(Received for publication, October 7, 1994)
From the
Most FF
type ATP synthases, including
that in Escherichia coli, use H
as the
coupling ion for ATP synthesis. However, the structurally related
F
F
ATP synthase in Propionigenium modestum uses Na
instead. The binding site for
Na
resides in the F
sector of the P.
modestum enzyme. We postulated that Na
might
interact with subunit c of F
. Subunit c of P. modestum and E. coli are reasonably
homologous (19% identity) but show striking variations around the
H
-translocating, dicyclohexylcarbodiimide-reactive
carboxyl (Asp
in E. coli). Several hydrophobic
residues around Asp
were replaced with polar residues
according to the P. modestum sequence in the hope that the
polar replacements might provide liganding groups for
Na
. One mutant from 31 different mutation combinations
did generate an active enzyme that binds Li
, the
combination being V60A, D61E, A62S, and I63T. Li
binding was detected by Li
inhibition of
ATP-driven H
transport, Li
inhibition
of F
F
-ATPase activity, and Li
inhibition of F
-mediated H
transport. The Li
effects were observed with membrane
vesicles prepared from a
nhaA,
nhaB mutant
background which lacks Na
/H
antiporters, and with purified, reconstituted preparations of
F
prepared from this background strain. Li
inhibition was observed at pH 8.5 but not at pH 7.0. H
thus appears to compete with Li
for the binding
site. Li
binding was abolished by replacement of
Glu
by Asp or Ser
by Ala. The side chains at
Ala
and Thr
may act in a supporting
structural role by providing a more flexible conformation for the
Li
binding cavity. Thr
does not appear to
provide a liganding group since H
transport in two
other mutants, with Gly or Ala in place of Thr
, was also
inhibited by Li
. We suggest that a X-Glu-Ser-Y or X-Glu-Thr-Y sequence
may provide a general structural motif for monovalent cation binding,
and that the flexibility provided by residues X and Y will prove crucial to this structure.
The FF
-type
H
-transporting ATP synthases catalyze the synthesis of
ATP during oxidative and photophosphorylation in a variety of
organisms, usually using a transmembrane electrochemical H
gradient as the driving force for ATP synthesis (Senior, 1988).
These enzymes are composed of two sectors termed F
and
F
. The F
sector is easily released from the
membrane and, when isolated, functions as an ATPase. The F
sector traverses the membrane and functions in H
transport. The complete coupled enzyme functions as a reversible,
H
-transporting ATP synthase. A typical
F
F
complex is found in Escherichia
coli, where F
is composed of five types of subunits in
an
stoichiometry, and
F
is composed of three types of subunits in an a
b
c
stoichiometry (Foster and Fillingame, 1982).
In the bacterium Propionigenium modestum, a profound variant of the
FF
-type ATP synthase is found which uses
Na
instead of H
as the coupling ion
for ATP synthesis (Laubinger and Dimroth, 1987, 1988). The size and
composition of P. modestum subunits are very similar to that
in E. coli. The enzymes also share many sequence homologies, i.e. the sequence identity between the most conserved subunit
is 69% and the identities between the F
subunits a, b, and c are 25, 29, 19%, respectively
(Ludwig et al., 1990; Esser et al., 1990; Kaim, et al., 1992). The F
F
complex of P. modestum, but not F
alone, is activated by
Na
or Li
, which suggests that the
cation binding site is in the F
moiety (Laubinger and
Dimroth, 1987). This conclusion is further substantiated by the
properties of a hybrid enzyme composed of the F
from E.
coli and the F
from P. modestum, formed both in vitro (Laubinger et al., 1990) and in vivo (Kaim et al., 1992; Kaim and Dimroth, 1993). The hybrid
enzyme shows Na
activation of ATPase activity and
functions as a Na
pump. At Na
concentrations < 1 mM, H
and
Na
compete for the binding and transport by the P.
modestum F
F
(Laubinger and Dimroth, 1989).
In addition, both Na
and Li
inhibit
passive H
translocation by P. modestum F
(Kluge and Dimroth, 1992). Dicyclohexylcarbodiimide
(DCCD) (
)reacts specifically with the Asp
of E. coli subunit c, or a Glu at the equivalent
position of other F
F
complexes, and thereby
blocks H
translocation through F
and
coupled ATPase activity (Hoppe and Sebald, 1984; Fillingame 1990).
Subunit c of P. modestum F
also reacts
with DCCD, presumably by covalently binding at the Glu
residue (Laubinger and Dimroth, 1988). The reaction with DCCD is
blocked when Na
or Li
is bound to the P. modestum F
(Kluge and Dimroth, 1993a, 1993b,
1994).
Subunit c is thought to fold in the membrane in a
hairpin-like structure of two -helices. Asp
of E.
coli subunit c is postulated to protonate and deprotonate
during each cycle of F
-mediated H
transport (Fillingame, 1990), and Glu
of P.
modestum subunit c is now thought to be a key component
of the Na
-binding site (Kluge and Dimroth, 1993a,
1993b, 1994). Comparison of the amino acid sequences of E. coli and P. modestum subunit c reveals striking
variations around the DCCD-reactive carboxyl group (Fig. 1).
Several hydrophobic residues in E. coli subunit c,
which are also hydrophobic in other species (Hoppe and Sebald, 1984),
are found as polar residues in P. modestum subunit c.
These polar residues could serve as liganding groups to bind
Na
. The E. coli residues with polar
replacements include Ile
, Met
,
Ala
, and Ile
. The E. coli Pro
, which is conserved in most other bacteria, is
replaced by Gly and the nearby invariant Val
by Ala.
Genetic studies of E. coli subunit c indicate that a
Pro is required in either of the two membrane-spanning helices (Fimmel et al., 1983; Fraga, 1990). In the P. modestum subunit, Pro is found at the equivalent of E. coli position 24 and Gln at the equivalent of position 28; this pattern
is also seen in the chloroplast subunit c.
Figure 1:
Helical hairpin
model for folding of subunits c from E. coli and P. modestum. The rectangles encase the proposed
transmembrane helices. Identical residues in the protein of both
species are indicated by the closed spheres. Residue
interchanges of Ala for Gly, or between Leu, Ile, and Val are indicated
by hatched spheres. The polar loop region of the protein,
including Arg in E. coli subunit c,
faces the cytoplasmic side of the inner membrane. Single-letter
abbreviations indicate residues surrounding the conserved
Asp
of E. coli subunit c which were
replaced by equivalent residues from the P. modestum sequence.
In this study,
we made a series of P. modestum-like substitutions in E.
coli subunit c in the hope of generating an E. coli enzyme that would bind Na. From a combination of
mutations, we ended up generating an active enzyme that is inhibited on
binding Li
.
Plasmid pYZ209, containing the eight structural genes of unc operon, i.e. unc DNA from HindIII (870) to BglII (10,172), was constructed by ligation of the SphI fragment of DNA (bases 3216-10,172 coding the uncAGDC genes) from plasmid pAP55 (Brusilow et al., 1983) into the unique SphI site of plasmid pYZ126, which is the uncE239 equivalent of plasmid pDF163. Plasmid pMO142 (Zhang et al., 1994), the wild type equivalent of plasmid pYZ209, is the control plasmid used in this study.
Standard HMK buffer contains 10 mM HEPES-KOH, pH 7.5, 5
mM MgCl, and 300 mM KCl. Modified HMK
buffers were prepared in which part of KCl was replaced by LiCl or NaCl
at the concentrations specified, and the pH adjusted to 7.0, 7.8, or
8.5. These buffers were used in the ATP-driven ACMA quenching and
ATPase assays. Fluorescence measurements were made as described in
Zhang and Fillingame(1994). ATPase activity was assayed at 25 °C
with 1 mM Tris-ATP [
-32] adjusted to pH 7.5
with KOH. Protein was determined with a modified Lowry assay as
described (Fillingame, 1975).
A total of 33 mutants were generated by
oligonucleotide-directed mutagenesis. These mutants fall into several
groups according to their ability to complement the chromosomal L4amber
or G23D subunit c mutations as scored by growth on a succinate
carbon source (Table 1). Replacement of Ile with a
polar residue Thr improves the function of D61E mutant (see E275, group 1) and also compensates for an otherwise
deleterious effect of the A62S mutation (compare E276 and E274). An additional change of V60A (see group 2) greatly
enhances the effect of I63T substitution and restores growth via
oxidative phosphorylation to a wild type level (see E272 and E239). The V60A, A62S, I63T (E240) triple mutant,
with Asp at position 61, grew less well. These results indicate that
the longer side chain of the Glu
residue can be
accommodated by a more polar microenvironment and/or more flexible
structure. Two other mutants containing substitutions of V60A, D61E,
A62S, I63G, or I63A (E277 and E278) were also
generated. Both displayed hearty growth on a succinate carbon source
which suggests a need for a flexible rather than polar residue at
position 63.
Single residue replacements of Ile or
Met
with Gln generate an active enzyme (see E249 and E244 under groups 3 and 4). However, these mutations
abolished growth when they were combined with any of the other
substitutions (E245-248 in group 3 and E260-263 in group 4). Pro
is essential for
function in the absence of compensatory substitutions of Pro at
position 20 of helix-1, i.e. a second mutation of A20P in
helix-1 can partially suppress mutation of P64L or P64A in helix-2
(Fimmel et al., 1983; Fraga, 1990). However, the possibly
similar exchange of Pro
with Ala
of helix-1,
as is found in the aligned sequence of the P.modestum subunit c, gave rise to nonfunctional proteins (see group
5). On the other hand, exchange of Pro
of helix-2 with
Ala
of helix-1 did restore some function to the mutant
enzyme containing a P.modestum-like pocket, i.e. A60E61S62T63 (
)(compare E266 in group 6 to E264 in group 2).
Figure 2: ATP-driven quenching of ACMA fluorescence by wild type and mutant membranes at pH 8.5. Membranes were diluted to 0.25 mg/ml in HMK buffer, pH 8.5, prepared with or without 50 mM LiCl or NaCl replacing a portion of the KCl. ACMA was added to 0.3 µg/ml and the fluorescence quenching initiated by addition of Tris-ATP to 0.9 mM. SF6847 was added to 0.3 µM.
All four substitutions in the
A60E61S62T63 mutant may be necessary for the Li binding. The quenching response of membranes from the
V60E61S62T63, A60D61S62T63 and A60E61A62T63 mutants was not inhibited
by Li
. To address the question of whether the change
of I63T was absolutely required for the Li
effect, two
other mutants were constructed containing mutations of A60E61S62G63 and
A60E61S62A63. ATP-driven ACMA quenching by these two membranes was also
inhibited by LiCl (as shown in Fig. 3, B and C), but not by Na
. Thr
is thus
not required for Li
binding. Rather, a smaller and
perhaps more flexible residue may be required in place of the
Ile
residue.
Figure 3:
pH dependence of Li
inhibition of ATP-driven quenching of ACMA fluorescence. Membranes were
diluted to 0.25 mg/ml in HMK buffer at the pH and LiCl or NaCl
concentration specified. The other conditions are as described in Fig. 2.
The effect of Li ions on
the quenching response of A60E61S62T63, A60E61S62G63 and A60E61S62A63
membranes was markedly influenced by the pH of the assay buffer, as
shown in Fig. 3. The Li
inhibition of
ATP-driven fluorescence quenching decreased as pH decreased. At pH 7.8,
50 mM Li
inhibited quenching of A60E61S62T63
membranes by only 35% in contrast to the 80% inhibition at pH 8.5. At
pH 7.0, 50 mM Li
did not affect the quenching
response at all. These results suggest that H
may
compete for the Li
-binding site.
The effects of the Li on the ATPase activities of
membranes prepared from the overproducing diploid cells are shown in Fig. 4. Li
significantly inhibited the ATPase
activity of A60E61S62T63 mutant membranes. Half-maximal inhibition was
observed at 5-10 mM LiCl and 90% inhibition at 50 mM LiCl. ATPase activity was completely abolished by 100 mM Li
(not shown). Mutant or wild type membrane
ATPase activity was little affected by 50 mM NaCl. The wild
type membrane ATPase activity was negligibly affected by Li
at concentrations up to 50 mM. Li
and
Na
also have negligible effects on the ATPase activity
of purified F
in HMK assay buffer (data not shown),
providing further support that the Li
inhibition is
mediated through the F
sector.
Figure 4:
ATPase activity of A60E61S62T63 mutant
membrane is inhibited by Li. Membranes (50 µg of
protein) were diluted into 1.0 ml of HMK buffer, pH 8.5, containing
LiCl (
) or NaCl (
) at the concentration indicated, replacing
a portion of the KCl. The ATPase activities were assayed at 25 °C
after addition of Tris-[
-
P]ATP to 1
mM.
The inhibition of
A60E61S62T63 membrane ATPase activity by Li is also pH
dependent as shown in Fig. 5. Strong inhibition by Li
was observed in the alkaline pH range but only moderate
inhibition at neutral pH. The results again suggest that Li
and H
may compete for the same binding site. The
Li
effects on ATPase activity and ATP-driven
H
translocation show a similar dependence on pH.
ATP-driven quenching of ACMA appears to be less sensitive to
Li
inhibition, but this is probably because ATPase
activity can be inhibited substantially without significant reduction
of the quenching response (Miller et al., 1990; Zhang and
Fillingame, 1994). This rationale likely accounts for the observed
Li
inhibition of ATPase activity at pH 7.0, but lack
of effect on ATP-driven quenching at the same pH.
Figure 5:
Comparison of Li
inhibition of A60E61S62T63 membrane ATPase activity at different pH
values. Membranes (50 µg of protein) were diluted into 1.0 ml of
HMK buffer at the pH and LiCl concentration specified. The other
conditions are as described in Fig. 4.
Figure 6:
Effect of Li and
Na
on pH gradient-driven quenching of ACMA
fluorescence by F
-containing proteoliposomes.
K
-loaded proteoliposomes (0.33 mg of phospholipid)
were diluted into 3.2 ml of assay buffer, pH 8.4, containing LiCl or
NaCl at the concentration indicated. ACMA was added to 0.3 µg/ml
and the fluorescence quenching initiated by addition of valinomycin to
10 nM to generate a K
diffusion potential.
SF6847 was added at 0.3 µM. DCCD in 16 µl of ethanol
was added to the diluted liposomes at a final concentration of 50
µM and the mixture incubated 10 min at 25 °C before
the addition of ACMA and valinomycin.
We have generated an E. coli FF
-ATPase that binds Li
by replacement of 4 residues in subunit c with residues
found in the equivalent positions of P. modestum subunit c. These substitutions include the DCCD-reactive Asp
and the immediately surrounding residues. Na
has
no effect on the A60E61S62T63 enzyme. Li
binds to the E. coli enzyme with an affinity close to that reported for the P. modestum enzyme (Kluge and Dimroth, 1993a). The K
for Li
inhibition of the E.
coli A60E61S62T63 ATPase was 5-10 mM at pH 8.5. In
both the P. modestum and E. coli enzymes,
Li
and H
appear to compete for the
same site. ATP-driven H
transport and ATPase activity
for the E. coli A60E61S62T63 enzyme gradually decreased as the
Li
concentration was increased, and the inhibition by
Li
was reversed by increasing H
concentration.
Our results show that Li inhibits both ATP-driven H
uptake and
valinomycin induced H
influx into
F
-containing liposomes. ATPase-coupled H
transport and ATPase activity seem to be somewhat more sensitive
to Li
inhibition. This could indicate that
Li
binds preferentially to a form of the carrier in
the active transport cycle. The form of the carrier mediating passive
H
transport may differ from the forms of the carrier
in the active transport cycle, as was discussed previously (Fillingame,
1990).
In other experiments (not shown), we were unable to
demonstrate a Li effect on H
efflux
from F
-stripped inverted membrane vesicles. In these
experiments, H
transport by F
was
estimated by the extent of dissipation of a
pH (acid interior)
established by electron transport coupled H
transport
(for method, see Zhang and Fillingame, 1994). These measurements were
done under conditions where the membrane potential was dissipated. The
explanation for lack of a Li
effect may relate to the
observations of Kluge and Dimroth(1992) that Na
or
Li
inhibition of
-driven H
influx into F
liposomes is dependent upon the side to
which these ions are added. H
uptake was abolished by
ion addition to the outside of the proteoliposomes, whereas the same
concentration of ion within the liposomes had no effect. To bind ion,
the inhibitory site may have to move to the positively charged side of
the membrane. The ion-binding site can obviously move to the outside of
these vesicles, in the absence of a membrane potential, if F
is bound and ATP present in catalytic sites.
All our
experiments were performed with membranes prepared from a nhaA,
nhaB background. This proved to be necessary
because both Li
and Na
do reduce the
ATP-driven quenching response of mutant membranes showing low ATPase
activity if the membranes have active antiporters. For example, 10
mM Na
or 10 mM Li
significantly reduced the ATP-driven ACMA quenching responses of
both the A60E61S62T63 and A60D61S62T63 mutant membranes, when the
mutant alleles were expressed in the AN346 background. We attribute
this to Na
/H
antiporter activity that
is also insensitive to 0.5 mM amiloride (Taglicht et
al., 1991).
All four substitutions in the A60E61S62T63 subunit c proved necessary for Li binding. The
V60E61S62T63, A60D61S62T63, and A60E61A62T63 subunit c mutant
enzymes were active but were not inhibited by Li
. The
side chains of both Ala
and Thr
in the
Li
binding A60E61S62T63 enzyme are smaller than those
of Val
and Ile
in the wild type protein. They
may act in a supporting structural role by providing a more flexible
conformation in the Li
binding cavity. On the other
hand, a polar residue appears to be required at position 62. We suggest
that the Li
-binding site could be formed, at least in
part, by the Glu
and Ser
side chain oxygens,
with perhaps the Glu
peptide carbonyl oxygen acting as an
additional liganding group (Fig. 7). A water molecule may also
contribute to the Li
binding since most of
metal-binding sites contain one or two water molecules (Toney et
al., 1993; Badger et al., 1994). The experiments of Kluge
and Dimroth(1994) indicate that P. modestum subunit c binds Na
in the absence of other F
subunits. We of course do not know whether the binding site is
formed in a single subunit c or by a multimer.
Figure 7:
A possible conformation of the
Li binding site in a monomer of subunit c.
The polypeptide backbone from a GluSer sequence in a normal
helix
was used as a template to generate the structure. The side chain
conformations in this structure were minimized by a combination of
molecular dynamics and mechanics with 1.9-2.1 Å distance
restraints for Na
/Li
binding to the
two Glu
carboxylate oxygens and the Ser
oxygen. Li
was then placed at an optimal
distance (1.9-2.0 Å) within that structure. Hydrogen atoms
are not shown.
Na has no effect upon and appears not to bind to
the A60E61S62T63 subunit c modified E. coli enzyme.
This may relate to several aspects of ion binding. The ionic diameter
of Na
(approximately 1.90 Å) is larger than that
of Li
(approximately 1.30 Å) (Hille, 1990). It
is well known that cavity size of a metal-binding site is in good match
to the ionic diameter of the cation most strongly bound, as shown for
example by the relationship between the structure of crown ethers and
their ability to complex various cations, i.e. 18-crown-6 has
a high affinity for K
, 15-crown-5 for
Na
, and 12-crown-4 for Li
(Weber and
Vogtle, 1981). Conceivably, additional small changes in the cation
binding cluster geometry, by introduction of smaller or more flexible
residues around position 61, might enable Na
to bind
to the protein. A second reason for absence of Na
binding could be a requirement for an extra liganding group. An
example is found in the structural analysis of dialkylglycine
decarboxylase (Toney et al., 1993). Dialkylglycine
decarboxylase specifically requires K
for activity.
However, in the absence of K
, the smaller
Na
can fit tightly in the binding site with
coordination to five oxygen ligands. With the larger K
(ionic diameter: 2.66 Å) bound, the cation binding cavity
diameter enlarges by about 0.8 Å, and the number of coordination
oxygens increases from five to six by enclosing an additional hydroxyl
group to the cation coordination sphere. In the case of subunit c, the A60E61S62T63 enzyme may lack a liganding group
necessary for Na
coordination, or because of
additional structural barriers, such a liganding group may not be able
to approach the cation bind site. The Pro
and Gln
replacements might provide the liganding groups in the P.
modestum enzyme.
Coincident with this work, the sequence of the
16-kDa proteolipid subunit of the Na translocating
ATPase of Enterococcus hirae was published (Kakinuma et
al., 1993), and it shows a Thr residue next to the proposed DCCD
reactive Glu
. Val and Gly are found at this position in
other ``V-type'' 16-kDa subunits. The side chain oxygens of
Glu
and Thr
may contribute at least some of
liganding groups in the Na
-binding site of this
enzyme.
In summary, these results demonstrate that the residues
around the DCCD-reactive carboxyl of subunit c at least
partially determine the cation specificity of F. We suggest
that a X-Glu-Ser-Y or X-Glu-Thr-Y sequence may provide a general structural motif for monovalent
cation binding and that the flexibility provided by residues X and Y will prove crucial to this structure. A definition
of the P. modestum and E. hirae Na
-binding sites will clearly have to await
direct mutagenesis experiments with these enzymes. In the case of P. modestum F
F
, we would now predict
that a Ser
Ala mutation in subunit c will
result in loss of Na
pumping with retention of
H
pumping.