From the Department of Biochemistry, University of Western Ontario, London, Ontario N6A 5C1, Canada
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ABSTRACT |
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The subunit of Escherichia coli
ATP synthase has been expressed and purified, both as the intact
polypeptide and as
', a proteolytic fragment composed of residues
1-134. The solution structure of
' as a five-helix bundle has been
previously reported (Wilkens, S., Dunn, S. D., Chandler, J.,
Dahlquist, F. W., and Capaldi, R. A. (1997) Nat.
Struct. Biol. 4, 198-201). The
subunit, in conjunction with
-depleted F1-ATPase, was fully capable of reconstituting
energy-dependent fluorescence quenching in membrane vesicles that had been depleted of F1. A complex of
with the cytoplasmic domain of the b subunit of
F0 was demonstrated and characterized by analytical
ultracentrifugation using bST34-156, a form of
the b domain lacking aromatic residues. Molecular weight determination by sedimentation equilibrium supported a
b2
subunit stoichiometry. The sedimentation
coefficient of the complex, 2.1 S, indicated a frictional ratio of
approximately 2, suggesting that
and the b dimer are
arranged in an end-to-end rather than side-by-side manner. These
results indicate the feasibility of the b2
complex reaching from the membrane to the membrane-distal portion of
the F1 sector, as required if it is to serve as a second stalk.
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INTRODUCTION |
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The proton-translocating ATP synthases couple the generation of
ATP to the protonmotive force present across membranes involved in
energy transduction (for reviews, see Refs. 1-4). These complex enzymes consist of a peripheral F1 sector, which catalyzes
ATP synthesis and hydrolysis, and an integral F0 sector,
which catalyzes movements of protons across the membrane. In the
relatively simple ATP synthase of Escherichia coli,
F1 contains five types of subunits in a stoichiometry of
3
3
, while F0
contains three types of subunits in a stoichiometry of
ab2c9-12. Subunit
interactions at the interface of the two sectors are responsible for
coupling their catalytic activities.
Recent work has strongly indicated that hydrolysis of ATP by
F1 is accompanied by rotation of the and
subunits
relative to the
3
3 hexameric ring
(5-11), consistent with proposals from Paul Boyer's laboratory (12).
The high resolution structure of the mitochondrial F1 (13)
reveals that the N and C termini of
form an antiparallel
coiled-coil running up the center of the
3
3 ring; this structure appears to
function as an asymmetric spindle, which, by rotating, plays the major
role in directing conformational changes at the catalytic sites. In the
intact ATP synthase, the rotation of
and
should be coupled to
proton conduction through F0. The a and
c subunits provide those residues that are essential for
proton conduction.
In all systems, or the analogous mitochondrial protein called
oligomycin sensitivity conferral protein
(OSCP),1 is essential for the
coupling of the catalytic activities of the two sectors. The
subunit (reviewed in Ref. 14) has no significant effect on steady-state
ATP hydrolysis rates by isolated F1-ATPase but does alter
unisite hydrolysis (15).
binds to F1 through
interactions with the external surface of the N-terminal third of the
subunit (16-20). In some systems,
alters the proton permeability of F0 (21). This effect is not seen in
E. coli, but here
is essential for the interaction of
F1 and F0, implying a link between
and
F0 (22). The physical and functional nature of the
-F0 interaction is currently the subject of intense
interest. In recent work, an interaction of
or OSCP with the
b subunit of F0 has been demonstrated (23-25).
Nearest neighbor analysis by chemical cross-linking had not revealed
cross-links between b and
in the E. coli
system (26), but they had been reported for the corresponding subunits
in the chloroplast (27) and mitochondrial (28) enzymes. The importance
of the cytoplasmic domain of b to the
F1-F0 interaction has also been demonstrated
through proteolysis (29-31) and direct binding (32) studies.
E. coli purified following pyridine treatment of
F1-ATPase was shown to be an elongated monomer (33), but
the low yield of the preparation limited the scope of work that could
be carried out. The current studies were undertaken to produce
recombinant
in quantities appropriate for high resolution
structural analysis and to permit the characterization of interactions
of
with other subunits in ATP synthase. Here we describe the
preparation of recombinant
and a proteolytic fragment called
'
as well as the hydrodynamic analysis of a complex of
with the
cytoplasmic domain of the b subunit of F0. The
solution structure of
' has been previously reported (34).
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EXPERIMENTAL PROCEDURES |
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Construction of Plasmids--
Recombinant DNA procedures were
carried out as described by Sambrook et al. (35) using
E. coli strain MM294 (36) as the host. Plasmid pHN2 (37),
which carries the tac promoter,
lacIq, and the unc transcription
terminator, was used as the vector. The E. coli uncH DNA
sequence encoding the subunit was amplified, and the translation
initiation region was altered using the expression cassette polymerase
chain reaction procedure of MacFerrin et al. (38).
The upstream primer, CGCGGAATTCTGGAGGATTTTAAAATGTCTGAATTTATTACGG, contained an EcoRI cloning site, a Shine-Dalgarno sequence,
an A/T-rich spacer region, and the first 19 bases of the
uncH coding sequence. The downstream primer,
GCATCCCGGGTTAAGACTGCAAGACGTCTG, contained a SmaI
cloning site and the last 20 bases of the uncH coding
sequence. The PCR product was cut with EcoRI and
SmaI and then ligated into pHN2 that had been digested with
the same enzymes, to produce plasmid pJC1. The uncH gene in
pJC1 was sequenced and compared with the published sequence (39) to
confirm that no mutations had been introduced within the coding
region.
Purification of --
Strain MM294/pJC1 was grown at 30 °C
in LB broth with vigorous shaking. When the cells reached a density
that gave an A600 of 0.8, isopropylthiogalactoside was added to a concentration of 1 mM, and growth was continued for 3-4 h. After harvesting and washing, the cell pellet was suspended in 10 volumes of cold 50 mM Tris-HCl, pH 8.0, 10 mM MgCl2.
All subsequent steps were carried out at 4 °C. Phenylmethylsulfonyl
fluoride was added to a concentration of 1 mM, and the
cells were broken by one passage through a French pressure cell at
20,000 p.s.i. Cell debris, ribosomes, and membranes were removed by
centrifugation for 1.5 h at 40,000 rpm in a Beckman Ti-60 rotor.
Soluble proteins were fractionated by ammonium sulfate precipitation
and column chromatography. Material precipitating between 20 and 32%
of saturation was collected, redissolved in one-half the original
volume, and again brought to 32% saturation with ammonium sulfate. The
precipitate was collected, redissolved in 5 ml of buffer containing 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 1 mM dithiothreitol (TED buffer), and dialyzed overnight
against 1 liter of the same buffer. The sample was applied to a column
of DEAE-Sepharose (1.5 × 30 cm) equilibrated with TED buffer, and
the proteins were eluted with a linear gradient of 0-400
mM NaCl in TED buffer. The
subunit eluted as the major peak midway through the gradient. The protein was precipitated by the
addition of ammonium sulfate to 40% of saturation, redissolved in a
small volume of TED buffer, and applied to a column (1.5 × 90 cm)
of Sephadex G-75 Superfine. The column was developed at a flow rate of
3 ml/h. Pure
eluted as the major peak. The yield was between 10 and
20 mg/liter of LB culture.
Analytical Ultracentrifugation-- Analytical ultracentrifugation was performed at 20 °C using a Beckman model XL-A analytical ultracentrifuge. The buffer contained 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, and 0.5 mM dithiothreitol. In sedimentation velocity experiments, the rotor speed was 60,000 rpm, and scans were taken at 10-min intervals. Data were analyzed using the software provided by Beckman. Observed sedimentation coefficients were calculated using the time derivative (dc/dt) analysis of Stafford (42), fitting the peak of the g(s*) versus s* plot to a Gaussian distribution. Molecular weight determinations using sedimentation equilibrium experiments were carried out using six-sector cells at various rotor speeds. Data sets were individually fitted to determine molecular weights. Partial specific volumes were calculated by standard methods (43).
Other Materials and
Methods--
bST34-156 was purified using
techniques similar to those employed for other forms of the
b cytoplasmic domain (32, 40), namely ammonium sulfate
precipitation, DEAE-Sepharose ion exchange chromatography, and size
exclusion chromatography on Sephacryl S-200. Fractions used in
analytical ultracentrifugation experiments had absorbance at 280 nm of
less than 0.005/mg/ml. The expression and purification of two other
forms of the b cytoplasmic domain,
b34-156 or b53-156,
have been described previously (40). -Depleted F1-ATPase
was the generous gift of Drs. S. Wilkens and R. Capaldi of the
University of Oregon (Eugene, OR).
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RESULTS |
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Design of pJC1 and Expression of --
Previous plasmids
carrying uncH, which encodes
, and its natural
translation initiation region gave poor expression of
, even when
transcription was from a high level
promoter.2 Two factors that
may have contributed to this low expression were corrected in the
construction of pJC1. First, evidence that an mRNA secondary
structure encompassing the region of uncH from the
Shine-Dalgarno to residue 36 of the coding sequence strongly reduces
expression has been presented by Pati and co-workers (47). In the
construction of plasmid pJC1, the expression cassette polymerase chain
reaction strategy of MacFerrin et al. (38) was used to replace C and G residues in the spacer region between the
Shine-Dalgarno and the initiation codon with A or T to weaken this
secondary structure, making the translation initiation region more
accessible to ribosomes. Second, in pJC1 the unc
transcriptional terminator was placed downstream of the uncH
sequence. We have previously shown that the terminator placed
immediately after a coding sequence can increase expression by
stabilizing the transcript against exonucleolytic attack (48).
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Interaction of b and --
Preliminary experiments conducted by
techniques such as size exclusion chromatography indicated that any
interaction between
and bsol (32), the
cytoplasmic domain of b, would be relatively weak.2 To see such an interaction, we carried out
sedimentation velocity experiments in the analytical ultracentrifuge.
This technique has the advantages that relatively high concentrations
may be analyzed, the sample is not significantly diluted during the
experiment, and information about the size and shape of the complex can
be obtained.
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Characterization of a (bST34-156)2
Complex by Sedimentation Velocity--
To eliminate the contribution
of the excess free b subunit to the observed s
value, we produced a form of b lacking aromatic residues
(see "Experimental Procedures" for details). The only tryptophan
and tyrosine residues present in b34-156 and b53-156 were added at their N termini during
plasmid construction to make them detectable at 280 nm, so removing
them would not be expected to affect the interaction with
. In the
new polypeptide, a leader sequence Met-Ser-Thr was fused to residues
Glu34 to Leu156 of b; since the
N-terminal methionine is ordinarily removed, this polypeptide was
called bST34-156, to indicate the Ser-Thr leader and to distinguish it from the polypeptide previously denoted b34-156. Lacking aromatic residues,
bST34-156 is transparent at 280 nm but can
still be observed at wavelengths below 250 nm due to absorbance by the
peptide bond and certain side chains. Sedimentation equilibrium
experiments showed bST34-156 to be essentially
dimeric (see observed and inferred molecular weights in Table
II) but with a slight tendency to become
monomeric at low protein concentrations. In sedimentation velocity
analysis, a low concentration of bST34-156 (0.5 mg/ml) sedimented at 1.47 S, and the sedimentation coefficient
increased to 1.71 at higher concentrations (Fig.
3,
). Again, this pattern suggests some degree of dissociation into monomers at low protein concentration, so the value observed at the higher concentrations is more likely an
accurate reflection of the sedimentation coefficient of the dimer.
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Sedimentation Equilibrium Analysis of the
bST34-156- Interaction--
Sedimentation equilibrium
experiments were carried out to confirm the subunit stoichiometry of
the complex as (bST34-156)2
. In
this experiment, the starting concentrations of
and
bST34-156 were 1 and 4 mg/ml, respectively;
this represents a 2.8-fold molar excess of
bST34-156 dimer over
. With these high
concentrations and the low rotor speeds used (12,000-18,000 rpm),
substantial concentrations of all species were maintained even near the
meniscus so that complex formation would be favored. As in the
sedimentation velocity analyses, the distribution of
and complexes
containing
could be determined at 280 nm without any direct
contribution from bST34-156; representative
data are shown in Fig. 4. The apparent
concentration gradient of
in the presence of the b
domain (
) was significantly steeper than when the subunit was present alone (
), indicative of formation of a complex with higher molecular weight. The results were analyzed in two ways (Table II).
Determination of the molecular weight that gave the best fit for a
single component provided a value of just over 40,000, which can be
compared with the inferred weight of 46,708 for the (bST34-156)2
complex.
Alternatively, the data could be fitted to two noninteracting
components,
and a second component of unknown molecular weight. The
best fit for the second component was about 45,000, which closely
approaches the expected value for the
(bST34-156)2
complex.
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Summary of Properties of
(bST34-156)2--
Information about both
the shape of the complex and the affinity of the subunits can be
obtained from the ultracentrifugation data shown in Fig. 3 and Table
II. Given a stoichiometry of two copies of
bST34-156 to one of
, which was indicated by the sedimentation equilibrium results, the sedimentation
coefficient allows calculation of a frictional ratio of 2.03-2.13
(Table III). In comparison, the
frictional ratio of
, a moderately extended protein, was 1.42, while
that of the highly extended bST34-156 was 1.76. These results indicate that the complex is more extended than either
the b dimer or the
subunit alone.
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DISCUSSION |
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The subunit of E. coli could be expressed either as
an inclusion body or as a soluble protein, depending on the temperature of growth. Previously, bovine OSCP was expressed as an inclusion body
that could be solubilized with guanidine hydrochloride (23, 49), while
yeast OSCP was expressed as a soluble protein (50). The expressed
subunit of spinach chloroplast F1-ATPase was partially soluble, while the solubility of
of Synechocystis
depended, like the E. coli subunit, on the temperature of
induction (51). Generally, it would appear that
does not fold as
efficiently as most proteins of its small size. We have concentrated on
purifying the soluble form, obtaining preparations with very high
activity for reconstitution of energy coupling. These results imply
that essentially all of the
is folded correctly, which was
essential for the ultracentrifugal analysis.
The proteolytic sensitivity of the expressed, soluble subunit after
breaking the cells is not surprising, given the ease with which the
subunit is cleaved while it is incorporated into F1-ATPase
(18). In this case, the cleavage turned out to be useful, since
'
was more amenable to structural analysis by NMR than was the entire
subunit (34). Intact
was obtained by removing as much contaminating
protein as feasible during the early stages of the purification. The
cleavage of
and the defined structure of
' imply the existence
of the subunit as a two-domain protein: a well folded N-terminal domain
and a less folded, or less stable, C-terminal domain, which is
particularly susceptible to proteolytic attack.
Evidence for the interaction of the cytoplasmic domain of b
with or OSCP has appeared recently. Collinson and co-workers (23)
found that an insoluble complex was formed from mixtures of
b', the cytoplasmic domain of mitochondrial b,
and OSCP. Sawada and co-workers (24) have demonstrated the interaction
of E. coli b and
, primarily through in vivo
studies (24). Rodgers et al. (25) found that the addition of
cytoplasmic b domain to 15N-labeled
subunit
caused a broadening of NMR signals from the well folded N-terminal
domain, which implied slower tumbling of the entire b-
complex compared with
alone. The site of the interaction could not
be determined, although the effect was much reduced when the
was
replaced by
'. Because of the qualitative nature of these
demonstrations, it has been possible to extract little information
regarding the structure of the b-
complex from these
experiments.
In contrast to the mitochondrial system, the complex of E. coli with the cytoplasmic domain of b was soluble,
permitting an analysis of size and shape. Sedimentation velocity and
equilibrium experiments using the bST34-156
construct showed that they produce an extended complex with a subunit
stoichiometry of b2
. While this stoichiometry
might be expected, given the fact that the cytoplasmic domain of
b forms dimers in solution (32), it was possible that
might displace one of the subunits of the b dimer to produce
a b
heterodimer. The molecular weight of such a complex
containing the bST34-156 construct would be
32,956. In contrast, we determined that the average molecular weight of species containing
in mixtures of that subunit and
bST34-156 was in excess of 40,000. Since this
average reflects contributions from both the complex and
alone, it
is more appropriate to fit the data obtained to a two-component system,
where one component is assigned the mass of
and the best molecular
weight for the second component is obtained through the fitting
process. The results of such an analysis gave a molecular weight of
45,000, in good agreement with the value of 46,708 expected for the
b2
complex.
The interaction of with the cytoplasmic domain of E. coli
b appears to be rapidly reversible and relatively weak, since no
interaction could be detected by size exclusion chromatography. The
Kd calculated from the sedimentation rate of
in the presence of subsaturating levels of
bST34-156 was in the range of 5-10
µM, if b concentrations are expressed as the dimer. However, our results suggest that a small fraction exists as
monomer at the low protein concentrations used, so this must be taken
as a rough estimate of affinity. Although somewhat weak, the
b2-
interaction nevertheless appears to be
essential for the proper binding of E. coli
F1-ATPase to F0, since
is essential for
this assembly and it seems unlikely to interact with any other subunit
of F0.
The shape of the (bST34-156)2
complex was revealed by sedimentation velocity ultracentrifugation to
be highly extended, with a frictional ratio of 2.1. The high value of
this frictional ratio reveals the complex to be at least as asymmetric
as bST34-156, the more asymmetric of its parts.
If the b dimer and
were to interact side-by-side, one
would expect the complex to be less asymmetric with a lower frictional
ratio. Instead, the b dimer and
appear to interact in
more of an end-to-end manner, making it a very elongated structure.
This result enhances the feasibility that
, a subunit classically
known to be involved in binding F1 to the F0
sector (22), may be located near the top of F1-ATPase, where it interacts with N-terminal sequences of the
subunits (16,
20). The results thus support a model of F1-F0
structure in which b reaches well up the side of the
F1 sector to make contact with
, forming a second stalk
(5, 34), which may function as a stator to hold the
3
3 hexamer while the
subunit rotates inside. The weakness of the interaction, however, suggests to us that
the complex must be stabilized in the entire enzyme, possibly through
interactions of b with other subunits in the F1
sector.
From data presented here, the first 52 residues from the N terminus of
b are not essential for the interaction of b and
. The lack of effect of b domains on the NMR signal of
' (25) and the effect of proteolytic digestion of
on the binding
of F1 to F0 (18) suggest that b will
interact with the C-terminal part of the
subunit. Further analysis
of the regions of b and
involved in the interaction is
currently under way in this laboratory.
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ACKNOWLEDGEMENTS |
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We thank Jennifer Bestard for constructing
plasmid pJB3, Faye Males for technical assistance, Derek McLachlin and
Matt Revington for helpful discussions, and Stephan Wilkens and Rod
Capaldi for the gift of -depleted F1-ATPase.
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FOOTNOTES |
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* This work was supported by Medical Research Council of Canada Grant MT-10237. The XL-A analytical ultracentrifuge was obtained with the support of the Academic Development Fund of the University of Western Ontario.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.: 519-661-3055;
Fax: 519-661-3175; E-mail: sdunn{at}julian.uwo.ca.
1 The abbreviations used are: OSCP, oligomycin sensitivity conferral proteins; bsol and bsyn, forms of the cytoplasmic domain of the b subunit containing residues Val25-Leu156 and Tyr24-Leu156, respectively; b34-156 and b53-156, forms of the cytoplasmic domain of the b subunit containing residues Glu34-Leu156 and Asp53-Leu156, respectively, preceded by the leader sequence Ser-Tyr-Trp, assuming removal of the initiating methionine; bST34-156, a form of the cytoplasmic domain of the b subunit containing residues Glu34-Leu156 preceded by a leader sequence of Ser-Thr, assuming removal of the initiating methionine; sobs, the sedimentation coefficient observed under the experimental conditions.
2 S. D. Dunn, unpublished observations.
3 S. Wilkens and R. A. Capaldi, personal communication.
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REFERENCES |
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