From the Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642
Received for publication, February 10, 2003
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ABSTRACT |
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The stator in
F1F0-ATP synthase resists strain
generated by rotor torque. In Escherichia coli the
b2 ATP synthesis by oxidative phosphorylation occurs on the enzyme
F1F0-ATP synthase. In Escherichia
coli the enzyme consists of eight different subunit types in
stoichiometry
This report is concerned with the structure and function of the stator,
a topic also of much current interest, which has been reviewed recently
in Refs. 5 and 6. The stator (b2 In this work we took a direct approach and tested the binding of a
synthetic peptide, consisting of Purification of Synthetic Peptides--
Peptide
Fluorescence Binding Assays--
Tryptophan fluorescence
titrations were carried out as described in Refs. 7 and 8 with
individual conditions given in the figure legends. Unless noted
otherwise the buffer was 50 mM HEPES/NaOH, 5 mM
MgSO4, pH 7.0. Binding-induced changes in Trp fluorescence
were plotted versus peptide concentration, and from the
resulting curves, Kd values were calculated by
nonlinear regression. In all cases the fluorescence signal due to the
peptide alone was negligible.
Circular Dichroism Measurements--
Measurements were made at
23 °C on a Jasco CD spectropolarimeter Model J710. Peptide was
dissolved at 250 µM in 0.3% NH3 solution in water.
Binding of a Synthetic Peptide Comprised of Residues
A further experiment in Fig. 1B showed that binding of
Two other peptides were synthesized. One was " Effect of pH and Mg2+ Ions on Binding of Peptide
Binding of the Synthetic Peptide
In Fig. 2A we show the titrations of
With wild-type and six of the mutant Predicted and Measured Structure of the N-terminal Residues 1-22
of This work establishes that the N-terminal 22 residues of The peptide The facts that the Kd of binding of The ability to use synthetic peptides to study the stator function at
the top of ATP synthase should greatly expedite future experimentation.
One can, for example, readily study the effects of mutations or
deletions of residues in the N-terminal region of subunit complex comprises the stator,
bound to subunit a in F0 and to
3
3 hexagon of F1. Proteolysis
and cross-linking had suggested that N-terminal residues of
subunit
are involved in binding
. Here we demonstrate that a synthetic
peptide consisting of the first 22 residues of
("
N1-22") binds specifically to isolated wild-type
subunit with high affinity (Kd = 130 nM), accounting for a major
portion of the binding energy when
-depleted F1 and
isolated
bind together (Kd = 1.4 nM). Stoichiometry of binding of
N1-22 to
at
saturation was 1/1, showing that in intact
F1F0-ATP synthase only one of the three
subunits is involved in
binding. When
N1-22 was incubated with
subunits containing mutations in helices 1 or 5 on the
F1-binding face of
, peptide binding was impaired as was
binding of
-depleted F1. Residues
6-18 are predicted to be helical, and a potential helix capping box occurs at residues
3-8. Circular dichroism measurements showed that
N1-22 had
significant helical content. Hypothetically a helical region of
residues
N1-22 packs with helices 1 and 5 on the
F1-binding face of
, forming the
/
interface.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3
3
ab2cn. Proton movement through the membrane sector of the complex, mediated by
subunits a and c, is believed to generate
rotation of subunits c,
, and
, which collectively
form the "rotor." In turn this rotation is believed to act on the
catalytic sites, three in number, at
/
interfaces of the
alternating
3
3 hexagon to generate ATP. A
"stator," consisting of subunits b2
, is
necessary to resist the rotor strain. In the reverse direction, ATP
hydrolysis in the catalytic sites drives rotation of the rotor, which
then generates uphill transport of protons across the bacterial plasma
membrane to form the electrochemical gradient essential for nutrient
uptake, locomotion, and other functions. Again rotor strain must be
resisted by the stator for efficient function. The mechanisms by which catalysis, proton gradient formation, and subunit rotation are functionally integrated are subjects of active investigation
(1-4).
) interacts with the
3
3 catalytic unit via
/F1 interactions and with the proton-translocating
machinery via b2/a interactions.
and b2 interact together via their C-terminal
regions. There may also be interaction between
b2 and
or
subunits. In two recent
reports we have studied the binding of the
subunit to
F1 (7, 8). Using novel tryptophan fluorescence assays, our
work established quantitative parameters for
binding to
F1, demonstrated that helices 1 and 5 of the N-terminal
domain of the
subunit form the F1-binding surface on
, and showed that the cytoplasmic domain of the b subunit
has a very large effect on the affinity of
binding to
F1. In this report we move to study the
-binding surface on F1.
subunit (and its mitochondrial homolog
oligomycin sensitivity conferral protein) is known from electron
microscopy studies to bind at the "top" of F1
(9, 10). Proteolysis (11) and cross-linking (12) experiments have
suggested that the extreme N-terminal residues of
subunit could be
involved in binding of
. Removal of the first 15 residues of
by
trypsin or of the first 19 residues by chymotrypsin was sufficient to
greatly reduce
binding to F1 (11). X-ray
crystallography studies have not yet been able to determine the
structure of these
subunit residues (4, 13, 14).
subunit residues 1-22, to the
isolated wild-type
subunit. We found that the peptide did bind to
wild-type
with high affinity and stoichiometry of 1 mol/mol,
producing the same change in fluorescence signal of natural residue
-Trp-28 in wild-type
as was produced by binding of
-depleted F1 to
subunit. Moreover fluorescence
responses upon binding of the peptide to isolated mutant
subunits
were similar to those produced upon binding of
-depleted
F1. The peptide showed
-helical structure by circular
dichroism, indicating that a predicted helix at residues 6-18 of
subunit may be important for binding of
.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Subunit--
Purification of wild-type,
mutant, and proteolytically cleaved
subunit (
') was as in Refs.
7 and 8.
N1-221 was purchased from
United States Biological (immunological grade) (mass = 2562.0 Da). Fresh batches of peptide were dissolved daily in 0.3%
NH3 solution in water and used for 1 day only. Experiments
showed that, over longer time periods, precipitation occurred. Peptide
concentration was determined by Lowry protein assay and agreed closely
with that calculated from peptide weight.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-22 to
Isolated Wild-type
Subunit--
As noted in the Introduction,
there is evidence suggesting that the N-terminal region of
subunit
of F1 is involved in binding
subunit. In E. coli F1, the N-terminal amino acid of
is Met with
a free amino group (11). A synthetic peptide comprising residues
1-22 (called "
N1-22") with free N and C termini was synthesized by a commercial source. The sequence is
MQLNSTEISELIKQRIAQFNVV. When the peptide was mixed with isolated
subunit it was seen (Fig. 1A)
that the fluorescence signal of the single Trp in
(
-Trp-28) was
considerably enhanced, by 50%, and blue-shifted by 4 nm. Exactly the
same fluorescence changes were seen on addition of
-depleted
F1 (
3
3
) to isolated
subunit (Fig. 1A). Therefore the synthetic peptide mimicked
-depleted F1 in binding to isolated
subunit. Using
this fluorescence signal, we had established previously by titration
that
-depleted F1 and wild-type
interacted with
Kd of 1.4 nM (7). In Fig. 1B
we carried out a titration of
N1-22 peptide with fixed
concentration of wild-type
. It is seen that binding saturated at a
maximal stoichiometry of 1 mol peptide/mol
subunit, and the
Kd was 130 nM (mean of five
experiments). Together these data demonstrate an unexpectedly tight and
specific association between the peptide and
.
View larger version (14K):
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Fig. 1.
Enhancement of fluorescence of wild-type
subunit upon addition of
N1-22 peptide and determination of
Kd of binding. A, fluorescence
enhancement of signal of
-Trp-28 in wild-type
subunit on
addition of saturating amount of
N1-22 peptide compared with
addition of
-depleted F1. Dotted line,
fluorescence of
subunit alone; open circles,
plus
N1-22 peptide; solid line,
plus
-depleted
F1 (fluorescence of F1 subtracted).
B, titration of wild-type
subunit and
' with
N1-22.
' is the proteolytic fragment of
containing residues
1-135 only.
exc = 295 nm;
em = 325 nm.
Filled circles, wild-type
; open circles,
'.
N1-22 to
', a proteolytic fragment of
consisting of residues
1-135 only, which contains the N-terminal helical domain of
(15), occurred with the same binding stoichiometry and affinity. Therefore the peptide binds to the N-terminal domain of
just as
F1 does (7).
N1-22Cam" in
which the C-terminal Val residue of
N1-22 was amidated. This peptide proved very difficult to dissolve in aqueous buffer and was not
further investigated. A second peptide was the 11-mer sequence
GTQLSGGQKQR with free N and C termini. This peptide, from the
ATP-binding cassette signature sequence of P-glycoprotein, with
no resemblance to
N1-22, dissolved readily in water but produced no
change whatsoever in fluorescence signal of isolated wild-type
subunit.
N1-22 to Wild-type
Subunit--
The experiments in Fig. 1 were
performed at pH 7.0 to mimic physiological conditions. In previous work
we had shown that binding of
-depleted F1 to wild-type
subunit was both Mg2+- and pH-sensitive (7). We
repeated these assays with
N1-22 peptide and wild-type
, and the
results are shown in Table I alongside
the previous data for comparison. It is evident that binding of
N1-22 to
was not strongly Mg2+-sensitive in
contrast to the situation with F1. There was a small pH
sensitivity in absence of Mg2+ but much lower than with
F1. Interestingly, however, at high pH (9.4) the two sets
of data converged, and particularly in absence of Mg2+ we
can conclude that, at pH 9.4, the N-terminal residues 1-22 of
provide all of the binding energy for
binding. At lower pH and in
presence of Mg2+ other interactions come into play with
F1 present.
Effect of pH and Mg2+ ions on Kd of binding of
N1-22 peptide to wild-type
subunit
-depleted F1 to wild-type
(taken from Ref.
7) are shown for comparison. MES, 4-morpholine ethane
sulfonic acid.
N1-22 to Isolated Mutant
Subunits--
In Ref. 8 we introduced eight mutations on the
F1-binding face of the
subunit. Two new Trp residues
were introduced at positions
-11 and
-79. These proved valuable
for monitoring binding of
to
-depleted F1. Most of
the mutations impaired binding affinity between
-depleted
F1 and
. We used these mutants for further
characterization of binding of peptide
N1-22 to
. The titration
curves are shown in Fig. 2, and the
calculated Kd values are summarized in Table II. We
have previously reported titration curves and Kd
values for binding of
-depleted F1 to each of the mutant
subunits; these values are also shown in Table
II.
View larger version (16K):
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Fig. 2.
Titration of mutant subunits with
N1-22 peptide.
A,
Y11A (inverted triangles) and
Y11W/W28L
(upright triangles). B,
A14D
(squares) and
A14L (diamonds). C,
N75A (inverted triangles) and
N75E (upright
triangles). D,
V79A (squares) and
V79W/W28L (diamonds). Concentration of the
subunits
was 2 µM.
exc = 295 nm;
em = 325 nm except for
Y11W/W28L and
V79W/W28L where
em = 360 nm.
Comparison of binding of N1-22 peptide versus
-depleted
F1 to isolated wild-type and mutant
subunits
-depleted F1 are taken from
Refs. 7 and 8. Values for
N1-22
peptide were calculated from titrations of the type shown in Figs. 1
and 2. All values are means of duplicate or triplicate experiments.
N1-22 with
Y11A
and
Y11W/
W28L mutant subunits. In the former the fluorescence
signal is that of the native
-Trp-28; in the latter the signal is
that of introduced
-Trp-11 with the natural
-Trp-28 mutated away (8). With
Y11A, the fluorescence was enhanced as it is when
-depleted F1 is added to
Y11A (8); however, with
Y11W/W28L the fluorescence was quenched, which is different to the
substantial enhancement of fluorescence seen when
-depleted
F1 is added to
Y11W/W28L (8). Due to limited solubility
of the peptide in assay buffer, titration curves could not be extended
beyond 30 µM peptide; however, we feel that the
calculated Kd values in Table II are reasonably
reliable. Fig. 2B shows titration of
N1-22 with
A14L
and
A14D mutant
subunits. It is seen that there is no signal
change, and therefore no Kd values can be assigned.
This could indicate that no binding occurred under these conditions,
but it may be noted that no signal change is seen when
-depleted
F1 is added to
A14L and
A14D mutants even under
conditions where binding does occur (8). Fig. 2C shows
titration of
N1-22 with the
N75A and
N75E mutants.
Enhancement of the fluorescence signal, similar to when
-depleted
F1 is titrated with these mutant
subunits (8), was
seen. Fig. 2D shows titration of
N1-22 with the
V79A
and
V79W/W28L mutant
subunits. In the former case substantial
enhancement of fluorescence is seen; in the latter case a quench of
fluorescence occurs. Both situations mimic what is seen when
-depleted F1 is added to these mutant subunits. It
should also be noted that all of the fluorescence responses were
accompanied by blue shifts on addition of
N1-22 as was seen on
addition of
-depleted F1. Therefore, in seven of eight
mutants, addition of
N1-22 peptide elicited a fluorescence response
similar to that for
-depleted F1, the only
exception being mutant
Y11W/W28L where fluorescence was quenched
rather than enhanced.
subunits the
Kd value for binding of peptide
N1-22 was higher
than that for
-depleted F1 (Table II, columns 2 and 3).
(With the remaining two mutants,
A14L and
A14D,
Kd could not be calculated due to lack of signal.)
From the ratio of the Kd values (Table II, column 4)
the
G0 values (Table II, column 5) were
calculated to be 10-20 kJ/mol.
Subunit--
As noted in the Introduction the structure of the
N-terminal residues 1-22 of the
subunits in
F1F0-ATP synthase is unknown. Several secondary
structure prediction
algorithms2 indicate that
residues
6-18 form an
-helix. Residues
3-8 conform to the
sequence of a typical helix capping box (17) with both H-bonding and
hydrophobic elements. We analyzed the helicity of
N1-22 peptide by
circular dichroism measurement (18). The results (Fig.
3) indicated helix content of 25%. Since
short peptides in aqueous medium often do not form ordered structure
(19, 20), this is a significant result.
View larger version (13K):
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Fig. 3.
Circular dichroism spectrum of
N1-22 peptide.
N1-22 peptide was
dissolved in 0.3% NH3 solution in water at 250 µM concentration.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit of F1 provide an important part of the binding
surface for the
subunit on F1F0-ATP
synthase and contribute substantial binding interactions to maintain
stator function. Our conclusion is based on the following evidence. 1)
The peptide
N1-22 consisting of the N-terminal 22 residues of
subunit binds to wild-type
subunit with high affinity
(Kd = 130 nM), implying specific
interaction. 2) Upon binding
N1-22 produces the same changes
in fluorescence signal of residue
-Trp-28 (enhancement of signal and
blue shift) as does binding of
-depleted F1 to
subunit, showing that
N1-22 brings about the same
environmental changes of
-Trp-28. 3) An unrelated peptide produced
no effect. 4)
N1-22 bound to six mutant
subunits with reduced
affinity just as
-depleted F1 does. These mutations were
shown previously to be located on the F1-binding surface of
. 5) The fluorescence response of inserted
-Trp-79 upon binding
of
N1-22 (quench of fluorescence and blue shift) was very similar
to that seen with
-depleted F1. With inserted
-Trp-11, the fluorescence response was different (quench with
peptide versus enhancement with
-depleted F1); however, the decrease in binding affinity was of
similar order of magnitude as in other mutants and wild type. Together the data establish that the
N1-22 peptide mimics F1 in
its binding to
subunit.
N1-22 bound to isolated
subunit with 1/1
stoichiometry at saturation. Since the stoichiometry of binding of
to F1 is 1/1 (7), this indicates that in intact
F1F0-ATP synthase only one of the three
subunits is involved in
subunit binding. Our work suggests that the
N-terminal region of
forms an
-helix. Previous work has shown
that the F1-binding surface on
is composed of helices 1 and 5 of the N-terminal domain of
(8). Therefore we propose that in
intact enzyme the
-helical N-terminal residues of one of the three
subunits packs on this binding surface of
, forming the
/
interface. Within the proposed helical region of
are several
conserved residues, notably
-Glu-7,
-Ile-8,
-Leu-11,
-Phe-19, that might make specific interactions with
. In this
model, two of the
subunit N termini would not be involved in
binding and would be available for other functions. The 20 S
proteasome structure provides a possible analogy in which protruding
helical N termini of the heptameric
subunits adopt different
spatial conformations and perform disparate functions (16).
N1-22 to
was lower than that for
-depleted F1 by 100-fold
(Kd of 130 versus 1.4 nM) and
also that the pH and Mg2+ sensitivity of binding was
different in the case of the peptide indicate that other interactions
between F1 and
also contribute to binding. However, it
is clear from the surprisingly high affinity of binding seen with the
peptide that the N-terminal 22 residues of
contribute a major
component (
75%) of the binding energy.
, a project that
we have found difficult to approach by conventional procedures because
genetic manipulations that disrupt stator function also interrupt
assembly of the enzyme in cells. It should also prove facile to extend
these studies to understanding the cooperative effect of the
b subunit on binding of
subunit to F1
(8).
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ACKNOWLEDGEMENTS |
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We thank Christina DeVries for excellent technical assistance and Kirti Patel for assistance with CD measurements.
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FOOTNOTES |
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* 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.
Present address: Dept. of Cell Biology and Biochemistry, Texas
Tech University Health Sciences Center, Lubbock, TX 79430.
§ To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, University of Rochester Medical Center, Box 712, Rochester, NY 14642. Tel.: 585-275-2777; Fax: 585-271-2683; E-mail: alan_senior@urmc.rochester.edu.
Published, JBC Papers in Press, February 20, 2003, DOI 10.1074/jbc.C300061200
2 V. A. Eyrich and B. Rost, the META-PredictProtein server (cubic.bioc.columbia.edu/predictprotein/submit_meta.html).
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ABBREVIATIONS |
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The abbreviation used is:
N1-22, synthetic
peptide consisting of residues 1-22 of
F1F0-ATP synthase
subunit.
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