(Received for publication, May 12, 1995; and in revised form, June 16, 1995)
From the
The asymmetry of Escherichia coli F -ATPase
(ECF
) has been explored in chemical modification
experiments involving two mutant enzyme preparations. One mutant
contains a cysteine (Cys) at position 149 of the
subunit, along
with conversion of a Val to Ala at residue 198 to suppress the
deleterious effect of the Cys for Gly at 149 mutation (mutant
G149C:V198A). The second mutant has these mutations and also Cys
residues at positions 381 of
and 108 of the
subunit (mutant
G149C:V198A:E381C/
S108C). On CuCl
treatment of
this second mutant, there is cross-linking of one copy of the
subunit to
via the Cys at 381, a second to the
subunit
(between
Cys
and
Cys
), while the
third
subunit in the ECF
complex is mostly free (some
cross-linking to
); thereby distinguishing the three
subunits as
,
, and
, respectively. Both mutants have ATPase activities
similar to wild-type enzyme.
Under all nucleotide conditions,
including with essentially nucleotide-free enzyme, the three different
subunits were found to react differently with N-ethylmaleimide (NEM) which reacts with Cys
,
dicyclohexyl carbodiimide (DCCD) which reacts with Glu
,
and 7-chloro-4-nitrobenzofurazan (NbfCl) which reacts with
Tyr
. Thus,
reacted with DCCD but
not NEM or NbfCl;
was reactive with all three
reagents;
reacted with NEM, but was poorly
reactive to DCCD or NbfCl. There was a strong nucleotide dependence of
the reaction of Cys
in
(but not in
) with NEM, indicative of the important role that
the
subunit plays in functioning of the enzyme.
An FF
type ATPase is found in the plasma
membrane of bacteria, as well as chloroplast thylakoid and
mitochondrial inner membranes, and this enzyme functions to synthesize
ATP in response to a light or respiratory chain-driven proton gradient.
It is a reversible enzyme, also working as an ATPase, using the
hydrolysis of ATP to establish a pH gradient for subsequent use in ion
transport processes. The simplest F
F
type
ATPases structurally are those from bacteria. The enzyme from Escherichia coli (ECF
F
) (
)is made up of a total of eight different subunits, five in
the ECF
part (
,
,
,
, and
) and
three in the F
part (a, b
,
c
) (Futai and Kanazawa 1983; Walker et al. 1984). The F
part of the enzyme from bacteria,
mitochondria, and chloroplasts is remarkably similar, particularly with
respect to
,
, and
subunits (Cross, 1988; Senior, 1990;
Boyer, 1993). Earlier chemical studies had pointed to an intrinsic
asymmetry of F
, which is expected given a stoichiometry of
three copies each of
and
subunits but only one copy of
,
, and
subunits. For example, it was shown that one of
the
subunits, which contain the catalytic sites, could be
cross-linked to the
subunit by the water-soluble carbodiimide EDC
(Lötscher et al., 1984a). DCCD was found
to react with only two of the three
subunits, excluding the one
that is linked to the
subunit (Lötscher et al., 1984a, 1984b; Tommasino and Capaldi, 1985; Stan-Lotter
and Bragg, 1986a). Also, it has been shown that NbfCl and DCCD can be
reacted with different
subunits (Stan-Lotter and Bragg, 1986b).
In addition, clear asymmetry of the F
was observed on
examination of F
by cryoelectron microscopy (Gogol et
al., 1989a; Wilkens and Capaldi, 1994), as well as in studies of
negatively stained specimens of the enzyme (Boekema and
Böttcher, 1992).
The recent high resolution
x-ray structure of MF (Abrahams et al., 1994)
confirms and provides more details of the asymmetry of the enzyme
molecule in relation to the interaction of small subunits and
nucleotide occupancy of catalytic sites. In the crystal form examined,
one of the
subunits (
) contains ADP +
Mg
, and this
is close to and may make contact
with the N-terminal
-helical part of the
subunit. A second
subunit, containing bound AMP-PNP + Mg
(
), is linked via the DELSEED region to a short
-helix (residues 82-99 in the numbering system of
ECF
) in the central part of the
subunit. The third
subunit, which is empty, (
), has several
interactions with the C-terminal
-helical part of the
subunit.
Unanswered, as yet, is whether differences in the structure
of -
subunit pairs such as observed by Abrahams et
al.(1994) are due to the association of small subunits, nucleotide
binding, or both interactions. This is particularly relevant as a
crystal form of rat liver F
has been reported which is
claimed to be symmetrical with respect to features of the
and
subunits and appears to have the small subunits, including
and
, scrambled (Bianchet et al., 1991; Pedersen et
al., 1995).
We are using a combination of molecular biological
and biophysical approaches to examine structure-function relationships
in ECFF
(e.g. Aggeler and Capaldi,
1992; Turina and Capaldi, 1994a, 1994b) Here, we describe studies in a
mutant
G149C:V198A, a functioning enzyme with a Cys residue in the
catalytic site region (Iwamoto et al., 1993), thereby allowing
probing of the conformation of this region under different nucleotide
conditions. We have used this mutant, and a second mutant which
includes the above mutations along with a Cys introduced into the
DELSEED region of the
subunit (through which different
subunits can be cross-linked to
and
subunits, respectively)
to study the asymmetry of the enzyme in relation to different
nucleotide occupancies of catalytic sites.
Prior to modification by NbfCl,
ECF was reacted with CuCl
to induce formation
of cross-links and the reaction stopped by addition of 1 mM EDTA. NEM (200 µM) was then added (for 1 h) to modify
accessible Cys residues prior to reaction with NbfCl. Excess NEM was
removed by transfer to a buffer containing 50 mM
Tris-H
SO
, 1 mM EDTA, and 10% (v/v)
glycerol (Tris pH 7.5 buffer) by column centrifugation. Cross-linked
ECF
(2-5 µM) was reacted with 500
µM NbfCl (10 mM ethanolic stock) in Tris pH 7.5
buffer for 1 h at 30 °C then unreacted NbfCl was removed by passage
through a centrifuge column equilibrated in MOPS pH 7.0 buffer. The
Nbf-modified F
was split into two aliquots, one of which
was further modified by reaction with [
C]DCCD
(200 µM) for 3 h at room temperature, after which time
unreacted [
C]DCCD was quenched by addition of 10
mM NaOAc, pH 5.2. Aliquots were subjected to modified SDS-PAGE
where the pH of the separating gel was lowered from 8.6 to 8.0 in order
to increase the stability of the Tyr
-Nbf adduct.
where P = Cys-1-[
C]NEM
adduct, P
= Cys-2-[
C]NEM
adduct, C = concentration of protein (ECF
), B
= initial concentration of ligand
([
C]NEM), k
, k
= second-order reaction constants for
Cys-1, Cys-2, respectively, and t = time.
The double mutant G149C:V198A was obtained by Futai and
colleagues as an enzymatically active revertant of the deleterious
mutation
G149C (Iwamoto et al., 1993). For our purposes,
these two mutations were inserted into the plasmid pRA100 as described
under ``Experimental Procedures.'' The ATPase activity of
ECF
isolated from the mutant was 8-14 µmol of ATP
hydrolyzed/min/mg as assayed at pH 7.5 in the presence of 2 mM ATP and 5 mM MgCl
, an activity in the same
range as that of the wild-type enzyme when measured under the same
conditions. After removal of the
subunit by trypsin cleavage, the
activity of the mutant
G149C:V198A increased to 40-55
µmol of ATP hydrolyzed/min/mg, again the same value obtained with
wild-type ECF
.
Figure 1:
Kinetics of incorporation of
[C]NEM into
Cys
of mutant
ECF
(
G149C:V198A) under different nucleotide
conditions. ECF
(2 µM) in MOPS pH 7.0 buffer
containing EDTA (A, crosses), ATP + EDTA (B, circles), AMP-PNP + Mg
(C, triangles), or ADP + Mg
+ P
(D, diamonds) was
incubated with 25 µM [
C]NEM for
varying times up to 2 h. Aliquots were withdrawn at the times
indicated, quenched with 10 mML-Cys, and
electrophoresed on a 10-18% linear gradient SDS-polyacrylamide
gel. The incorporation of radioactivity into
Cys
(mol [
C]NEM/mol F
) was
determined in gel slices of
subunits (described under
``Experimental Procedures''). The curve fits represent
modification of 2 Cys residues according to .
One possible explanation of the
observed asymmetry described above is that 1 mol of nucleotide is
tightly bound in catalytic sites rather than in non-catalytic sites
after two passages through centrifuge columns. To avoid this ambiguity,
labeling studies were also conducted with ECF freed of
nucleotide according to Senior et al.(1992). In this
procedure, the enzyme is twice precipitated from solution in
(NH
)
SO
and then subjected to gel
filtration in a buffer containing 50% glycerol, 4 mM EDTA.
After such treatment, the
G149C:V198A mutant was found to retain
less than 0.3 mol of nucleotide/mol of enzyme. Reaction of this
essentially nucleotide-free enzyme with [
C]NEM
still gave the same labeling profiles as that obtained with enzyme
retaining from 1 to 2 mol of tightly bound nucleotide (result not
shown).
The profile of [C]NEM reaction with
Cys
of ECF
in the presence of 5 mM ATP (trace B) or 5 mM AMP-PNP + 5 mM MgCl
(trace C), conditions in which all three
catalytic sites would be occupied by nucleotide, are also shown in Fig. 1. The rates of incorporation of NEM under these conditions
were 84 M
s
and 16 M
s
in ATP, and 81 M
s
and 17 M
s
in AMP-PNP,
respectively in Fig. 1. The data are similar to those obtained
for ECF
without added nucleotides (trace A),
except for an approximately 2-fold lower rate of incorporation of the
second mole of NEM.
[C]NEM modification of
ECF
in the presence of 5 mM ADP + 5 mg
Mg
+ 5 mM P
, both added
directly or generated by catalytic turnover of added ATP (Fig. 1, trace D), also resulted in incorporation of
only 2 mol of reagent. However, with all three catalytic sites occupied
by ADP, the rates of NEM incorporation were considerably slower, i.e. 10 and 1 M
s
leading to incorporation of less that 1 mol of reagent under the
conditions described in Fig. 1(i.e. 25
µM, 2 h of incubation). The stoichiometry of incorporation
and calculation of kinetic constants (notably k
),
under this nucleotide condition, was therefore determined using a
higher concentration of [
C]NEM (200
µM) when 2 mol of reagent could be reacted with the
enzyme.
In different experiments, the absolute rates of NEM incorporation varied by a factor of around 20% with the relative differences observed under different nucleotide conditions always maintained. From Fig. 1, it is clear that the conformation of one or both of the NEM reactive, catalytic site regions is significantly different when ADP is bound compared with when ATP is bound or when the catalytic sites are empty.
Figure 2:
Kinetics of incorporation of
[C] NEM into
Cys
of mutant
ECF
F
(
G149C:V198A) under different
nucleotide conditions. F
F
(5 µM)
in MOPS pH 7.0 buffer containing AMP-PNP + Mg
(triangles), or ATP + Mg
(ADP
+ P
+ Mg after catalysis) (squares), was
incubated with 200 µM [
C]NEM for up
to 2 h. At the times indicated, the incorporation of radioactivity into
Cys
of
subunits (mol
[
C]NEM/mol F
) was determined as
described in Fig. 1.
The
mutant G149C:V198A:
E381C/
S108C had an ATPase activity of
12-16 µmol of ATP hydrolyzed/min/mg, within the range
obtained for wild-type enzyme. ECF
for this mutant was
reacted first with CuCl
to generate disulfide bonds between
Cys
residues and the
and
subunits,
respectively (Fig. 3). The cross-linked enzyme was then reacted
with [
C]NEM which becomes incorporated into
Cys
along with any
Cys
not
involved in disulfide bond formation with
or
. The rate of
reaction of NEM with the different
subunits was followed by
slicing and counting
C incorporated into the cross-linked
products as well as into the non-cross-linked
subunit, as a
function of time. As shown in Fig. 4A, there was no
incorporation of [
C]NEM into the
-
cross-linked product under any of the nucleotide conditions tested.
Therefore, the
subunit, to which the short central
-helix of
is bound, is the one most shielded from maleimide reaction. There
was incorporation of up to 1 mol of
C into the
-
cross-linked product, the rate of which was nucleotide dependent (Fig. 4B). NEM modification of Cys
in
this
subunit occurred at the fast rate observed for the reaction
of Cys
in the mutant
G149C:V198A when ATP +
EDTA was present, but occurred at the slower of the two rates in this
mutant when ADP + Mg
+ P
was
present. The nucleotide dependent switching in rates of modification of
Cys
in the
subunit linked to
is, therefore,
more than 50-fold. In the time course of the labeling experiment, two
NEM molecules were bound into the free
subunit, 1 mol into
Cys
and the second into the (free) Cys
.
There was no significant nucleotide dependence of the rates of
modification of these sites in the free
subunit (Fig. 4C). Experiments with the mutant
E381C have
shown no alteration in the rate of [
C]NEM
modification of the Cys at 381 under different nucleotide conditions
(results not shown).
Figure 3:
Disulfide bond formation between
Cys
and
,
, and
subunits of mutant
ECF
(
G149C:V198A:E381C/
S108C). Coomassie
Brilliant Blue-stained 10-18% linear gradient SDS-PAGE of
untreated (lane 1) and cross-linked mutant (lane 2)
ECF
. Cross-links were induced by passage of ECF
(7.5-12.5 µM) through two consecutive
CuCl
-containing centrifuge columns then the reaction
stopped by the addition of 1 mM EDTA. 40 µg aliquots were
incubated in dissociation buffer in the absence of DTT prior to
SDS-PAGE.
Figure 4:
Kinetics of incorporation of
[C]NEM into cross-linked
subunits of
mutant ECF
(
G149C:V198A:E381C/
S108C).
Cross-linked mutant ECF
in MOPS pH 7.0 buffer containing
ATP + EDTA (circles), or ADP + Mg
+ P
(diamonds), was incubated with 200
µM [
C]NEM for varying times up to
2.5 h. At the times indicated, the incorporation of radioactivity into
Cys
(mol [
C]NEM/mol
F
) was determined as described in Fig. 1for
-
(A),
-
(B), and free
subunit (C).
Figure 5:
Correlation of ATPase activity with
incorporation of [C]NEM into
subunits of
mutant ECF
(
G149C:V198A). ECF
, 1
µM or 2 µM, was equilibrated in MOPS pH 7.0
buffer containing ATP + EDTA (circles) or ATP +
Mg
(ADP + P
+ Mg after
catalysis) (squares), and incubated with 25 µM [
C]NEM (ATP + EDTA) or 200 µM [
C]NEM (ATP + Mg
for up to 2 h. The incorporation of radioactivity into
149 (mol [
C]NEM/mol
F
) was determined at various times from gel slices of the
subunit isolated by SDS-PAGE. In a parallel experiment, the
residual ATPase activity (% basal activity) was determined at the same
time intervals under the same conditions. The dashed and dotted lines plot for full inhibition at 1 and 2 mol of NEM
incorporated, respectively.
Figure 6:
DCCD modification of mutant ECF (
E381C/
S108C) under different nucleotide conditions and
correlation of ATPase activity with incorporation of
[
C]DCCD. A, mutant ECF
(2 µM) was equilibrated in MOPS pH 7.0 buffer
containing EDTA (crosses), ATP + EDTA (circles),
AMP-PNP + Mg
(triangles), or ATP + Mg
(ADP + P
+ Mg after
catalysis) (squares), and incubated with 200 µM DCCD. Aliquots were withdrawn for assay of residual ATPase
activity at various time intervals up to 3 h. B, in a
parallel experiment, aliquots of ECF
(2 µM)
were equilibrated in MOPS pH 7.0 buffer containing ATP + EDTA (circles), AMP-PNP + Mg
(triangles), or ATP + Mg
(ADP
+ P
+ Mg after catalysis) (squares), and
incubated with 200 µM [
C]DCCD. At
the times indicated in A, and at 2 and 3 h, aliquots were
withdrawn and the reaction was quenched by addition of 10mM NaOAc, pH 5.2. The incorporation of radioactivity into
Glu
(mol [
C]DCCD/mol
F
) was determined as described in Fig. 1and plotted
against the corresponding ATPase activity.
Table 1gives data on incorporation of DCCD into the different
subunits assessed by CuCl
-induced disulfide bond
formation in the
E381C/
S108C mutant. In these experiments,
enzyme was reacted for 1 h in EDTA-containing buffer, by which time
between 1.3 and 1.6 mol of [
C]DCCD had become
incorporated into the enzyme with more than 90% inhibition of activity.
The modification of ECF
by the hydrophobic carbodiimide
occurred predominantly in the
subunit linked to
and in the
free
subunit, with no major preference between the two.
Incorporation of DCCD into the
linked to
was low, as
expected from previous studies, and may in part represent background
labeling, given that there was a proportionally small labeling of
and
subunits in our experiments (result not shown).
Importantly, the same distribution of label, mainly into -
and into the free
subunit, was observed whether the DCCD reaction
occurred first followed by disulfide bond formation to link
subunits to their partner small subunits, or if cross-linking was
performed first, followed by DCCD labeling.
Fig. 7shows the labeling of ECF from the mutant
E381C/
S108C with NbfCl. The reagent,
detected by its fluorescence, binds predominantly to the free
subunit (seen both in the
subunit band and in the
-
cross-linked product), with a small amount of reaction in
-
,
but no labeling of that
linked to the
subunit.
Figure 7:
NbfCl modification of cross-linked mutant
ECF (
E381C/
S108C) followed by incorporation of
[
C]DCCD. A, cross-links were
induced by passage of mutant ECF
(7.5-12.5
µM) through two consecutive CuCl
-containing
centrifuge columns and the reaction stopped by the addition of 1 mM EDTA. Cross-linked mutant ECF
was transferred to a
Tris pH 7.5 buffer, and incubated with 500 µM NbfCl for 1
h at 30 °C. Excess NbfCl was removed by column centrifugation and
an 80-µg aliquot subjected to SDS-PAGE in the absence of DTT. Lane 1, Coomassie Brilliant Blue-stained gel; lane 2,
fluorogram of the same gel. B, relative intensities of
NbfCl incorporation into the different
subunits as observed upon
UV illumination (302 nm) (A, lane
2).
DCCD
reaction of NbfCl-labeled ECF led to incorporation of
[
C]DCCD into the
subunit linked to
,
and into the free
subunit, to the same extent as with enzyme that
had no prior reaction with NbfCl (Table 1).
The studies reported here exploit the recently described
mutant of ECF,
E381C/
S108C, in which CuCl
induces high yield cross-linking between one
subunit and
, and a second
subunit and the
subunit. As a
consequence, it is possible to distinguish the three
subunits by
their interaction with the small subunits. Additionally, a mutant was
constructed that contained a Cys introduced into the catalytic site
region (
Cys
) as well as Cys at
residue 381
and at
residue 108. Chemical modification studies were conducted:
(i) with NEM to modify Cys
, (ii) with DCCD, which reacts
with
Glu
, and (iii) with NbfCl, which reacts at
Tyr
.
With DCCD, the modification was introduced
both prior to, and following, induction of disulfide bond formation
between subunits and
and
subunits, respectively. Up
to 2 mol of reagent were incorporated under both conditions with the
same distribution of reagent between the three different
subunits. This result establishes that the asymmetrical distribution of
DCCD is not induced by the incorporation of the reagent, but reflects
an intrinsic asymmetry of the enzyme. Furthermore, the data with DCCD
are reassurance that cross-linked enzyme fairly reflects the structure
of ECF
, which is not significantly altered by disulfide
bond formation between
with
, and
with
subunits.
The key finding of the work presented here is that the three
different subunits have different conformations (shown
schematically in Fig. 8), in the absence of nucleotides in
catalytic sites and even when both catalytic and non-catalytic
nucleotide-binding sites are empty. Therefore, there must be an
intrinsic asymmetry of F
induced by interactions of the
-
pairs with the small subunits
and
. One
subunit, that which interacts directly with the short central
helix of the
subunit (residues 82-99 in E. coli)
(
), is reactive to the hydrophobic carbodiimide
DCCD but does not react with NbfCl. A Cys introduced at residue 149 in
place of Gly in this copy of the
subunit is shielded from
reaction with NEM. The
subunit linked to the
subunit
(
) does not appear to bind DCCD at Glu
and has very poor reactivity to NbfCl. Cys
in this
subunit is the most reactive of the three to NEM. The third
subunit, the free
subunit (
), reacts readily
with DCCD, Cys
is modified by NEM, and this copy of the
subunit is the primary site of reaction of NbfCl.
Figure 8:
Schematic representation of the different
conformations of the three subunits of ECF
,
designated
,
, and
, relative to interactions of the
-
subunit pairs with the single-copy subunits
and
.
forms a cross-link between
Cys
and
Cys
, reacts with DCCD but not with NbfCl,
and residue Gly
Cys (P-loop) of this
subunit
is shielded from reaction with NEM;
forms a
cross-link between
Cys
and
Cys
,
incorporates NEM into Cys
, but reacts only very poorly
with DCCD and NbfCl;
is the primary site of
reaction with NbfCl, reacts with DCCD, and Cys
of this
subunit is modified by NEM.
Asymmetry of
the enzyme, as reflected by the different reactivities of the three
subunits, is retained after binding of nucleotides into catalytic
(and non-catalytic) sites under conditions where all sites would be
occupied (i.e. 5 mM nucleotide). With either ATP
+ EDTA, AMP-PNP + Mg
, or ADP +
Mg
+ P
bound, DCCD reacted with
and
but not
, while NEM reacted with
,
but not
. The recent
structure determination of Abrahams et al.(1994) establishes
an asymmetry of F
under conditions different from those
used here, in this case for enzyme with ADP in one catalytic site,
AMP-PNP in a second catalytic site (that which is in the
subunit
linked to the short
helix of
and, therefore
in our terminology), and a third catalytic site empty of
nucleotide. It is interesting to note that Weber et al.(1994)
have shown that the three catalytic sites have the same affinity for
ATP or ADP in the absence of Mg
(i.e. in
EDTA). Therefore, the catalytic sites can become equivalent without
loss of the asymmetry induced by binding of the small subunits.
The
chemical modification studies presented here provide interesting data
on nucleotide-dependent conformational changes occurring in
ECF. Binding of ATP to the enzyme under conditions that
prevent hydrolysis of the substrate (ATP + EDTA or AMP-PNP +
Mg
) does not greatly alter the reactivity of
Cys
compared with when catalytic sites are empty of
nucleotide. However, binding of ADP + Mg
+
P
has a profound effect. The reactivity of the enzyme to
DCCD, in contrast, is not sensitive to nucleotide conditions. It is
modulated by Mg
, probably indirectly, by interaction
of the cation with carboxyl groups, resulting in some shielding from
the carbodiimide. Importantly, the nucleotide-dependent conformational
change reflected in the altered reactivity of Cys
occurs
specifically in that
subunit linked to the
subunit.
Conformational changes have been detected in the
subunit during
ATP hydrolysis (Mendel-Hartvig and Capaldi, 1991; Aggeler et
al., 1992; Turina and Capaldi, 1994a) and ATP synthesis (Richter
and McCarty, 1987), in concert with translocation of this subunit
between an
and a
subunit (Wilkens and Capaldi, 1994). The
results presented here, then, add evidence to the proposal (Capaldi et al., 1994) that the
subunit plays an important role
in the functioning of ECF
F
.