From the Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642
Received for publication, November 26, 2002, and in revised form, January 16, 2003
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ABSTRACT |
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The stator function in ATP synthase was studied
by a combined mutagenesis and fluorescence approach. Specifically,
binding of ATP synthase is the membrane enzyme responsible for ATP synthesis
in oxidative and photophosphorylation of prokaryotes and eukaryotes,
and also for ATP-driven proton pumping to generate the transmembrane
proton gradient in bacterial membranes. In Escherichia coli,
ATP synthase consists of a complex of eight subunits,
The stator must be able to resist strain resulting from rotor torque,
thus its construction is of considerable importance. The dimer of
b-subunits forms an elongated helical connection between
subunit a and the C-terminal domain of the Purification of F1, Purification of E. coli Strains and Expression and Purification of the Cytoplasmic Domain of b
Subunit in Soluble Form--
The soluble form of the cytoplasmic
domain of the b subunit named
bST34-156 was expressed from plasmid pJB3 (20)
after transformation into strain DK8. The procedure followed
essentially that described in Ref. 20. Briefly, six 1-liter cultures of strain pJB3/DK8 were grown and harvested, cells were broken by a French
press, and the supernatant fraction was collected after centrifugation.
Ammonium sulfate was added to 60% saturation, the pellet was
resuspended and dialyzed, the protein was then purified by sequential
chromatography on Whatman DE-52 anion exchange resin and by gel
filtration on Sephacryl S-100. Buffers were described as in Ref. 20.
Yield of bST34-156 was 23 mg of protein/liter of culture.
A Potential F1-binding Surface on the
Mutagenesis and Purification of the
For each mutant, we purified Fluorescence Spectra of Purified Mutant
A tryptophan inserted in position Effect of Addition of
The engineered Fluorescence Titration of Binding of
Both mutations in position
Titrations with the
In position Effect of the Effect of Effect of the Soluble Cytoplasmic Domain of the b Subunit on
Binding of
We demonstrated that addition of bST34-156 to
wild-type or any of the mutant
Fig. 8 shows the effect of inclusion of
bST34-156 on the binding of wild-type and
mutant
With regard to wild-type
In contrast, with mutants Titration of Mutant The goal of this study was to investigate in detail the
interaction between the As target residues for mutational analysis we selected Surprisingly, despite losses in F1-binding energy of up to
15 kJ/mol, all mutations of residues in helices 1 and 5 were still fully or nearly fully functional in vivo and in
vitro. In a previous study (14) we determined that the To explore this possibility we included the soluble cytoplasmic domain
(bST34-156, Ref. 20) in The experiments presented here supplement and extend earlier studies on
OSCP. A study using deletion mutants of bovine OSCP indicated that
removal of the N-terminal 28 residues, corresponding approximately to
all of helix 1 in E. coli The quantitative binding assays described here for wild-type and mutant
-subunit to
-depleted F1 was
studied. A plausible binding surface on
-subunit was identified from
conservation of amino acid sequence and the high resolution NMR
structure. Specific mutations aimed at modulating binding were
introduced onto this surface. Affinity of binding of wild-type and
mutant
-subunits to
-depleted F1 was determined
quantitatively using the fluorescence signals of natural
-Trp-28, inserted
-Trp-11, or inserted
-Trp-79. The
results demonstrate that helices 1 and 5 in the N-terminal domain of
the
-subunit provide the F1-binding surface of
.
Unexpectedly, mutations that impaired binding between F1
and
were found to not necessarily impair ATP synthase activity.
Further investigation revealed that inclusion of the soluble
cytoplasmic domain of the b subunit substantially enhanced
affinity of binding of
-subunit to F1. The new data show
that the stator is "overengineered" to resist rotor torque during catalysis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3
3
ab2cn. It was defined in earlier times in terms of a membrane-peripheral F1 sector (
3
3
)
containing three catalytic sites, and a membrane-embedded F0 sector
(ab2cn), which carries out
transmembrane proton transport. Recent work has demonstrated that the
enzyme functions as a rotary motor. The centrally located "rotor"
consists of subunits
cn. At the top, it
rotates inside the
3
3 hexagon, and thus
modifies the activities of the catalytic sites; at the base, it rotates
against subunit a, thereby facilitating proton movement. In
this way, the energy of the proton gradient is transduced into the
energy of ATP synthesis/hydrolysis. Understanding the mechanism by
which this occurs is currently of major interest. To ensure that
subunits a and
3
3 remain
firmly fixed in relation to each other they are connected by a
peripheral structure, the "stator" stalk, consisting of subunits
b2 and
. For recent reviews of the structure
and function of ATP synthase, see Refs. 1-3.
-subunit, it
lies at one side of the
3
3 hexagon, and
its functional domains have been well characterized (4-6). There may
exist functional interactions between b2 and the
3
3 hexagon (7). Currently only partial
high-resolution structure has been reported for the b
subunit (8, 9). The
-subunit has been shown by electron microscopy
to bind to the very top ("crown") of the
3
3 hexagon (10), and the homologous
mitochondrial OSCP1 subunit
also binds at the top of the molecule, with its C-terminal domain at
the side (11). The N-terminal region of
-subunit (residues
1-15
or
1-19) was shown to be necessary for
binding by proteolysis
experiments (12), and cross-linking between an inserted Cys at residue
2 and natural Cys residue(s) in
(13) provided further evidence
that the N terminus of
, which is not seen in high-resolution x-ray
structures, binds to
. In our laboratory, quantitative determination
of the affinity of binding between
-subunit and the
3
3
complex (also called
"
-depleted F1"), using a novel fluorescence assay
dependent upon the fluorescence of the single natural Trp in
,
revealed a Kd of 1.4 nM in the E. coli enzyme (14). This is equivalent to a binding energy of ~50
kJ/mol, approximately equivalent to rotor torque (15, 16). A similar
Kd value was reported for the chloroplast enzyme
(17) using a fluorescent probe attached to
. We were able to show
that a fragment of
containing only N-terminal residues
1-1342 (called
') bound
with the same affinity as intact
, demonstrating that all the
determinants of binding lie in the N-terminal region of
(14). The
structure of the N-terminal domain of
(residues 2-106 in the
fragment
') has been determined to high-resolution by NMR (18).
Examination of this structure in combination with homology searches of
the sequences of other species of
and OSCP suggested possible
residues that might be involved in binding interaction at the
F1/
interface. Using the quantitative binding assay that
we had developed, in combination with mutagenesis, we were able here to
test these ideas. From the results, this paper presents the
identification of the surface on the
-subunit that provides
interactions with F1. In addition we show that inclusion of
the soluble cytoplasmic domain of the b subunit
substantially enhanced the affinity of binding of
-subunit to
F1.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Subunit,
Preparation of
-depleted F1, Fluorescence Binding
Assays, Assay of ATP-driven Proton Pumping in Reconstituted Membrane
Vesicles, Routine Procedures--
These were all as described
previously (14). For measurement of binding of purified
-subunit to
-depleted F1, the
-depleted F1 was the
"
W107 F1" from strain pSWM86/DK8 as previously
described (14).
W107 F1 contains only the Trp at residue
-107, it has normal functional properties, and has a low Trp
background signal in the
-binding assay. For the ATP-driven proton
pumping assay, the
-depleted F1 was from strain
pSWM92/DK8 (
W28L), which is easier to completely deplete of
-subunit than wild-type F1 (14). Growth yields on
limiting glucose were measured as in Ref. 19.
-Mutagenesis--
Strains pSWM86/DK8 and
pSWM92/DK8 were described previously (14). Strain AN2015 has the
genotype uncH241, argH, pyrE, entA, recA. It contains the
mutation
W28stop, and was a kind gift of G. B. Cox and F. Gibson. For mutagenesis of
, the oligonucleotide-directed mutagenesis procedure in which an EcoRI-SmaI
fragment from plasmid pJC1 (20) was moved into the M13mp19 template was
used (14). After mutagenesis the same fragment was moved back to pJC1,
creating a new mutant plasmid that was transformed into strain AH3 for expression of mutant
(14). For the
Y11W/
W28L and
V79W/
W28L mutants the initial EcoRI-SmaI
fragment in M13mp19 was taken from pSWM101, containing the
W28L
mutation. New mutations, oligonucleotides and resultant mutant plasmids
were as follows:
Y11A, GTAGCTCGCCCCGCGGCCAAAGCAGC, plasmid pSWM104, introduces SacII site;
Y11W,
GGTAGCTCGCCCATGGGCCAAAGCAGC, plasmid pSWM105, introduces
NcoI site;
A14L,
CCCTACGCCAAGCTAGCTTTTGACTTTGC, plasmid pSWM107, introduces
NheI site;
A14D,
CCTACGCCAAAGATGCATTTGACTTTGC, plasmid pSWM106, introduces
NsiI site;
N75E,
CGAAAACGGTCAGGAGCTCATTCGGGTTATGGC, plasmid pSWM113, introduces
SacI site;
N75A,
CGAAAACGGTCAAGCGCTGATTCGGGTTATG, plasmid pSWM112, introduces
Eco47III site;
V79A,
GAACCTGATTCGGGCCATGGCTGAAAATGG, plasmid pSWM114, introduces
NcoI site;
V79W,
GAACCTGATTCGGTGGATGGCGGAAAATGGTCG, plasmid pSWM115, introduces
EciI site; and
G150D,
GTCTGTAATGGCGGACGTTATCATCCG, plasmid pSWM119, introduces
EciI site.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Subunit--
Fig. 1 shows the NMR
structure of the N-terminal domain of the
-subunit of E. coli ATP synthase (18) as determined using the proteolytic
fragment (
') consisting of residues 1-134 (intact
has 177 residues). As noted in the Introduction, because proteolytic removal of
the 43 C-terminal residues has no effect on the affinity for binding to
-depleted F1 (14), it is highly likely that the domain
shown in Fig. 1 contains most, if not all, of the amino acid residues
involved in binding of
to F1. Comparison of the sequences of
or OSCP from 90 species by BLAST search (21) showed
that the highly conserved residues are clustered on a relatively small
portion of the
surface, as demonstrated in Fig.
2. Because
sequences are in general
not highly conserved, for example, in comparison with those of
- or
-subunits (22), it appeared probable that this conserved part of
, formed by helices 1 and 5 (colored dark blue and
yellow, respectively, in Fig. 1), constituted the
F1-binding site. We decided to test this hypothesis by
mutagenesis of four conserved residues,
-Tyr-11,
-Ala-14,
-Asn-75, and
-Val-79. From the BLAST search (above) we found the
following degrees of conservation of these residues. At position
-11, Tyr occurred in 95% of species and Phe in 5%; at
-14, Ala
occurred in 90%; at
-75 Asn occurred in 78% and Asp in 6%; and at
-79 a hydrophobic residue (Val, Leu, or Ile) occurred in 92% of
species. Fig. 2A shows the location of these residues on the
hypothesized binding surface.
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Fig. 1.
High resolution (NMR) structure of the
N-terminal domain of -subunit. The protein backbone from
residue
-Ser-2 (helix 1, dark blue) to residue
-Thr-106 (helix 6, red) is shown. Helices are numbered
1-6. Residue
-Trp-28, located in helix 2 (light blue),
is depicted in "spacefill" representation. Adapted from Ref. 18 and
displayed using PyMOL (31).
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Fig. 2.
Sequence conservation on a proposed binding
surface of -subunit. A, the surface of the
N-terminal domain of
is shown in the same orientation as in Fig. 1.
Strongly conserved or conservatively replaced residues are colored in
red; partially conserved residues are colored in
pink. The remaining residues of
(gray) show a
high degree of variability. Residues 11 and 14 in helix 1 and 75 and 79 in helix 5 were mutated in this work. Residue 85 was mutated in
previous work and is discussed in the text. B, series of
views obtained by rotating the molecule in 90° steps. The central
depiction shows the molecule from the same angle as in A
above and in Fig. 1.
-Subunit--
Two mutations
were introduced in each of the four conserved positions, namely:
Y11A,
Y11W;
A14D,
A14L;
N75A,
N75E;
V79A,
V79W. The
Ala mutations were designed to give an estimate of the contribution of
the natural residue to subunit-subunit binding energy. The introduced
Trp residues could give new probes for assaying the interaction of
with F1 and in both cases they were combined with the
W28L mutation to remove the only naturally occurring Trp in
. The
remaining mutations were designed to perturb binding of
to
F1. The mutation
G150D was included as a control. This mutation is known to completely impair ATP synthase function (23), but
because it occurs in the C-terminal domain of the
-subunit it is
expected to interfere with binding of
to the b subunit (24) and not with binding to F1.
-subunit following previously published
procedures (14, 20), except in the cases of
A14D and
G150D, where
the concentration of saturated ammonium sulfate necessary to
precipitate
was 50% rather than 32%. Yields ranged from 0.5 to 3 mg/liter culture. Purity of the mutant
was checked by SDS gels, and
in each case was the same as with wild-type as previously shown in Ref.
14. Each purified mutant
was assayed to determine the affinity of
binding to F1 using the fluorescence assay developed
previously (14). This assay depends on the fact that binding of
to
F1 that has been depleted of
produces a large
enhancement of fluorescence of the natural
-Trp-28 residue. As noted
above, for the
Y11W and
V79W mutants, we removed the natural
-Trp-28 (by combining with
W28L) and used the new Trp as the
probe. For each mutation we also assayed the effect on function
in vivo, by measuring growth of mutant cells on succinate plates or in limiting glucose medium, and we measured effects on
ATP-driven proton pumping in vitro by reconstitution with
membrane vesicles and
-depleted F1 using purified mutant
.
--
Fig.
3, A-E, shows the
Trp fluorescence spectra of the purified mutant
-subunits in the
absence of F1. Except for the
Y11W/W28L and
V79W/W28L
mutants (dashed curves in Fig. 3, A and
D, respectively), the fluorophor is the natural Trp in
position
-28. Several of the spectra of mutant
(
A14L,
V79A, and
G150D) resemble closely that of wild-type
(dotted lines in Fig. 3, A-E) with a maximum at
326 nm, indicating a relatively unpolar environment for the tryptophan
side chain. The wavelength position of the spectra for
N75A and
N75E (Fig. 3C) is also very similar; however, the fluorescence intensity is 20-30% higher, suggesting that the
mutations caused minor changes in the environment of
-Trp-28. In the
A14D mutant, the spectrum of
-Trp-28 was red-shifted by 2 nm, in
the
Y11A mutant by 5 nm, indicative of an increased polarity
experienced by the fluorophor. In general, however, the mutations
appeared not to perturb the tertiary structure to any large extent,
by this criterion.
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Fig. 3.
Tryptophan fluorescence spectra of purified
mutant -subunits. Spectra (
exc = 295 nm) were
measured in 50 mM HEPES, 5 mM
MgSO4, pH 7.0, and corrected. In all panels the wild-type
spectrum is shown as the dotted line. A,
solid line,
Y11A; dashed line,
Y11W/W28L.
B, solid line,
A14D; dashed line,
A14L. C, solid line,
N75A; dashed
line,
N75E. D, solid line,
V79A;
dashed line,
V79W/W28L. E, solid
line,
G150D.
-11, in the
Y11W/W28L mutant,
has an emission maximum of 337 nm (Fig. 3A), suggesting clearly a more polar environment than for
-Trp-28 in wild-type. A
tryptophan in position
-79 (Fig. 3D) has nearly aqueous
surroundings, as indicated by fluorescence maximum at 350 nm of the
V79W/W28L mutant. These data are consistent with the predicted
location from the structure (Figs. 1 and 2).
-depleted F1 on the
Fluorescence Spectra of Trp in
-Subunits--
Wild-type and most of
the mutant
preparations contained
-Trp-28 as the sole Trp. The
response of the
-Trp-28 fluorescence upon binding of
-depleted
F1 to wild-type
is shown in Fig. 4A. As described previously
(14), there was an increase in fluorescence intensity of about 50%,
combined with a slight blue-shift (~4 nm), indicative of a change to
a more unpolar environment upon binding. The same change was seen in
the
V79A and
G150D mutants (data not shown). In the
N75A and
N75E mutants the fluorescence signal of unbound
was higher to
begin with (Fig. 3C), but the final signal after binding to
-depleted F1 was very similar to wild-type
, as in
Fig. 4A (data not shown). In the
Y11A mutant, which had a
lower intensity and a more red-shifted spectrum before addition of
F1, the fluorescence increase upon F1 binding
was about 40%, and the blue-shift was by 6 nm. Mutants
A14D and
A14L did not show any change in fluorescence under the experimental conditions used (discussed below).
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Fig. 4.
Tryptophan fluorescence spectra of purified
wild-type and mutant -subunits after addition of
-depleted
F1. Spectra are shown before (dotted lines)
and after (solid lines) addition of 2-3-fold excess of
-depleted F1. All spectra are corrected for the
contribution by F1 and unbound
. A, wild-type
. B,
Y11W/W28L. C,
V79W/W28L.
-Trp-11, in the
Y11W/W28L mutant, experiences a
very pronounced fluorescence increase upon F1 binding, by 80-90% at 330 nm, combined with a blue-shift of 4 nm (Fig.
4B). In contrast, the fluorescence of the engineered
-Trp-79, in the
V79W/W28L mutant, is quenched upon F1
binding, by about 50% at 350 nm, accompanied by a blue-shift of 14 nm
(Fig. 4C). The blue-shifts of all three Trp residues
(
-Trp-11,
-Trp-28, and
-Trp-79) reflect a more unpolar
environment of the tryptophan side chains upon F1 binding,
suggesting that the fluorophors are better shielded from the medium in
this state. This, in turn, provides good evidence that all three
fluorophors are actually located at, or close to the
F1-binding surface on the
-subunit. These data
indicated that for all except the
A14D and
A14L mutants a direct
fluorescence assay was available for binding of
-subunit to
-depleted F1, and that in the cases of
A14D and
A14L a competition assay would be required.
-Subunit to
-depleted
F1 and Determination of Binding Affinities of Mutant
-Subunits--
Binding of mutant
to
-depleted F1
was measured by monitoring the tryptophan fluorescence of
, either
because of the natural
-Trp-28 or because of the engineered
-Trp-11 or
-Trp-79. For each mutant, initially an F1
concentration of 0.05 µM was chosen. If this
concentration was too high to obtain a reliable Kd value (because the resulting titration curve was "stoichiometric," for example, the curve for wild-type
in Fig.
5A), the titrations were
repeated using 0.01 µM F1. If the initial
F1 concentration was too low (i.e. the titration
curves did not reach saturation), the experiments were repeated using
0.5 µM F1. Typical titration curves are shown
in Figs. 5 and 6 and the resultant
calculated Kd values are given in Table
I. In all cases where a binding
stoichiometry could be determined reliably (i.e. with stoichiometric or close-to-stoichiometric binding curves), it was very
close to 1 (0.9 to 1.2) mol of
per mol of F1.
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Fig. 5.
Fluorescence titrations to assess binding of
-Tyr-11 and
-Ala-14 mutant
to
-depleted
F1. Pure
-subunit was mixed with
-depleted
F1, and the resulting fluorescence enhancement at 325 nm
(after subtraction of the contribution of
alone and of
F1 alone) was plotted against the concentration of
.
F1 concentrations are given in the figure. A,
open circles/dotted line, wild-type
;
inverted triangles/solid line,
Y11A;
triangles/dashed line,
Y11W/W28L.
B, inverted triangles/solid line,
Y11A; triangles/dashed line,
Y11W/W28L;
squares,
A14D; diamonds,
A14L.
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Fig. 6.
Fluorescence titrations to assess binding of
-Asn-75 and
-Val-79 mutant
to
-depleted
F1. Pure
-subunit was mixed with
-depleted
F1, and the resulting fluorescence changes at 325 nm
(A and B) or 360 nm (C) were plotted
(after subtraction of the contribution of
alone and of
F1 alone) against the concentration of
. F1
concentrations are given in the figure. A, open
circles/dotted line, wild-type
; inverted
triangles/solid line,
N75A;
triangles/dashed line,
N75E. B,
V79A. C,
V79W/W28L.
Effect of mutations in -subunit on affinity of binding of
to
-depleted F1
-11 resulted in a significantly decreased
binding affinity. Based on the results obtained with the
Y11A
mutant, the
-Tyr-11 side chain in wild-type
contributes about 12 kJ/mol of binding energy (Table I). In this case, tryptophan is not a
good substitute for tyrosine, because the
Y11W mutation also causes
a loss of close to 12 kJ/mol in binding energy (calculated by comparing
Y11W/W28L to
W28L mutant).
A14D and
A14L mutants, even using 0.5 µM F1 and up to 2.5 µM
, did
not result in significant changes in the
-Trp-28 fluorescence (Fig.
5B). One possible explanation is that the binding affinity
is so low that even at the highest concentrations used, binding is
negligible. In this case, Kd would be >5
µM (and the loss in binding energy >20 kJ/mol).
Alternatively, it is possible that binding occurs, but that the
mutations affect the interaction between
and F1 in such
a way that the fluorescence of
-Trp-28 does not respond to binding.
To address this question, we performed competition titration
experiments, which showed that a 6-10-fold excess of
A14D or
A14L mutant
-subunit did not significantly reduce binding of
wild-type
. On the basis of these results, we can calculate that the
Kd for
A14D or
A14L mutant is at least 0.1 µM. Thus, from either calculation, both mutations at
residue
-14 perturb interaction between
and F1 considerably.
-75, neither mutation
N75A nor
N75E has a
significant effect on the F1 binding affinity (Fig.
6A and Table I). Specifically the results obtained with the
N75A mutant suggest that the natural asparagine side chain does not
contribute binding energy. In contrast, the valine side chain of
residue
-Val-79 makes a moderate contribution of about 6 kJ/mol
(Table I). A tryptophan in this position strongly interferes with
binding to F1, with a loss of binding energy of more than
11 kJ/mol (comparing
V79W/W28L to
W28L). As expected, the
mutation
G150D in the C-terminal region of
, used as a control
here, had no effect on F1 binding affinity.
Mutations on ATP Synthesis in Vivo--
Plasmids
containing mutant uncH (
-subunit) genes were
transformed into strain AN2015, which contains the chromosomal mutation
-Trp-28
stop and is therefore unable to grow by oxidative
phosphorylation. All plasmids carrying mutations in positions 11, 14, 75, and 79 of
were able to restore oxidative phosphorylation, as
demonstrated by their growth yields on limiting glucose (Table
II) and their ability to grow on plates
containing succinate as the sole carbon source (not shown). In
contrast, the mutation
G150D prevented ATP synthesis by the ATP
synthase in vivo (Table II).
Effect of mutations in -subunit on growth of cells in vivo and
ATP-driven proton pumping in vitro
(uncH) gene was transformed into strain AN2015, which
contains a nonsense mutation
W28stop. For the latter, 500 µg of
KSCN-stripped membranes were reconstituted with 100 µg of
-depleted F1 plus 10 µg of wild-type or mutant
-subunit, and ATP-induced quench of acridine orange fluorescence was
measured.
Mutations on ATP-driven H+ Pumping in
Vitro--
KSCN-stripped membranes were reconstituted with wild-type
or mutant
together with
-depleted F1 and proton
pumping was initiated by addition of ATP. It was found that none of the
mutants at positions 11, 14, 75, or 79 of
caused significant
impairment of ATP-driven H+ pumping (Table II). These data
indicate that, in the presence of intact F0, functional
binding of the mutant
-subunits to F1 did occur. From
the considerations presented in the Introduction, one likely mechanism
for such an effect would be through involvement of the b
subunit dimer, and specifically the cytoplasmic domain of b
that interacts with
. In contrast, the
G150D mutant prevented formation of a proton gradient upon ATP hydrolysis (Table II) as
expected from previous work (23). As already noted, this mutation
occurs in the C-terminal region of
and is expected to interrupt
interaction of
with b.
to
-depleted F1--
For these
experiments the soluble cytoplasmic domain of the b subunit
that we used was purified bST34-156 (20). It consists of residues 34 through 156 (C terminus) of the b
subunit with an additional Ser-Thr- sequence at the N terminus. The
purified protein showed a single major band on SDS gels with the
expected mobility (20). Functional integrity of
bST34-156 was evaluated using the method of
Dunn (25). In this assay, inhibition of reconstitution of
ATP-dependent proton pumping by F1 in stripped membranes is measured. Inhibition comes about as a result of
competition between F0 and added b subunit
cytoplasmic domain for a limited number of F1 molecules. We
found that reconstitution was inhibited by
bST34-156 in a dose-dependent
manner. The ratio of bST34-156/F1 giving 50% inhibition of reconstitution was 2 µg/µg, the same as
in Ref. 25. Theoretically bST34-156 should
contain no Trp residue. Calculation of the Trp content of our
preparation, from the fluorescence spectrum in 6 M
guanidine hydrochloride, revealed a Trp content of 0.1 mol/mol. The
fluorescence because of Trp contamination in
bST34-156 was corrected routinely.
-subunits in the absence of
F1 had no effect on the Trp fluorescence of the
-subunit. In initial experiments with F1 present, we
chose to use a mutant
with a high Trp signal and a relatively low
affinity for binding to F1, namely
Y11W/W28L. Fig.
7A demonstrates an experiment
in which bST34-156 and
-depleted
F1 were added at a constant concentration and the amount of
added
Y11W/W28L subunit was varied. This fluorescence titration showed that addition of bST34-156 to the
binding assay had a very large effect, reducing Kd
for binding of
Y11W/W28L to F1 from 0.5 µM
to <5 nM. In Fig. 7B we determined the apparent Kd for bST34-156. Here, with
constant concentrations of
-depleted F1 and
Y11W/W28L
subunit present, the concentration of bST34-156
was varied. The apparent Kd for the bST34-156 dimer (the physiological form, Refs.
4 and 5) was 150 nM, with two independent experiments
showing excellent agreement.
View larger version (13K):
[in a new window]
Fig. 7.
Effect of bST34-156 on binding
of Y11W/W28L mutant
to
-depleted F1. The
signal used was the fluorescence increase of residue
-Trp-11 upon
binding of
Y11W/W28L mutant
to
-depleted F1.
Contributions of mutant
alone,
-depleted F1, and
bST34-156 were subtracted. A,
titration of
-depleted F1 with
Y11W/W28L mutant
in the presence of 10 µM
bST34-156. B, titration of equimolar
(100 nM) concentrations of
-depleted F1 and
Y11W/W28L mutant
with bST34-156
(plotted as bST34-156 dimer).
-subunits to
-depleted F1. We used a
concentration of 4 µM bST34-156
in these experiments to be sure of saturation while reducing the impact
of the Trp fluorescence because of contaminants in
bST34-156 to a minimum. Presence of this
contamination restricted the concentration range accessible for
titration experiments to
100 nM F1 for all
mutants except
Y11W/W28L, with its larger signal, where 20 nM F1 could be used. Nevertheless, it is clear
from Fig. 8, A-C, that in mutants
Y11W/W28L,
Y11A,
V79A, and
V79W/W28L, inclusion of
bST34-156 in the binding assay brought about a
substantial increase in binding affinity between the
-subunit and
F1. The titration curves (Fig. 8) are all fully or close to
"stoichiometric," thus we can only stipulate that in these
cases that the Kd was reduced to <5 nM
(except for
Y11W/W28L where we can say Kd was reduced to <1 nM). Still, by comparison with the
Kd values obtained in the absence of
bST34-156 (shown in Table I), it is clear that
the changes are large, indeed
500-fold in the case of
Y11W/W28L
mutant.
View larger version (15K):
[in a new window]
Fig. 8.
Effect of bST34-156 on binding
of mutant -subunits to
-depleted F1. Mutant
-subunit was added to
-depleted F1
(concentration given in the figure) in the presence of 4 µM bST34-156. Trp fluorescence
intensity changes at 325 nm (A-C,
squares) or 360 nm (C, diamonds) were
plotted against the concentration of
. A, open
circles/dotted line, wild-type
; inverted
triangles/solid line,
Y11A. B,
Y11W/W28L. C, squares/solid line,
V79A; diamonds/dashed line,
V79W/W28L.
, the above mentioned technical constraints
prevented us from determining whether the binding affinity for
F1 was increased by inclusion of
bST34-156 (the Kd in the
absence of bST34-156 was already 1.4 nM, Table I). If we assume that a 500-fold increase in
affinity occurs for wild-type, as it did for
Y11W/W28L, this would
give a Kd for wild-type
in the presence of
bST34-156 of
3 pM.
A14D and
A14L the titration curves (not
shown) looked the same as those in Fig. 5B
(squares and diamonds) even when
bST34-156 was present. From this we can
conclude that either these mutant
-subunits do not bind
significantly to F1 even in the presence of
bST34-156, or that if they do bind, the binding
does not engender a change in the fluorescence signal of
-Trp-28.
-Subunits with KSCN-stripped Membranes and
-depleted F1 in the Reconstituted Proton-pumping
Assay--
Table I demonstrated that the mutant
A14D and
A14L
subunits did reconstitute ATP-driven proton pumping in membrane
vesicles when added back under saturating conditions with
-depleted
F1 to KSCN-stripped membranes. To investigate the binding
properties of these mutant
-subunits in the presence of intact
F0 further we titrated them in this assay with constant
amounts of stripped membranes and
-depleted F1. Whereas
we had shown before (14) that such a titration cannot be used to
determine absolute values of Kd for binding of
to F1, nevertheless, we expected that by comparison of the
mutants with wild-type we could get qualitatitve information regarding
the binding of the
A14D and
A14L proteins. Fig.
9 indicates that the titration curves
obtained with these (and other) mutants are not very dissimilar from
wild-type. We therefore conclude that the failure to see enhancement of
fluorescence when these mutant subunits are added to
-depleted
F1 in the presence of bST34-156
(above) is most likely because of the fact that the mutations affect
the environment of residue
-Trp-28 and prevent enhancement of its
fluorescence signal upon binding.
View larger version (17K):
[in a new window]
Fig. 9.
ATP-driven proton pumping in stripped
membranes reconstituted with mutant -subunit and
-depleted
F1: titration with mutant
. Membrane vesicles were
stripped of F1 by KSCN treatment, then reconstituted with
-depleted F1 and varying amounts of mutant or wild-type
-subunit. ATP-driven proton pumping was monitored by quenching of
acridine orange fluorescence, and the maximal percent quench was
plotted as a function of
concentration in the cuvette. Open
circles/dotted line, wild-type
; inverted
triangles/solid line,
Y11A; open
squares/solid line,
A14D; open
diamonds/dashed line,
A14L; filled
diamonds/dashed line,
V79W/W28L.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit and F1 in ATP synthase,
and specifically to identify the F1-binding surface on the
-subunit. Based on the location of conserved or conservatively
replaced residues in one particular region of the high resolution NMR
structure of
, we hypothesized that the F1-binding
surface might be formed by helices 1 and 5 (see Figs. 1 and 2). Our
results confirmed the hypothesis. However, several mutations in
that clearly disrupted binding of
to F1 did not lead to
impaired function, an unexpected finding. Additional studies showed
that inclusion of the soluble cytoplasmic domain of the b
subunit substantially enhanced the binding affinity between
and
F1 and compensated for loss of binding affinity caused by
mutations.
-Tyr-11 and
-Ala-14 in helix 1 and
-Asn-75 and
-Val-79 in helix 5. The
first evidence that these residues are at or close to the F1-binding site came from tryptophan substitutions in
positions 11 and 79, whose fluorescence signals responded strongly to
binding of F1 (Fig. 4, B and C).
Further evidence came from the titrations in Figs. 5 and 6, and from
competition binding experiments. Calculated binding affinity
measurements (Kd values, see Table I) indicated that
three of the four residues are directly involved in binding. The
tyrosine side chain of residue
-Tyr-11 contributes about 12 kJ/mol
binding energy, possibly because of
-
or
-cation interactions.
Increasing the size of the side chain in position
-Ala-14, either by
itself (
A14L mutant) or in combination with introduction of a
negative charge (
A14D mutant) reduces the affinity for
F1 significantly, probably by affecting interprotein
surface complementarity. The valine side chain in position
-Val-79
contributes about 6 kJ/mol binding energy, very likely because of
hydrophobic interactions. The lack of binding energy contribution of
the side chain of residue
-Asn-75 suggests this residue is not
directly involved in binding. However, it might not necessarily be
taken as an argument against its location at the F1-binding
surface; in an analysis of the interaction between the human growth
hormone and its receptor (26) it was found that only one-quarter of the
residues at the protein-protein interface had a significant impact on
the binding energy. Whereas this might be an extreme case, at many
protein-protein interface amino acid side chains can be found that
should have the potential to participate in binding, but in fact play
no or only a very minor role (see examples summarized in Ref. 27).
G29D
mutation reduces the binding affinity between
and F1
moderately, corresponding to a loss in binding energy of about 7 kJ/mol. We ascribed the strong functional impairment of this mutant ATP
synthase to the interruption of binding of
to F1. This
led us to conclude that the stator resistance function was finely
balanced. However, the results of the present study showed that much
larger losses of binding energy between
and F1 were
well tolerated. Thus, we must now conclude that not all of the binding
energy necessary to affix the stator stalk to F1, to resist
the elastic strain generated by rotational catalysis, must necessarily
be derived from
/F1 interactions. In all likelihood,
interactions between the b subunits and
and/or
F1 also contribute.
binding assays and found that
it substantially decreased the Kd of binding of
to F1. Due to technical limitations of the fluorescence
assays, absolute values for this Kd in presence of
bST34-156 could not be obtained, but an enhancement of
500-fold in affinity was evident from the results, equivalent to an
additional binding energy of
15 kJ/mol. The additional binding energy
could come from interactions between b subunit and
or
, and/or from b-induced conformational changes in
.
Interestingly, the Kd for interaction between
bST34-156 and isolated
(in the absence of
F1) was 5-10 µM (20) but the
Kd measured here for binding of
bST34-156 in the presence of isolated
and
-depleted F1 was 150 nM, indicating a considerable
cooperativity between the stator subunits. Overall, the new data
indicate that the wild-type stator stalk is "overengineered,"
i.e. it is equipped with excess binding energy. This might
explain why only very few impairing point mutations in
have been
found (23, 28).
, impaired binding of OSCP to
F1 (29). Also a study of rat OSCP showed that the strongly
conserved residue
-Arg-85 (Arg or Lys in 95% of sequences, residue
Arg-94 in OSCP) contributed significantly to F1-binding energy (30). This residue occurs in the loop following helix 5 (see
Fig. 2). Interestingly, despite the loss of binding energy, the
R85A
and
R85Q mutants were still functional in vitro (30), and
the authors concluded, as we do here, that other interactions provide
binding energy. In E. coli, the mutation
R85Q reduced the membrane-bound ATPase activity, by 50%, but had little effect on
growth characteristics (28).
binding to
-depleted F1 in the presence or absence of the soluble cytoplasmic domain of the b subunit will
allow us in future work to assess further aspects of stator subunit interactions. The influence of specific residues of the b
subunits on
binding affinity can readily be studied by mutagenesis,
for example. In addition, we can now investigate, by mutagenesis and other techniques, the binding site for
on F1, which
appears to consist to a significant extent of one or more of the
extreme N termini of the three
subunits, for which a high
resolution structure is not yet available.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Alma Muharemagic and Christina DeVries for excellent technical assistance. We also thank Dr. Stanley D. Dunn (University of Western Ontario) for plasmid pJB3 and for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM25349 (to A. E. S.).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. Tel.: 585-275-2777;
Fax: 585-271-2683; E-mail: alan_senior@urmc.rochester.edu.
Published, JBC Papers in Press, January 29, 2003, DOI 10.1074/jbc.M212037200
2 E. coli residue numbers are used throughout.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
OSCP, oligomycin-sensitivity conferral protein (the mitochondrial homolog of
E. coli );
bST34-156, soluble
cytoplasmic domain of b subunit (20).
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