(Received for publication, May 18, 1995; and in revised form, July 5, 1995)
From the
Alanine-scanning mutagenesis was applied to the subunit of
the F
-F
ATP synthase from E. coli.
Nineteen amino acid residues were changed to alanine, either singly or
in pairs, between residues 10 and 93. All mutants, when expressed in
the
deletion strain XH1, were able to grow on succinate minimal
medium. Membranes were prepared from all mutants and assayed for
ATP-driven proton translocation, ATP hydrolysis ±
lauryldiethylamine oxide, and sensitivity of ATPase activity to N,N`-dicyclohexylcarbodiimide (DCCD). Most of the
mutants fell into 2 distinct classes. The first group had inhibited
ATPase activity, with near normal levels of membrane-bound
F
, but decreased sensitivity to DCCD. The second group had
stimulated ATPase activity, with a reduced level of membrane-bound
F
, but normal sensitivity to DCCD. Membranes from all
mutants were further characterized by immunoblotting using 2 monoclonal
antibodies. A model for the secondary structure of
and its role
in the function of the ATP synthase has been developed. Some residues
are important for the binding of
to F
and therefore
for inhibition. Other residues, from Glu-59 through Glu-70, are
important for the release of inhibition by
that is part of the
normal enzyme cycle.
The F-F
ATP synthase from Escherichia coli (for reviews, see (1, 2, 3) ) is composed of eight different
types of polypeptide subunits which are coded by eight genes found in a
single operon called unc (or atp). The enzyme can be
physically separated into a membrane-bound portion called F
and a water-soluble portion called F
. F
consists of five types of subunits:
(uncA),
(uncD),
(uncG),
(uncH), and
(uncC) in a stoichiometry of 3:3:1:1:1. F
consists of three types of subunits: a (uncB), b (uncF), and c (uncE) in a
stoichiometry of 1:2:10 ± 1 (4) .
In the intact
F-F
complex, the net synthesis of ATP at sites
in F
is in response to an electrochemical proton gradient
across the membrane, which drives proton movement through
F
. This proton movement is coupled to net synthesis of ATP
at sites in F
, probably via a series of conformational
changes. Isolated F
catalyzes the hydrolysis of ATP, but
not its synthesis. The molecular events linking the movement of protons
through the membrane-bound portion of the ATPase to the synthesis of
ATP at distant sites, remain essentially unknown. Recently, x-ray
crystallographic studies of F
from bovine mitochondria (5) has provided structural information about
,
, and
parts of
. In the resulting model, the amino and carboxyl termini
of
penetrate the central cavity of the
hexamer, in an asymmetric fashion.
One interpretation is that the
subunit rotates with respect to
and
, thereby sequentially interacting with different
catalytic sites, as indicated by earlier studies(6) .
With
the E. coli enzyme, two additional subunits, and
,
are necessary for the proper binding of the
complex to F
. In
addition,
has the interesting property of acting as an intrinsic
inhibitor of ATP hydrolysis by the soluble
F
(7, 8) . The mechanism by which
acts upon the remaining F
subunits to cause inhibition is
unknown. The removal (or displacement) of
from the complex
results in about a 6-fold increase in ATP hydrolysis
activity(9, 10) . This effect is not dependent upon
the presence of the
subunit. The
subunit is thought to
inhibit by reducing the rate of product release(10) , the
rate-limiting step in hydrolysis. It is known to bind to isolated
subunit(11) , but can be cross-linked to
,
, and
subunits(12, 13, 14, 15) .
A series of deletion mutants has been constructed from the carboxyl
terminus of and analyzed(16) . A truncated form
containing only the first 93 residues, plus 2 serine residues at the
new carboxyl terminus, was capable of inhibiting ATP hydrolysis and of
promoting the binding of F
to F
. A shorter
subunit (80 residues) was also capable of the binding function, but did
not have inhibitory properties. These data seem to suggest that an
``inhibitory domain'' exists, composed at least partially of
residues between 80 and 93, and that it is distinct from a binding
domain in the first 80 residues. Other studies (17, 18) indicated that about 15 amino acids can be
lost from the amino terminus without a total loss of function.
Nucleotide-dependent conformational changes in have been
detected by protease sensitivity (19) and by cryoelectron
microscopy using monoclonal antibodies(20) . An interesting
recent result is that second-site suppressors of a c subunit
mutation (Q42E) were mapped to
-Glu-31. Quite possibly, both
and
might be intimately involved in the coupling of proton
translocation to ATP synthesis, as has been reviewed
recently(21) . To probe the interactions of
with other
subunits, and its role in function, we have applied alanine-scanning
mutagenesis(22) .
Figure 1:
A,
the gene for the subunit, uncC. The darkened regions of the gene indicate segments that have been replaced by synthetic
DNA containing additional restriction sites(27) . Sites used in
this study have been indicated. B, deoxyoligonucleotides used
for cassette mutagenesis. For each pair of oligonucleotides, the two
restriction sites used for ligation are indicated at the appropriate
ends. The residues that are mutagenized are indicated above the
oligonucleotides, and the corresponding new codons are underlined. The following symbols indicate 50% mixtures of
bases at a designated position: M = A + C, K = T + G, S = G + C, R = A + G, Y = C +
T.
Mutations were constructed as outlined in Fig. 1.
Amino acid residues in were converted to alanine to maximize the
possibility that the effects of mutagenesis would be confined to local
changes. Residues were selected for mutagenesis based on the following
criteria. First, partially conserved, and generally polar, residues
were selected. Second, the locations of the mutations were generally
confined to the first 85 amino acids, the size of
that was shown
to be sufficient for inhibition of F
ATPase
activity(16) .
Eighteen mutants, with either single or
double alanine substitutions in , were analyzed for growth
properties following transformation of the
deletion strain XH1.
The mutants that were constructed and their growth properties are shown
in Table 1. Only 2 mutants, S10A and F16A, grew poorly on minimal
succinate medium, as compared to wild type. The measurement of growth
yields in minimal glucose medium confirmed those results: S10A (48%),
F16A (67%), T77A (85%), and all of the rest were >90% of the wild
type level. The results of ATP-driven proton translocation assays by
membrane vesicles followed the same pattern: S10A and F16A had no
activity, E70A and T77A had 65% and 60% of the wild type activity,
respectively, and all of the rest were >90%.
ATP hydrolysis by
membrane vesicles from these mutants was measured under three sets of
conditions, and the results are shown in Table 1. The standard
assay was the coupled enzyme assay at pH 7.5. This assay was repeated
in the presence of 1% LDAO, which is known to release the inhibition of
F ATPase activity by
(9, 31) . The
second assay reveals two properties of the mutants. First, it yields
the level of uninhibited ATPase activity of the membranes, which
reflects the total amount of F
bound. Second, when compared
with the first assay it shows the fold stimulation upon LDAO addition,
which reflects the fold inhibition by
. Finally, a third assay was
performed after preincubation with DCCD, a specific inhibitor of proton
translocation by F
. Decreased sensitivity of ATPase
activity to DCCD reflects ATP hydrolysis that is not obligatorily
coupled to proton translocation.
Results of the standard ATP
hydrolysis assays revealed that membranes from S10A and F16A had
undetectable levels of membrane-bound ATPase. Membranes from the other
mutants ranged from 51% to 132% of the wild type levels of ATPase
activity, suggesting 2 classes of mutants. This was confirmed by assays
in the presence of LDAO, or after preincubation with DCCD. Six mutants
showed stimulation of ATP hydrolysis by LDAO of less than 2.4-fold,
compared to the wild type value of 3.1-fold. These mutants were T77A,
R85A, D81A/R85A, T82A/R85A, T43A, and K46A. This group also tended to
have lower levels of bound F ATPase, averaging 61% of wild
type, and to have normal sensitivity to DCCD, averaging 60% (95% of the
wild type value).
A second class consisted of E70A, S65A, E59A/E60A,
K54A/E60A, E59A, and E31A. These mutants showed higher than normal
stimulation of ATPase activity by LDAO, with values ranging from 3.5-
to 4.6-fold. These mutants showed a more normal level of membrane-bound
F ATPase, averaging 85% of the wild type level. However,
the membrane-bound ATPase activity was less sensitive to DCCD,
averaging only 48% (76% of the wild type level). Six additional
mutants, E21A, Q24A, E29A, R51A, K54A, and R93A had properties that
were intermediate between the other two classes and were more similar
to the wild type.
Membranes from all mutants were subjected to
immunoblotting using two previously described monoclonal antibodies to
(23) . Both monoclonal antibodies failed to recognize
F16A, and S10A was recognized only weakly. The only significant
difference between the two antibodies was that
I failed to
recognize E21A and Q24A.
A sequence alignment and secondary
structure prediction was obtained using the EMBL PHD
system(32) , and results of the secondary structure predictions
are summarized in Fig. 2. Sequences from seven subunits
were found to have greater than 30% identity with that of E.
coli, and those were used in the secondary structure prediction.
Six regions of
-strand were predicted in the first 78 amino acids,
and three regions of
-helix were predicted at the carboxyl
terminus(79-138), consisting of 5, 16, and 21 residues. Two other
regions were weakly predicted to be
-strands: residues 15-16
and 32-34. Each of the 8 predicted
-strands shown in Fig. 2has 2 hydrophobic residues separated by a single residue.
These residues are underlined. This trend exists in each of the eight
sequences and is consistent with amphipathic
-strands, such
as those which would form a
-barrel or
-sandwich. The only
exception is the second predicted
-strand, in which the 2
hydrophobic residues are adjacent. Further analysis of 21 bacterial
sequences retrieved by BLAST analysis (33) revealed that
this pattern of hydrophobic residues was present in each sequence (not
shown).
Figure 2:
PHD
prediction of secondary structure of . The lines labeled AA contain the amino acid sequence of
, with the corresponding
numbers above. PHD indicates the profile network prediction,
where E indicates
-strand, H indicates
-helix, and blank spaces indicate a loop prediction. Rel indicates the reliability index of prediction, on a scale
from 0 to 9 (most reliable). SUB indicates a subset of the
predictions for which the expected accuracy will be >82%. In this
line, loop predictions are indicated by L. Where Rel < 5,
no prediction is made, and these are indicated by dots. Underlined
residues are the hydrophobic residues in the predicted
-strands. Other sequences used in the prediction are Vibrio
alginolyticus, Bacillus PS3, Bacillus
megaterium, Bacillus firmus, Rhodospirillum
rubrum, Synechocystis PCC 6803, and Synechococcus PCC 6301.
The subunit has a central role in energy transduction
by the E. coli F
-F
ATP synthase. The
goal of the present work was to identify by mutagenesis individual
amino acids in
that are involved in specific roles. Since amino
acid replacements can cause a variety of nonlocal effects on protein
structure, alanine-scanning mutagenesis (22) was used in this
study.
Two mutants, S10A and F16A, seemed to be defective in
assembly of the ATP synthase. Residue 10 is predicted to be in the
first turn following the first -strand. The mutant S10C has
previously been characterized (13) and has been cross-linked
to
via the thiol group. Our results are consistent with the
location of residue 10 being near the
-
interface. In
addition, introduction of alanine at position 10 creates a hydrophobic
patch at residues 8-11 (VVAA) that may promote misfolding or
-
aggregation. Others (18) have observed that a
deletion of residues 7-14 in
caused aggregation.
The
mutant F16A is even less functional. Residue 16 resides in the second
predicted -strand at a position that is always Phe, Tyr, or Trp.
Other studies have shown that amino-terminal deletions in
of 16
residues are much more severe than are deletions of only 15 residues (17) . Our results also indicate a critical role for residue
16.
Residues Thr-77, Asp-81, Thr-82 and Arg-85 seem to be important
for the binding of to other subunits. Each of the four mutants
showed reduced levels of membrane-bound F
ATPase activity,
but this activity was not significantly inhibited by
as shown by
the low stimulation by LDAO (approximately 1.5 versus 3.1 for
wild type). It is not unexpected that mutations that alter
binding to other subunits, presumably
(11) , would reduce
both the binding of F
to F
in membranes and
also the inhibition of ATPase activity by
. These residues extend
from the eighth predicted
-strand to the
-helical region.
This region has been implicated previously in the inhibitory role of
by analysis of deletions from the carboxyl terminus(16) .
It has also been shown that an
-monoclonal antibody has an epitope
that is buried in F
and part of that epitope is between
residues 78 and 85 (18) . Two other mutants analyzed in this
study have similar properties: T43A and K46A. These residues lie in a
stretch of 15 amino acids between the fourth and fifth predicted
-strands. It is possible that these residues make up part of the
epitope for
-mAb-1, described previously(18) .
A second
group of mutants has an opposite effect on the inhibitory properties of
. This group also contains residues from two distinct regions:
residue 31 and residues 59-70. With these mutants a relatively
high level of F
is bound to membranes, averaging 85% of
wild type, but the ATPase activity is highly inhibited. Upon addition
of LDAO, the activity is stimulated up to 4.6-fold. It appears that
these mutants, E31A, E59A, E60A/K54A, E59A/E60A, S65A, and E70A, form
stable F
-F
complexes, but that the F
is locked in an abnormally inhibited state.
Residue Glu-31 is
located in the fourth predicted -strand and has been mutagenized
previously(27) . This residue has also been identified as the
site of partial suppressors of the c subunit mutant
Q42E(34) . In the present study, E31A was found to reduce the
sensitivity of F
ATPase to DCCD (76% of wild type).
Mutations at the nearby residue His-38 were shown (27) to
reduce the sensitivity of F
ATPase to DCCD. This evidence
tends to support either of the possibilities offered(34) , that
this region of
interacts directly with the polar loop of c or that it interacts with ``stalk'' subunits, which
interact with c. A reduced sensitivity to DCCD by membrane-bound
F
ATPase is also seen with most of the other members of
this group, especially S65A and E70A (70% and 62% of wild type,
respectively). Residue 65 is predicted to lie in a loop between the
sixth and seventh
-strands, and residue 70 is part of the
predicted seventh
-strand. The double mutant E59A/E60A also has
reduced sensitivity to DCCD, and these residues are found in a loop
between the fifth and sixth predicted
-strands.
The two
monoclonal antibodies have been characterized previously with respect
to binding of in F
and F
-F
,
I (5A3-C11), and
II (13A7-E9) (23) . Our results
reveal a single difference between these two antibodies:
I does
not recognize the mutants E21A and Q24A. Since it was determined
previously (23) that
I does not recognize
in
F
-F
complexes, but that
II does weakly, it
is likely that residues 21 and 24 of
are shielded by the binding
of F
to F
.
The results of the present study
contribute to the growing picture of the role of in the structure
and function of the F
-F
ATP synthase. Previous
work by others has indicated a two-domain structure for
(16) , separated by a ``hinge'' region near
residues 80-85(18) . Conformational changes in
have
been observed via differences in protease sensitivity and in response
to ligands at nucleotide binding sites(19) . DCCD has been
shown to alter the nucleotide dependence of trypsin
cleavage(35) . Movement of
relative to other F
subunits has been observed via specific cross-linking reagents,
also in response to ligands at nucleotide binding
sites(6, 12, 13) . The effects of several
mutations at the strictly conserved residue His-38 have also been
studied. H38C has been shown to be accessible to Ellman's
reagent, and the modified
remains inhibitory(36) . Two
other mutants H38I and H38R were found to have decreased sensitivity to
DCCD, but
seemed to be highly inhibitory(27) .
A
schematic model for the secondary structure of is shown in Fig. 3, based on the PHD secondary structure predictions,
hydrophobicity considerations, and the results of our mutagenesis. The
model is oriented such that the top would be near the
and
subunits in F
, and the bottom would be near the c subunits of F
. The model includes 8 antiparallel
-strands, 3
-helical segments, and one significant region of
undesignated secondary structure. It is proposed that 6
-strands
are organized into an anti-parallel
-barrel, and that 2 additional
-strands form a
-hairpin. Three of the
-strands in the
barrel are proposed to interact with
: primarily strands 2 and 8,
and to a small extent strand 1. The other 3
-strands in the
barrel, 5, 6, and 7, are proposed to form a relatively exposed face
that interacts with one or both of the
-helical segments. The
-hairpin, formed by
-strands 3 and 4, is proposed to be close
to F
subunits. A region of undesignated secondary
structure, found between
-strands 4 and 5, and the short
-helix following
-strand 8 are both proposed to have
interactions with
. The sites of alanine mutations are also
indicated in Fig. 3.
Figure 3:
A secondary structural model of the
subunit. The black arrows represent
-strands, and the white cylinders represent
-helices. The sites of alanine
mutations are indicated by circles filled with the residue
number. Residue 108, the site of cross-linking to
(12) , is also indicated.
In view of the secondary structure model
and the previously described conformational changes of , the
following interpretation of these mutations is offered: residues
Ser-10, Phe-16, Thr-43, Lys-46, Thr-77, Asp-81, Thr-82, and Arg-85 are
proposed to be important for the binding of
to other subunits,
primarily
. Failure to bind properly, due to mutations at these
residues, leads to decreased inhibition by
, and to some loss of
F
from membranes. Specifically, the conformation of
that stabilizes bound products at the active site is affected, but the
functional coupling of F
and F
is not affected.
Residues Glu-31, Lys-54, Glu-59, Glu-60, Ser-65, and Glu-70 are
proposed to be important for the release of inhibition that is part of
the normal conformational cycle of
. Mutations at these residues
lead to a highly inhibited form of membrane-bound F
, and
ATPase activity is partially decoupled from proton translocation. These
residues might interact with carboxyl-terminal residues of
or, in
the case of Glu-31, with c subunits(34) . One prediction
is that some of these mutants might be resistant to proteolysis by
trypsin, independent of nucleotide(19, 35) .