From the Department of Biochemistry, University of Western Ontario, London, Ontario, Canada N6A 5C1
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ABSTRACT |
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An affinity resin for the F1
sector of the Escherichia coli ATP synthase was prepared by
coupling the b subunit to a solid support through a unique
cysteine residue in the N-terminal leader. b24-156, a form of b lacking the
N-terminal transmembrane domain, was able to compete with the affinity
resin for binding of F1. Truncated forms of
b24-156, in which one or four residues from
the C terminus were removed, competed poorly for F1
binding, suggesting that these residues play an important role in
b-F1 interactions. Sedimentation velocity
analytical ultracentrifugation revealed that removal of these
C-terminal residues from b24-156 resulted in a
disruption of its association with the purified subunit of the
enzyme. To determine whether these residues interact directly with
,
cysteine residues were introduced at various C-terminal positions of
b and modified with the heterobifunctional cross-linker
benzophenone-4-maleimide. Cross-links between b and
were obtained when the reagent was incorporated at positions 155 and
158 (two residues beyond the normal C terminus) in both the
reconstituted b24-156-F1 complex
and the membrane-bound F1F0 complex. CNBr
digestion followed by peptide sequencing showed the site of
cross-linking within the 177-residue
subunit to be C-terminal to
residue 148, possibly at Met-158. These results indicate that the
b and
subunits interact via their C-terminal regions
and that this interaction is instrumental in the binding of the
F1 sector to the b subunit of
F0.
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INTRODUCTION |
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In the process of oxidative phosphorylation or photophosphorylation, the electron transport chain generates a transmembrane proton gradient. The ATP synthase, or F1F0-ATPase, allows protons to flow down this electrochemical gradient and uses the energy obtained to synthesize ATP (for reviews, see Refs. 1-4). Under appropriate conditions ATP synthase can hydrolyze ATP to pump protons. The enzyme is composed of two sectors; the F0 sector is membrane-integral and is responsible for proton translocation, and the F1 sector is attached to the membrane via F0 and houses the catalytic sites for ATP synthesis. F1 is easily detached from the membrane and can be purified as a soluble protein with ATPase activity.
ATP synthases contain at least eight types of subunits. In the
relatively simple enzyme from Escherichia coli, the
F1 sector has the stoichiometry
3
3
1
1
1,
whereas F0 is composed of three subunits of stoichiometry
a1b2c9-12.
The a and c subunits, but not b,
contain residues essential for the translocation of protons across the
membrane (3). The 156-residue b subunit is believed to span
the membrane once at its hydrophobic N terminus, whereas the remainder
of the protein is very hydrophilic. b is thought to exist as
a dimer in the complex (5-7), and proteolysis studies have shown that
the hydrophilic region of b is required for the association
of F1 with the membrane (7-9). Removal of two residues
from the C terminus of b disrupts normal assembly of the
complex (10), as does mutation of Gly-131 to aspartate (11). Thus the
b subunit is essential for linking the F1 and F0 sectors and likely plays a key role in the coupling of
energy from proton translocation to ATP synthesis.
The crystal structure of the mitochondrial F1 has shown
that the and
subunits alternate in a hexagonal ring structure, with two long
-helices from
extending into a hole in the center of the ring (12). Recent evidence strongly suggests that
and probably
rotate relative to
and
during catalysis (13-16). Several studies have implied that the
subunit is located near the
top of the
cluster (17-20). Like b,
is required
for the binding of F1 to F0. Membranes of
mutant E. coli strains expressing truncated forms of
showed little ATPase activity (21), suggesting that F1
cannot bind to the membrane in the absence of
. In truncation and
mutagenesis studies using the mitochondrial (22, 23) and yeast (24)
homologues of
, called
OSCP1 for
oligomycin sensitivity
conferring protein, the C-terminal region
of OSCP was implicated in F0 binding.
The only subunit of F0 able to span the distance from the
membrane to the top of F1 is b. The hydrophilic
portion of b is dimeric, highly -helical, has an
elongated shape, and binds to F1 (25). Although chemical
cross-linking of E. coli ATP synthase has failed to reveal
b-
cross-links, such products have been obtained with the
chloroplast (26) and mitochondrial (27) enzymes. In the chloroplast
work, the cross-link produced by
1-ethyl-3,3-(dimethylaminopropyl)-carbodiimide was mapped to the
C-terminal part of b. The site in
was determined to be
within the cyanogen bromide fragment encompassing residues Val-1 to
Met-165 of the 187-residue polypeptide. Recent studies (28-31) have
demonstrated the interaction of E. coli b and
in the
absence of other subunits.
In the present work we examine the interaction between E. coli
b and F1. An F1 affinity resin has been
generated by linking the hydrophilic portion of b to a solid
support. By using binding assays based on this resin, analytical
ultracentrifugation, and chemical cross-linking, we have gathered
evidence to demonstrate an interaction between residues at the C
terminus of b and the C-terminal region of .
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EXPERIMENTAL PROCEDURES |
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Construction of Plasmids-- Molecular biological procedures were carried out as described by Sambrook et al. (32). Plasmid pMR2, which was used as an intermediate during the construction of pJB2, was generated from pSD80 (33) by elimination of an NdeI site outside of the multiple cloning region, followed by insertion of the PCR-amplified uncF gene from pSD51 (25) into the EcoRI and HindIII sites. The PCR primer was designed such that the initiating ATG codon is part of an NdeI site.
Plasmid pJB2, encoding the bMERC protein, was constructed as follows. pDM3 (34) was used as the template for PCR, using the mutagenic primer 5'-GCGCATATGGAACGTTGCTCGAATTCCCACTACG-3', the 5' end contains an NdeI site and the 3' end is complementary to the beginning of the gene encoding b24-156. The initial ATG codon is underlined. The second primer for PCR was the M13 forward sequencing primer. The resulting PCR product was inserted into the NdeI and HindIII sites of pMR2 to encode a protein beginning with the amino acid sequence MERCSNSH followed by residues Tyr-24 through Leu-156 of the b sequence. The entire open reading frame was sequenced to ensure that no mutation had arisen during the PCR. Plasmids expressing other variants of b24-156 (b24-155, b24-152, and the mutations D150C, K151C, E155C, and 158C) were based on pDM3 (34). In general, mutagenic PCR primers were designed to encode the desired mutation and were cloned into pDM3 using unique restriction endonuclease sites. Correct incorporation of the desired mutations into all of the above plasmids was determined by DNA sequencing. To introduce mutations into the full-length b protein, appropriate restriction endonucleases were used to cut the desired fragment from the pDM3-based plasmid and transfer it to pDM8 (34). The construction of plasmid pSD114 encoding b34-156 has been described previously (34).Expression and Purification of
Proteins--
b24-156 and
b34-156 were expressed and purified as
described (34). Cysteine-containing and truncated forms of b24-156 as well as bMERC
were expressed and purified in the same manner as
b24-156, except that proteins containing cysteine residues were purified in the presence of 1 mM
dithiothreitol (DTT). Plasmids encoding wild type or mutated
full-length b were transformed into the uncF E. coli strain KM2 (35). Expression of the proteins and preparation
of membranes were performed as described (34). The subunit was
expressed and purified as described previously (31). F1 was
purified by standard methods (36).
Production of the F1 Affinity Resin-- DTT was removed from bMERC by passing it through a Sephadex G-25 size exclusion column equilibrated with 50 mM triethanolamine HCl (TEA-HCl), pH 7.5, 1 mM EDTA. Sulfo-link Coupling gel, obtained from Pierce, was washed with 8 volumes of 50 mM TEA-HCl, pH 7.5, 5 mM EDTA before addition of 2.5 mg of bMERC per ml of resin. The mixture was incubated at room temperature with agitation for 1 h, and then excess buffer was removed and assayed for protein. Based on the amount of protein remaining in the solution, the amount of bMERC bound to the resin was inferred to be 2.2 mg per ml of resin. The resin was then incubated at room temperature in 50 mM Tris-HCl, pH 8.5, 1 mM EDTA containing 50 mM cysteine to block any unreacted thiol-binding sites. After 1 h, excess buffer was removed; the resin was washed with 1 M NaCl, and it was resuspended to its original volume in buffer at pH 7.4. The resin was stored at 4 °C. A control resin was prepared by incubating the Sulfo-link Coupling gel with 50 mM Tris-HCl, pH 8.5, 1 mM EDTA containing 50 mM cysteine instead of the bMERC protein.
Assays for F1 Binding by the Affinity Resin--
Two
assays for F1 binding were used. In the "soluble ATPase
activity" assay, 31.3 µg of F1 supplemented with 0.8 µg of the subunit was incubated with 20 µl of the affinity
resin in a final volume of 50 µl of buffer containing 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM ATP, 10% glycerol, and 0.15 mg/ml BSA (binding buffer).
The
subunit was added to the assay to make up for any deficiency of
that subunit in the F1-ATPase, since a fraction of
is
easily lost during purification of the complex (37). After gentle
agitation for 1 h at room temperature, the mixture was centrifuged
to pellet the resin. The ATPase activity of the supernatant was assayed
as described (37).
Analytical Ultracentrifugation--
Analytical
ultracentrifugation was carried out using a Beckman XL-A
ultracentrifuge at 20 °C. Buffer containing 50 mM
Tris-HCl, pH 7.5, 100 mM NaCl, and 1 mM EDTA
(centrifugation buffer) was used. In sedimentation velocity experiments
the rotor speed was 60,000 rpm, and scans were taken at 10-min
intervals. During analysis of , 1 mM DTT was added to
the centrifugation buffer; the buffer used during experiments in which
and forms of b were mixed contained 0.5 mM
DTT. The data were analyzed with the Beckman software using the time
derivative method of Stafford (38). The values of Cohn and Edsall (39)
were used to calculate partial specific volumes. Sedimentation
equilibrium experiments were performed at 20 °C and 20,000 rpm using
b24-152 at concentrations of 0.5, 1.0, or 2.0 mg of protein per ml in centrifugation buffer. Three molecular weight
determinations were done at each concentration.
Cross-linking of Membranes-- Membranes containing F1F0 complexes including either wild type or mutated b were diluted to 0.25 mg of total protein per ml with buffer containing 50 mM Tris-HCl, pH 8.0, 5 mM MgCl2, and 10% glycerol. Benzophenone-4-maleimide (BPM) (Molecular Probes, Eugene, OR) dissolved in dimethylformamide (DMF) was added to a final concentration of 1 mM, and the mixture was allowed to stand at room temperature for 30 min. Controls were performed in which only DMF was added to the membranes. The treated membranes were exposed to long wave ultraviolet light from an Ultra-Violet Products model TM-36 transilluminator for 5 min. As a control some BPM-modified samples were placed on the transilluminator but were removed before it was turned on. After illumination, SDS-PAGE sample buffer was added to the samples, which were then heated at 100 °C for 5 min and analyzed by SDS-PAGE followed by Western blotting.
Cross-linking of Reconstituted F1b-- Purified F1 and wild type or cysteine-containing b24-156 were passed separately through 1-ml centrifuge columns (40) containing Bio-Gel P-10 resin (Bio-Rad) equilibrated with 50 mM sodium phosphate, pH 7.5, and 1 mM EDTA. Tris-(2-carboxyethyl)phosphine (Molecular Probes, Eugene, OR) was added to all b24-156 samples in a 1.1-fold molar excess to ensure the reduction of any disulfide bonds. BPM in DMF was added in a 5-fold molar excess over b24-156; in control samples DMF alone was added. After incubation for 15 min at room temperature, an excess of DTT was added to consume unreacted BPM. The b24-156 was then mixed with the column-centrifuged F1 at a molar ratio of 1.2 F1 per b24-156 dimer, in the presence of 5 mM MgCl2. Cross-linking and analysis was carried out as described above for the membrane samples.
Cyanogen Bromide Cleavage and Analysis of Cross-linked Products-- Cross-linked b24-156E155C-F1 and b24-158158C-F1 were analyzed by SDS-PAGE and stained briefly with Coomassie Blue. After destaining for 10 min with 10% acetic acid, gel slices containing bands to be cleaved were excised from the gel and were dried by lyophilization. The dried gel slices were then treated with 2% CNBr in 70% formic acid. After 30 min the slices had swollen back to their original sizes. At this time the excess CNBr solution was removed, and the tubes containing the gel slices were sealed and incubated at 37 °C overnight. The pH of the slices was then adjusted by three successive 15-min incubations in 150 µl of 1.0 M Tris-HCl, pH 8.0, followed by 15-min incubations in 150 µl of 1.0 M Tris-HCl, pH 6.8, 150 µl of 67 mM Tris-HCl, pH 6.8, and 150 µl of SDS-PAGE sample buffer containing DTT. The slices were placed in the wells of a second SDS-polyacrylamide gel that had been pre-electrophoresed for 1 h at 100 V in the presence of 50 mM Tris-HCl, pH 8.0, 0.1% SDS, containing 0.1 mM sodium thioglycolate to scavenge free radicals in the gel. Electrophoresis was carried out in the presence of 0.1 mM thioglycolate. The gel was either stained with Coomassie Blue or Western blotted onto a polyvinylidene difluoride (PVDF) membrane. After blotting, the membrane was stained briefly with Coomassie Blue and destained with 30% methanol before the bands of interest were excised and analyzed by peptide sequencing.
Other Methods--
SDS-PAGE was performed by the method of
Laemmli (41) using 15% separating gels. The proteins were stained with
Coomassie Brilliant Blue R-250. Protein blotting onto PVDF membranes
was carried out using carbonate blot buffer (42). The anti-b
monoclonal antibodies 10-1A4 and 10-6D1 were generous gifts of Drs.
Karlheinz Altendorf and Gabriele Deckers-Hebestreit of
Universität Osnabrück, Germany. Anti- polyclonal
antiserum was raised against purified
subunit (43), and the
anti-
antibodies were affinity purified on a column containing
immobilized recombinant
(31). Antibodies were labeled with
125I by the IODO-GEN method (44). Protein concentrations
were determined by the method of Bradford (45) or Lowry et
al. (46). Peptide sequencing was performed at the Laboratory for
Macromolecular Structure at Purdue University (West Lafayette, IN)
using an Applied Biosystems 470A sequencer.
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RESULTS |
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F1 Affinity Resin-- To characterize better the binding between the F1 sector and the hydrophilic portion of b, we coupled this region of b to a solid matrix to form an affinity resin for F1. The Sulfo-link coupling gel from Pierce, to which proteins can be coupled specifically via thiol groups, was chosen as a matrix. Since the hydrophilic region of b has no cysteine residues, the site of coupling to the resin can be specified by site-directed mutagenesis of b. Because the C-terminal region was thought most likely to be involved in b-F1 contacts, we introduced a cysteine residue near the N terminus of the hydrophilic region. A construct encoding the polypeptide sequence MERCSNSHY24-L156 (bMERC) was produced as described under "Experimental Procedures." The first four amino acids of this sequence were taken from the E. coli enzyme 3-methyladenine-DNA glycosylase I, because of the polar nature of the sequence and the high expression of this enzyme in a recombinant system (47).
The purified bMERC was coupled to the Sulfo-link resin as described under "Experimental Procedures," at a final concentration of 2.2 mg per ml of resin. Upon incubation of the modified resin with the F1 complex followed by centrifugation, a significant amount of F1 sedimented with the resin, as determined by SDS-PAGE (Fig. 1A, first lane). Only a small amount of F1 co-sedimented with the resin that had been modified with cysteine (Fig. 1A, last lane). The F1 present in this pellet was probably not bound specifically to the resin but rather was trapped between and within the resin particles. As a control for the volume of trapped liquid, BSA was included in all incubations; similar amounts of BSA were observed to co-sediment with both resins under all conditions used (Fig. 1). Thus the bMERC-modified resin is able to bind F1 specifically. The faint band migrating at the position of b24-156 in the first lane represents a trace of bMERC eluted from the resin during the incubation in SDS sample buffer. Most likely this arose from instances in which only one subunit of the dimer became covalently coupled to the resin.
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Competition for F1 Binding by the SDS-PAGE Assay-- To test the ability of the hydrophilic portion of b to compete with the resin for binding to F1, the resin was incubated with F1 in the presence of increasing amounts of b24-156 (formerly known as bsyn; Ref. 33). After centrifugation and analysis of the pellet by SDS-PAGE, the amount of F1 bound to the resin was observed to decrease as the concentration of b24-156 increased, until essentially no F1 was bound by the resin at 6.5 µM of b24-156 dimer (Fig. 1A). Note that the amounts of soluble b24-156 trapped within the pelleted resin provide an internal representation of the amount of competitor added. These experiments demonstrate that b24-156 is able to compete with the bMERC-modified resin for binding to F1, establishing a simple competition assay for determining the relative affinity of any mutant form of b for F1.
Such experiments were carried out using b24-155 and b24-152, which lack one and four residues from the C terminus, respectively. It was found that these forms of b competed very poorly, relative to b24-156, with the bMERC-modified resin (Fig. 1B). At a dimer concentration of 10 µM, b24-155 showed a small amount of competition, whereas at the same concentration b24-152 showed no detectable competition by the SDS-PAGE assay. It is evident, however, that very weak competition is difficult to detect in this manner, as it requires seeing a small difference in band intensity.Competition for F1 Binding by the Soluble ATPase Activity Assay-- To determine weak competition more reliably and in a quantifiable way, the assay was modified such that soluble ATPase activity, rather than bound ATPase protein, was measured. Under the conditions used in these assays, more than 90% of the added enzyme was bound by the resin. Competition for F1 binding by b24-156, b24-155, and b24-152 was determined by the increase in ATPase activity remaining in the supernatant solution when these forms of b were added to the incubations. As expected, the amount of F1 in the supernatant solution increased sharply with increasing concentration of b24-156, whereas the ability of b24-155 and b24-152 to compete with the bMERC-modified resin was far weaker, although still detectable (Fig. 2). These results show that the C-terminal residues of b are essential for its proper interaction with F1-ATPase. The preparations of soluble b were tested directly for ATP hydrolysis activity to make certain that trace contamination with an enzyme such as alkaline phosphatase could not account for the increase in soluble ATPase activity. In no case could such a contaminant account for more than 2% of the observed soluble activity.
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Sedimentation Velocity Analysis--
Recent ultracentrifugation
results from this laboratory have provided evidence for the formation
of an elongated complex by two molecules of the hydrophilic region of
b and one molecule of the subunit (31). We performed
further centrifugation experiments to determine whether the reduced
binding of the C-terminal truncations of b to F1
could be due to loss of contacts with
. The isolated b24-156 and
subunits showed sedimentation
coefficients (Table I) which were similar
to those previously reported for bsol (25) and
(31). The increase in the s20,w upon mixture of equimolar amounts of dimeric b24-156
and
is confirmation that these proteins form a complex in solution.
Mixtures of
with b24-155 or with
b24-152 resulted in s values only
slightly above the weighted averages of the two components, indicating
that binding of b to
is significantly disrupted by removal of one or four residues from the C terminus. Thus the essential nature of these residues for binding F1 derives
from their role in binding
. The molecular mass of
b24-152 was determined by sedimentation
equilibrium, as described under "Experimental Procedures," to be
28,100 ± 1000 Da confirming that the dimeric nature of
b was unaffected by the C-terminal truncation. Unlike b24-156 (34), no aggregation of
b24-152 was observed at high
concentrations.
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Cross-linking of b and --
Individual cysteine residues were
introduced into full-length b at positions 150, 151, and
155. A fourth construct was made that encoded a protein, referred to as
b158C, having two residues, glycine and cysteine, attached
to the C terminus of b. It was anticipated that the glycine
would provide conformational flexibility to the C-terminal cysteine,
increasing the likelihood of obtaining a cross-link. Plasmids bearing
these mutated forms of b were all able to complement the
uncF strain KM2 for growth on minimal media with succinate
as the sole carbon/energy source, indicating that the
b-F1 interaction was not disrupted in the
mutants.
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Peptide Analysis of b24-156- Cross-links--
To
identify the region of
involved in the cross-links to
b24-156, a slice containing the cross-linked
product from each cysteine mutation was cut from an SDS-polyacrylamide
gel and treated with cyanogen bromide as outlined under "Experimental Procedures." CNBr cleavage of b24-156 should
give rise to fragments of 2, 12, and 126 residues, with the large
C-terminal fragment (residues Ala-31 to Leu-156) containing the site of
the cross-link. The
subunit should give rise to fragments between 10 and 49 residues in length. The difference between
b24-156 and its largest CNBr fragment is
readily apparent on SDS-PAGE (Fig. 6).
CNBr cleavage of each cross-linked product gave rise to a predominant
fragment that is markedly larger than the 126-residue fragment derived
from b24-156 alone (marked by an
arrow in Fig. 6).
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DISCUSSION |
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The results provided demonstrate that the residues at the C
terminus of b are essential for proper interaction with
F1, that the interaction of b with is
weakened when these residues are lacking, and that the C-terminal
region of b is proximal to that of
. Together these
subunits are believed to form a "second stalk" reaching from the
membrane to near the top of the F1 sector, which may
function to hold the
3
3 subunits
stationary during rotation of
and
driven by proton flow through
F0 (48, 49). Earlier truncation and mutagenesis studies
showed that the C terminus of b was essential for proper
assembly of ATP synthase (10) and implied that the C terminus of
played a role in interaction of F1 with the F0
sector (21-24).
Our present studies confirm and extend this earlier work by providing
additional information about the b interaction. The major
site of cross-linking of the E155C and 158C proteins to
was shown
to be C-terminal to Met-148. It is interesting that the cross-linked
subunit was not cleaved at Met-158 by cyanogen bromide treatment
and that no signal corresponding to this residue was observed during
the appropriate cycle of peptide sequencing. The most straightforward
interpretation of these results is that the major site of benzophenone
linkage was through the side chain of Met-158. The benzophenone moiety
may have formed a cross-link at C
of this methionine to
create a tertiary carbon center that would be relatively unreactive to
cleavage by CNBr. It is notable that methylene groups adjacent to
nitrogen or sulfur are particularly reactive to benzophenone (50).
Modeling of the C-terminal region of b as an -helix
reveals an amphipathic structure (Fig.7).
The cross-linking results imply that the hydrophobic face of this helix
on one of the b subunits interacts with
. We suspect that
the hydrophobic face of the second b subunit is in contact
with another region of b. Removal of one or more hydrophobic
residues might disrupt this interaction, causing a significant
conformational change in the soluble b protein. Such a
conformational change to a more asymmetric shape would explain why the
sedimentation coefficients of b24-155 and b24-152 were markedly lower than that observed
for b24-156 (Table I). In the absence of
,
the hydrophobic face of the first b subunit would not have
its normal partner and might interact nonspecifically with the
similarly exposed hydrophobic face of another
b24-156 dimer. This would explain why the
slight aggregation of b24-156 observed during
sedimentation equilibrium centrifugation at high concentrations (34) is
not seen with b24-152.
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Hydrophobic residues are conserved at positions corresponding to
E. coli residues 153 and 156 in the b subunit of
many organisms (51). It is tempting to suggest that the importance of
these hydrophobic residues in the b- interaction may
provide the explanation for the observation that low ionic strength
disrupts the binding of F1 to F0. However,
other regions of b must also be involved in
b-F1 interactions, since
b24-152 was able to compete to a minor extent
with the affinity resin for binding to F1 (Fig. 2). In this
regard it is also noteworthy that some bacteria have b
subunits that lack the hydrophobic residues at the C terminus. For
example, the b subunit of the thermophile PS3 ends at the residue corresponding to position 148 in E. coli b (52).
The difference in sedimentation coefficients observed between the
(b24-156)2 and the
(b34-156)2
complexes (Table I)
was surprising. One possible explanation is that flexibility in the
N-terminal region of the soluble b construct allows the hydrophobic residues Y24VWPPLMAAI33, present in
b24-156 but absent in
b34-156, to loop back and interact with
.
Such an arrangement would be more compact, and the complex would
therefore sediment faster than if the N-terminal helices were in a more
extended conformation. Because the N termini of the b
subunits are anchored in the membrane in ATP synthase, it seems
unlikely that residues 24-33 of b normally interact with
in the intact complex.
Takeyama and co-workers (10) showed that one aspect of the defective
ATP synthase assembly caused by deletion of residues from the C
terminus of b was the failure of F0 to form a
functional proton pore in vivo. In subsequent work, Brusilow
and co-workers (53, 54) demonstrated that the subunit was required
for the formation of the proton pore from the cloned F0
subunits. Here we have shown that the same region of b
implicated in the F0 assembly process is essential for
binding to
, strengthening the argument that the b-
interaction is critical for the assembly of functional F0.
At present, however, we have no evidence of how a signal arising from
the interaction may be transmitted from the C-terminal end of
b to the membrane, where interaction with the other
F0 subunits would occur.
The solution structure of residues 1-105 of ', a proteolytic
fragment consisting of residues 1-134 of
, has been solved by NMR
spectroscopy, but the structure of the entire 177-residue subunit could
not be determined (49). Although the C-terminal regions of both
proteins are predicted to be largely
-helical, in the absence of
concrete structural information from either region it is difficult to
propose specific interactions between amino acid residues in
b and
. The proposed site of cross-linking, Met-158, lies
before a predicted C-terminal helix encompassing residues
167-175
(55).
Our current results demonstrate that b and interact via
their C-terminal regions, and a recent study from our laboratory has
shown that the b2
complex is extended enough
to span the distance from the membrane to the N-terminal domain of
(31). Thus our work is consistent with the hypothesis that the
b and
subunits form a stator to prevent
3
3 from moving relative to
. Since the
stator must resist an appreciable torque (15), and since the
Kd of the b-
interaction is relatively high (5-10 µM; Ref. 31), it is likely that the
interaction of these subunits is stabilized by the presence of the
other parts of the ATP synthase complex. A further possibility is that
b makes direct contact with other subunits, such as
or
, to stabilize the b-F1 interaction. The
newly developed competition assay for b-F1
binding provides a simpler, more quantitative, and more sensitive way
of detecting minor changes in b-F1 affinity
compared with earlier methods. We are currently using this assay to
define other residues of b that affect
b-F1 interactions.
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ACKNOWLEDGEMENTS |
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We thank Drs. Gabriele Deckers-Hebestreit and Karlheinz Altendorf for providing the 10-1A4 and 10-6D1 monoclonal antibodies; Drs. Kimberly McCormick and Brian Cain for providing E. coli strain KM2; Matthew Revington for helpful discussions and construction of plasmid pMR2; Hanna Abou Alfa for helpful discussions; and Faye Males for technical assistance.
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FOOTNOTES |
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* This work was supported by Grant MT-10237 from the Medical Research Council of Canada. The Beckman XL-A analytical ultracentrifuge was acquired through support from 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.
Supported by an Ontario Graduate Scholarship.
§ 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 protein; bMERC, form of the b subunit containing residues Tyr-24 to Leu-156 with the N-terminal leader sequence MERCSNSH; b24-156, b24-155, and b24-152, forms of the b subunit containing the residues indicated with the N-terminal leader sequence MTMITNSH; b24-158 or 158C, form of the b subunit identical to b24-156 but with Gly and Cys added to the C terminus; DTT, dithiothreitol; TEA, triethanolamine; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; BPM, benzophenone-4-maleimide; PCR, polymerase chain reaction; DMF, dimethylformamide; PVDF, polyvinylidene difluoride.
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REFERENCES |
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