotting with monoclonal antibodies to the individual subunits of the
enzyme showed to involve
+
,
+
,
+
, and
+
(result not shown). The effect
of cross-linking on ATPase activity is listed above each lane. The
residual activity of both mutants was less than 10% of that of the
untreated enzyme. The only cross-linked products seen in wild-type
ECF
treated similarly were between the
and
subunits (results not shown, but see Mendel-Hartvig and Capaldi,
1991b). This cross-linking of
to
had no effect on ATPase
activity.
Figure 1:
Cross-linking of
ECF-ATPase from double mutants on CuCl
containing centrifuge columns. 1 mg of ATPase, precipitated with
ammonium sulfate, was dissolved in 0.2 ml of 50 m
M MOPS, pH
7.0, 0.5 m
M EDTA, and 10% glycerol and passed twice through a
centrifuge column in the same buffer. Half of the sample was then
applied on two consecutive columns containing 20 µ
M CuCl
and no EDTA. The other half was left untreated.
After addition of 5 m
M EDTA, ATPase activities were measured
and expressed as relative values with the untreated sample as 100%.
50-µg samples were electrophoresed on an 10-18%
polyacrylamide gel after addition of 20 m
M NEM and
dissociation buffer without reducing agent.
E381C/
S108C
without ( lane 1) and with CuCl
treatment ( lane
2);
S383C/
S108C without ( lane 3) and with
CuCl
treatment ( lane
4).
Cross-linking of to
via disulfide bond
formation must involve the introduced Cys in the DELSEED region and the
introduced Cys
of the
subunit. The yield of this
product is close to 100% in both mutants, based on the disappearance of
the
subunit. The cross-linking between
and
must
involve the Cys introduced into the DELSEED region and either
Cys
or Cys
, the two endogenous cysteines of
the subunit. Based on the recently published x-ray structure of
MF
(Abrahams et al., 1994), it is more likely that
Cys
is involved. The yield of the
-
cross-link
is 90-95% in the mutant
E381C/
S108C and somewhat less
in the mutant
S383C/
S108C. In wild-type ECF
, the
yield of the
+
cross-linked product is essentially 100%.
In the DELSEED mutants, the yield of
+
is significantly
less because of the presence of a
+
cross-link product
(discussed later). In the mutant
S383C/
S108C, the
+
cross-linked product migrates differently from
+
; this is not the case for the mutant
E381C/
S108C (Fig. 1).
Disulfide Bond Formation between
When ECF and
Inhibits
ATPase Activity and Is Nucleotide-dependent
from either double mutant was reacted with CuCl
in
the absence of the column centrifugation step, the same cross-link
products were obtained. However, the yield in which the disulfide bonds
were formed was now determined by the concentration of CuCl
used, and the length of time for which samples were incubated
before the reaction was stopped by addition of EDTA to chelate the
metal ion. Preliminary experiments showed that disulfide bond formation
between
and
was much greater than that between
+
,
+
, or
+
under the
conditions of cross-linking chosen. This made it possible to examine
both the nucleotide dependence of this specific cross-link and its
effect on ATPase activity (Fig. 2). As shown for the mutant
E381C/
S108C, the yield of the
+
cross-link
product was high in the absence of nucleotides, i.e. in
Mg
alone ( lane 1) and high in
Mg
+ADP, either added directly ( lane 3)
or as generated by addition of ATP+Mg
followed
by turnover of the enzyme ( lane 2). However, the yield of this
cross-link was very low (less than 10%) in the presence of
AMP
PNP+Mg
( lane 4) or with
ATP+Mg
when azide was added to prevent enzyme
turnover (result not shown). The cross-linking yield obtained with the
mutant S383C/
S108C was much lower (lessthan 10%). The
inhibition of ATPase activity was in proportion to the yield of
cross-linked products, i.e. in the range 70-75% in ADP
for the mutant
E381C/
S108C, but below 10% in the presence of
AMP
PNP. Fig. 2shows also the important finding that the
cross-linking could be reversed by adding DTT. This is true, whether
the cross-linking was conducted by adding CuCl
with or
without the gel filtration centrifugation step. The activity
determination reported above each lane in Fig. 2, and in
subsequent figures, is the rate of ATPase hydrolysis of the
cross-linked enzyme as a percentage of the activity of the same sample
after adding DTT to break the linkages.
Figure 2:
Cross-linking of ECF from
E381C/
S108C without the use of centrifuge columns. 2.5 m
M MgCl
was added to ATPase in 50 m
M MOPS, pH
7.0, 0.1 m
M EDTA, and 10% glycerol at 0.3 mg/ml. 0.2 ml
samples were supplemented with no nucleotides ( lanes 1 and
5), 2 m
M ATP ( lanes 2 and 6), 2
m
M ADP ( lanes 3 and 7) and 2 m
M AMP
PNP ( lanes 4 and 8), respectively,
incubated for 10 min at room temperature, and treated with 20
µ
M CuCl
for 3 h. 5 m
M EDTA was added,
and activities were measured after 2 h in the absence ( lanes
1-4) or presence of 10 m
M DTT ( lanes
5-8). 30 µg of enzyme was applied per lane after
addition of 40 m
M NEM and DTT-free dissociation buffer. ATPase
activities are expressed as percentage of DTT-treated samples. The
ATPase activities of the control samples in lanes 5-7 were 11 µmol of ATP hydrolyzed/min/mg. The activity of the
sample reacted with AMP
PNP and then diluted in the assay buffer
was 5 µmol of ATP hydrolyzed/min/mg.
Disulfide Bond Formation between
The nucleotide dependence and activity
effects of the and
Inhibits
ATPase Activity and Has the Opposite Nucleotide Dependence of the
+
Linkage
+
cross-link could be examined
independently of the
+
cross-link by using the single
mutants in which an introduced cysteine was present in the DELSEED
region, but the
subunit was wild-type. Fig. 3 A shows
cross-linking of the single mutants
E381C and
S383C using the
CuCl
gel filtration centrifugation procedure. The 90% yield
of cross-linking of
to
gave a 90% inhibition of ATPase
activity. Fig. 3 B shows cross-linking of
to
in the
mutant
E381C generated by addition of 50 µ
M CuCl
without a column step. The yield of
+
was low in Mg
+ADP ( lane
1), or in Mg
+ADP+P
( lane 2). In contrast, the yield of this cross-linked
product was high with Mg
+ATP added in the
presence of azide to prevent enzyme turnover ( lane 3) or with
Mg
+AMP
PNP added ( lane 4). Under
all nucleotide conditions, the inhibition of activity mirrored the
extent of
+
cross-linking, being around 10% with ADP in
catalytic sites (the experiment in Fig. 3), and 70% with ATP
bound. The overall yield of cross-linked products obtained with the
mutant
S383C was much lower than that for
E381C, when
experiments such as those in Fig. 3 B were conducted under the
identical conditions of CuCl
reaction.
Figure 3:
Cross-linking of ECF from
single mutants
E381C and
S383C. A, 0.7 mg of ATPase
from mutants
E381C ( lanes 1 and 2) and
S383C ( lanes 3 and 4) in 0.2 ml was passed, as
described in Fig. 1, twice through EDTA containing columns, followed by
two CuCl
-containing columns. The cross-linked material (1.6
mg/ml) was either incubated in the absence ( lanes 1 and
3) or presence of 20 m
M DTT ( lanes 2 and
4). 60 µg of ATPase was applied per lane. B, 2.5
m
M MgCl
was added to 0.2-ml aliquots of 0.3 mg/ml
ATPase from mutant
E381C in 50 m
M MOPS, pH 7.0, 0.1
m
M EDTA and 10% glycerol, followed by 2 m
M ADP
( lane 1), 2 m
M ADP + 2 m
M P
( lane 2), 2 m
M NaN
+ 2 m
M ATP ( lane 3), and 2 m
M AMP
PNP ( lane
4). Samples were treated for 4 h with 50 µ
M CuCl
at room temperature and 30 µg of protein
applied on a gel as described in Fig. 2. The ATPase activities of
samples used in the experiments in lanes 2 and 4 of
part A and the controls for lanes 1 and 2 of
part B were in the range 7-9 µmol of ATP
hydrolyzed/min/mg. The activity of the control for lane 3B, in
which the sample with azide had been diluted in the assay buffer, was
4.3 and that for lane 4B where AMP
PNP had been added was
4.9 µmol of ATP hydrolyzed/min/mg.
The Nucleotide Dependence of the
Our previous
work has shown that nucleotide-dependent conformational changes
involving the +
Cross-linked Product Depends on the
Subunit
subunit depend on the presence of the
subunit
(Aggeler and Capaldi, 1993; Turina and Capaldi, 1994a). To examine the
effect of the
subunit on
+
cross-linking,
ECF
from the mutant
E381C was depleted of the
subunit by affinity chromatography using a monoclonal antibody against
the
subunit (Dunn, 1986). A comparison of the gel profiles of
enzyme cross-linked under different nucleotide conditions by CuCl
(Fig. 4) shows that there was essentially no nucleotide
dependence of cross-linking of
to
in
-free
ECF
. This was confirmed by activity measurements which gave
around 90% residual activity for all samples.
Chemical Modification of the DELSEED Region Inhibits
ATPase Activity
To examine the relative exposure of the Cys
residues introduced into the DELSEED region, ECFfrom the
mutants
E381C and
S383C was reacted with
[
C]NEM and with the larger maleimide, CM.
[
C]NEM was found to label the introduced Cys at
either 381 or 383 in all three
subunits. Fig. 5 correlates ATPase
activity and the incorporation of NEM into
E381C. Incorporation of
close to 3 mol of the maleimide caused no inhibition of ATPase
activity. Modification of the Cys at 381 with CM at the same
concentration and for the same time intervals had a much more dramatic
effect on activity. With this bulky maleimide, inhibition occurred
rapidly in a time course that suggests that only 1 mol is necessary for
maximal inhibition (95%).
-free ECF
from the
mutant
E381C, and identical results were obtained. Therefore, any
observed effects of maleimide modification must be due to changes at
the interface between
and
, rather than the
and
subunits. In one set of experiments, the effect of modifying the free
subunit, i.e. the one not associated with either the
or
subunit, was examined. ECF
from the mutant
E381C/
S108C was reacted with CuCl
to induce
cross-linking of one
subunit to
and a second
to
in essentially 100% yield. The cross-linked sample was then reacted
with CM to modify the fraction of Cys of the free
subunit that
had not been cross-linked with the
subunit (estimated at around
60%). Cross-links were then broken by DTT treatment, and the effect of
incorporating the maleimide into the free
subunit was measured.
The reaction with CM gave 60% inhibition of the ATPase activity.
Figure 5:
Correlation of ATPase activity of
ECF from
E381C/
S108C with incorporation of
[
C]NEM. ATPase from the
/
double
mutant
E381C/
S108C was reacted at 0.4 mg/ml in 50 m
M MOPS, pH 7.0, 0.5 m
M EDTA, 5 m
M ATP, and 10%
glycerol with 25 m
M [
C] N-ethylmaleimide. The
incorporation of [
C]NEM into
Cys
was determined at 1, 10, 30, 60, and 150 min (mol
[
C]NEM/mol F
( open
circles)). In a parallel experiment the change of ATPase activity
due to the incorporation of 25 µ
M NEM ( circles)
or 25 µ
M CM ( squares) into
Cys
of
E381C/
S108C was determined at 1, 10, 30, 60, and 150
min.
Disulfide Bond Formation in
ECF
FF
F
was
isolated from both double mutants and examined for disulfide bond
formation upon CuCl
addition. Fig. 6 A shows data
for the mutant
S383C/
S108C on treatment with 20 µ
M CuCl
in the presence of ATP+Mg
and without the column centrifugation step (which is difficult to
perform with the vesicular enzyme preparations). Both the
-
and
-
subunit cross-links were formed. However, no
cross-linking between
or
plus
was obtained in the
intact ECF
F
. As with ECF
,
cross-linking between
and
, and
and
, caused
inhibition of ATPase activity in proportion to the yield of the two
cross-linked products. The cross-links were destroyed and activity
restored to greater than 90% by addition of 20 m
M DTT.
Fig. 6B shows disulfide bond formation induced by 20
µ
M CuCl
in the mutant
E381C/
S108C
under different nucleotide conditions. In the presence of
ADP+Mg
( lanes 1 and 2), the
yield of the
-
cross-linked product was much greater than
that of
-
, while in the presence of ATP+Mg
(as ATP+Mg
+azide in lane 5 and AMP
PNP+Mg
in lane 6), the
yield of
-
was much higher than that of
-
subunits.
Therefore, the same nucleotide dependence of cross-linked products is
seen in ECF
F
as in ECF
alone.
Figure 6:
CuCl induced cross-linking of
ECF
F
from the two
/
double mutants.
A, 0.12 mg of reconstituted ECF
F
from
S383C/
S108C in 0.2 ml of 50 m
M MOPS, pH 7.0, 2
m
M MgCl
2 m
M ATP, and 10% glycerol was
incubated with 20 µ
M CuCl
for 3 h at room
temperature. The sample was split in half and, after incubation for
another 3 h in the absence ( lane 2) or presence of 20 m
M DTT ( lane 3), 10 µl of 1
M NEM in
Me
SO was added to each 100-µl aliquot. Dissociation
buffer without reducing agent was added, and 60 µg of protein was
loaded on each lane of an 10-18% polyacrylamide gel. As a
control, CuCl
-treated ECF
(see Fig. 1) was
applied ( lane 1). B, 1.5 mg of reconstituted
ECF
F
from
E381C/
S108C was suspended
in 1.5 ml of 50 m
M MOPS, pH 7.0, 2 m
M MgCl
, and 10% glycerol. To 0.3-ml aliquots, 2 m
M ATP ( lanes 1 and 3), 2 m
M ADP + 2
m
M P
( lanes 2 and 4), 2 m
M NaN
+ 2 m
M ATP ( lanes 5 and
7), and 2 m
M AMP
PNP ( lanes 6 and
8) were added, respectively. After incubation for 10 min and
CuCl
treatment for 3 h, 100-µl samples were taken and
not reduced ( lanes 1, 2, 5, and 6)
or reduced with 20 m
M DTT for 2 h ( lanes 3,
4, 7, and 8) and then quenched with 100
m
M NEM. 80 µg of protein was loaded per lane. Only the top
portion of the 10-18% gel is shown. The ATPase activity of the
control lanes was 19 µmol of ATP hydrolyzed/min/mg for lanes 3 and 4, and 10 and 14 for lanes 7 and 8 which had seen azide and AMP
PNP,
respectively.
subunit, provide new data on the structure of ECF
and help
describe nucleotide-dependent conformational changes occurring in the
complex. Using single mutants, in which Cys residues have been
introduced into the DELSEED region, and double mutants with alterations
of the DELSEED region along with introduction of a Cys into the
subunit at position 108, it was possible to obtain disulfide bond
formation between
and
and/or
and
. The
cross-linking of
to
is not surprising, based on the recent
x-ray structure which shows the proximity of Cys
of
to the DELSEED region (Abrahams et al., 1994). The
cross-linking of
to
from a Cys at 108 of
to the
DELSEED region was predicted from earlier work (Lötscher and
Capaldi, 1984; Dallmann et al., 1992). Dallmann et al. (1992) have shown that the cross-link of
to
by the
water-soluble carbodiimide
1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide involves the
DELSEED region and that part of the
subunit COOH-terminal to
residue 96. The cross-link, which was shown to be an ester linkage, was
not seen in the mutant
S108C, implying that 1 of the 3 serines,
either at 106, 107, or most likely at 108, was involved. The data
presented here confirm that it is the region of
near residue 108
that binds to the
subunit.
+
and
+
cross-links can be obtained in
near 100% yield in the same ECF
molecule when the double
mutants
E381C/
S108C or
S383C/
S108C were used,
establishes that the small
-helix of the
subunit (around
residue 87) and the COOH-terminal part of the
subunit, interact
with different
subunits. Cross-linking of these mutants in the
high yields obtained here means that it is now possible to
experimentally distinguish the three
subunits in any F
molecule, based on the interaction of these catalytic subunits
with the small subunits. This will be very useful in future studies to
examine the affinity of the three different
subunits for ADP,
ATP, etc.
was conducted by
addition of CuCl
without the column procedure, the rate
and/or yield of the cross-linking of both
-
and
-
was much less in
S383C/
S108C than in
E381C/
S108C.
It appears, therefore, that the Glu at 381 is better positioned for
interaction with both the endogenous Cys of
(Cys
)
and Cys
of the
subunit than is the Ser at 383 of
the
subunit.
(Abrahams et al., 1994) has
provided details of the arrangement of the
,
, and
subunits and new insights into catalysis by the enzyme. Briefly, this
structure confirmed that the three
subunits are in different
conformations and are structurally different, with the asymmetry
related both to nucleotide occupancy of catalytic sites and
interactions of the
subunit. Thus, in one
subunit the
catalytic site is closed around an Mg
ADP
(
) and the
subunit is closest to the
NH
-terminal
-helical region of the
subunit. A
second
subunit has a partly open catalytic site containing
AMP
PNP+Mg
(
), and it is
this
subunit which interacts with the short
-helix of the
subunit containing Cys
. The third
subunit is
empty of nucleotide (
), and this
has
interactions with the long COOH-terminal
-helix of the
subunit. It is interesting then that there is nucleotide dependence of
(the rate of) cross-linking between the DELSEED region and the
and
subunits. The disulfide link between the DELSEED region of
that
subunit which interacts with the short central
-helix
of the
subunit is generated in higher yield with uncleaved ATP
(either ATP+azide, or AMP
PNP) in catalytic sites. This
suggests that the interaction between the region of
and
is
more stable with ATP than ADP in the catalytic site. In contrast, the
interaction between the
subunit and another
subunit is more
stable in ADP than ATP, based on the data presented here, and on our
previous studies of the interaction of the
with the core
ECF
complex (Mendel-Hartvig and Capaldi, 1991a; Aggeler
et al., 1992).
subunit in
determining the properties of the F
is now well
established. It has been shown that
-free ECF
has both
altered rates of unisite ATP hydrolysis, due to an increased off rate
of product, P
(Dunn et al., 1987), and a greatly
enhanced (10-fold increased) rate of multisite ATP hydrolysis (Dunn
et al., 1987; Aggeler et al., 1990; Mendel-Hartvig
and Capaldi, 1991a). The functional effect of the
subunit could
result from the direct interaction of the
with a
subunit,
or with the
subunit, or both. We have previously found that the
nucleotide-dependent conformational changes in the
subunit, as
measured by fluorescence probes bound to a cysteine introduced at
position 8 of the
subunit, are lost on removal of the
subunit (Turina and Capaldi, 1994a), as is the nucleotide dependence of
cross-linking from this site to the
subunit by the
tetrafluorophenylazide maleimide series of bifunctional reagents
(Aggeler and Capaldi, 1993). Here we show that the nucleotide
dependence of disulfide bond formation between the short central
-helix of
and
is lost on removal of the
subunit. Taken together, these data suggest that the
subunit may regulate the functioning of the F
, at least in
part, by controlling the conformation of the
subunit.
subunit to the
subunit through a single disulfide bond, from
either the Cys at position 381 or position 383 in
to a Cys at
position 108 of
, blocks ATPase activity. Similarly, the linkage
of the Cys at 381 or 383 in
to a Cys at position 87 in the
subunit completely inhibits enzyme activity. When the disulfide bonds
are broken, full activity is restored.
subunit is arranged as a loop linking two
-helices and is in
a domain distinct from that containing the catalytic site. As with much
of the
subunit, the DELSEED region appears unaltered (and the
structures can be superimposed) whether ATP or ADP are bound in
catalytic sites (Abrahams et al., 1994). It is unlikely,
therefore, that disulfide bond formation between
and
and
the DELSEED region causes conformational changes within the
subunit which alter nucleotide binding in the catalytic sites. Rather,
the most straightforward interpretation of the inhibitory effect of
covalent bond formation between
-
or
-
is that
these linkages block movements of the (
+)
subunits
relative to
and
that are necessary for catalytic
cooperativity. Based on our previous studies ( e.g. Gogol
et al., 1990; Wilkens and Capaldi, 1994) and on the features
of the structure of the F
moiety (Abrahams et al.,
1994), we believe that there are translocations of the
and
subunits between different
subunits driven by ATP hydrolysis (and
ATP synthesis in the reverse direction of enzyme functioning) which, as
first suggested by Boyer and Kohlbrenner (1981), cause alternation of
catalytic sites between the three nucleotide-binding states ( i.e. between
,
, and
). The chemical modification studies described here
are similarly best interpreted in terms of an effect on movement of
subunits relative to one another. Incorporation of a small molecule
(NEM) into the DELSEED region has no significant effect on the
functioning of the enzyme even when close to 3 mol of reagent are bound
and cannot therefore be inducing major conformational changes in the
subunit. In contrast, when the more bulky maleimide CM was bound
into the
subunit at position 381 at 1 mol equivalent ( i.e. 1 mol of CM/mol of ECF
), activity was dramatically
reduced. Inhibition of activity was observed when free
subunit
( i.e. that
not in contact with
or
) was
modified. Such a result would be expected if the incorporation of CM
prevents the free
subunit from ``switching'' to become
associated with the
or
subunits during catalytic site
alternation.
and
, and
and
, discussed above, CuCl
treatment of the double mutant led to disulfide bond formation
between
and
, and
and
. The cross-linking of
to
by disulfide bond formation has been observed before and
obtained in essentially 100% yield (Bragg and Hou, 1986; Tozer and
Dunn, 1986; Mendel-Hartvig and Capaldi, 1991b). It involves Cys
of the
subunit (Mendel-Hartvig and Capaldi, 1991b). In the
DELSEED mutants, there is competition between the
and
subunits for the
subunit. In contrast to the linkage of
to
, or
to
, there is no disulfide bond formation between
and
, or
and
in ECF
F
,
implying that the association of the
subunit in ECF
is not physiological.
,
soluble portion of the E. coli F
F
ATP
synthase; CM, N-[4-[7-(diethylamino)-4
methyl]coumarin-3-yl]maleimide; NEM,
N-ethylmaleimide; DTT, dithiothreitol; AMP
PNP,
5`-adenylyl-
,
-imidodiphosphate; LDAO,
N, N-dimethyldodecylamine- N-oxide; kb,
kilobase(s).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.