(Received for publication, September 1, 1995; and in revised form, November 30, 1995)
From the
The interaction faces of the and
subunits in the Escherichia coli F
-ATPase have been explored by a
combination of cross-linking and chemical modification experiments
using several mutant
subunits as follows:
S10C,
H38C,
T43C,
S65C,
S108C, and
M138C, along with a mutant
of the
subunit,
T106C.
The replacement of Ser-10 by a Cys
or Met-138 by a Cys reduced the inhibition of ECF by the
subunit, while the mutation S65C increased this inhibitory
effect. Modification of the Cys at position 10 with N-ethylmaleimide or fluoroscein maleimide further reduced the
binding affinity of, and the maximal inhibition by, the
subunit.
Similar chemical modification of the Cys at position 43 of the
subunit (in the mutant
T43C) and a Cys at position 106 of the
subunit (
T106C) also affected the inhibition of ECF
by the
subunit.
The various subunit mutants were
reacted with TFPAM3, and the site(s) of cross-linking within the
ECF
complex was determined. Previous studies have shown
cross-linking from the Cys at positions 10 and 38 with the
subunit and from a Cys at position 108 to an
subunit (Aggeler,
R., Chicas-Cruz, K., Cai, S. X., Keana, J. F. W., and Capaldi, R.
A.(1992) Biochemistry 31, 2956-2961; Aggeler, R.,
Weinreich, F., and Capaldi, R. A.(1995) Biochim. Biophys. Acta 1230, 62-68). Here, cross-linking was found from a Cys at
position 43 to the
subunit and from the Cys at position 138 to a
subunit. The site of cross-linking from Cys-10 of
to the
subunit was localized by peptide mapping to a region of the
subunit between residues 222 and 242. Cross-linking from a Cys at
position 38 and at position 43 was with the C-terminal part of the
subunit, between residues 202 and 286.
ECF treated
with trypsin at pH 7.0 still binds purified
subunit, while enzyme
treated with the protease at pH 8.0 does not. This identifies sites
around residue 70 and/or between 202 and 212 of the
subunit as
involved in
subunit binding.
Proton translocating FF
ATPases (also
ATP synthases) are found in the membranes of bacteria, chloroplasts,
and mitochondria, where they function in oxidative phosphorylation or
photophosphorylation to couple a transmembrane proton electrochemical
gradient to ATP synthesis. These complex enzymes are reversible and can
use ATP hydrolysis to establish a proton gradient for subsequent use in
substrate and ion transport processes (reviewed in Cross(1988), Senior
(1990), and Boyer(1993)).
As the name implies, FF
ATPases are made up of two parts, an extrinsic F
part
that contains the catalytic sites and a membrane-intrinsic F
part that contains the proton channel. Cryo-electron microscopy
shows these two parts separated by a stalk of 40-45 Å in
length (Gogol et al., 1987; Lücken et
al., 1990). In the bacterium Escherichia coli, the
F
part (ECF
) is made up of five different
subunits (
,
,
,
, and
) in the molar ratio
3:3:1:1:1. The F
part contains three different subunits (a,
b, and c) in the ratio 1:2:9-12 (Senior, 1990; Fillingame, 1992).
The structure of the F part in the enzyme from bovine
mitochondria has been obtained recently to 2.8 Å resolution by
x-ray crystallography (Abrahams et al., 1994). This shows
three
and three
subunits arranged in a hexagon, with the
subunit extending the length of the cavity within the hexagon,
projecting from one end of the structure into an extension that is part
of the stalk. The
subunit of MF
, which is the
equivalent of the
subunit in bacterial and chloroplast enzymes,
is also in the stalk but, in the crystal form studied, is probably
disordered and not resolved.
There is now considerable evidence that
the and
subunits play an important role in coupling
catalytic site events with proton translocation (e.g. Gogol et al., 1990; Nakamoto et al., 1993; Abrahams et
al., 1994; Capaldi et al., 1994; Zhang et al.,
1994) and that this coupling involves conformational changes and,
probably, translocations of one or both subunits (Aggeler et
al., 1992, 1993; Turina and Capaldi, 1994a, 1994b; Wilkens and
Capaldi, 1994). It is important, therefore, to obtain the detailed
structures of, and interaction sites between, these two subunits.
We
have recently determined the structure of the subunit in solution
by NMR spectroscopy (Wilkens et al., 1995). Here, we describe
experiments that identify the
subunit binding site on the
subunit, and that locate regions of the
subunit involved in the
subunit binding.
A number of mutants were used in the present study. Mutants
S10C (Aggeler et al., 1992),
H38C (Skakoon and Dunn,
1993),
S108C (Aggeler et al., 1992), and
M138C (this
study) were created in the unc operon containing plasmid
pRA100 (Aggeler et al., 1992). These mutants each showed
wild-type growth on limiting glucose. Mutants T43C and S65C were
created in the plasmid pEX2 containing the uncC, i.e.
subunit, gene (Skakoon and Dunn, 1993). The inhibitory
effect of the
subunit when bound to isolated ECF
(Sternweis and Smith, 1980) was used as a convenient measure of
the binding of the
subunit to the core complex
(
). Table 1summarizes the
activity effects of the various
subunit mutations. It includes
data for ECF
isolated from strains, in which the mutation
was in the unc operon, and data from experiments in which
wild-type ECF
has been depleted of endogenous
subunit
and then reconstituted with an excess of the mutant
subunit. The
ATPase activity of wild-type ECF
was 10 ± 0.5
units/mg (µmol of ATP hydrolyzed/min/mg of enzyme), under our assay
conditions at pH 7.5, with 2 mM ATP and 5 mM Mg
. The activities of ECF
isolated
from the mutants
H38C and
S108C were very similar to that of
wild-type, while that of enzyme from mutants
S10C and
M138C
were around 4- and 2-fold higher than the wild-type, respectively. The
ATPase activity of
and
-free ECF
was 51 ±
3 units/mg, an activation of around 5-fold, which could be reduced to a
basal of around 7 units/mg by addition of excess wild-type
subunit. Addition of purified
subunit carrying the H38C or S108C
mutations had similar effects to the addition of wild-type subunits. In
all cases, excess of the
subunit inhibited activity below that of
isolated ECF
, suggesting that there is some loss of
subunit during isolation of the intact ECF
complex.
Rebinding of
S10C gave a minimal activity of 13 units/mg. Addition
of a 9-fold excess of
subunit with the mutation T43C also failed
to inhibit the core ECF
complex
(
) to levels found using
wild-type, consistent with the mutant having a reduced affinity for the
core complex. Addition of mutant
S65C gave a higher inhibition,
giving a final ATPase activity that was only 70% of wild-type enzyme.
The activity data, therefore, indicate that mutations S10C and T43C
reduce, while the mutation S65C increases, the inhibitory effect of the
subunit on the core ECF
complex.
The mutants
S10C and T43C were examined further in experiments in which the
introduced Cys was reacted with various maleimides, and the effect of
this modification on activity was monitored. Fig. 1shows the
concentration dependence of the inhibition of ATPase activity of the
mutant
S10C before and after modification with different
maleimides. It can be seen that both the concentration for half-maximal
inhibition and the absolute extent of inhibition of this mutant were
altered significantly by NEM and dramatically by modification with FM.
Modification of the mutant T43C with FM had a much smaller, although
significant, effect on the binding affinity and maximal inhibition
(results not shown). These results indicate that the regions of the
enzyme around residue 10 and, to a lesser extent, around residue 43 are
important for interaction of the
subunit with the core ECF
and the resulting inhibition of enzymatic activity.
Figure 1:
Effect of
chemical modification of the subunit from the mutant S10C subunit
on its inhibitory function. Filled circles,
S10C labeled
with FM; open squares,
S10C labeled with NEM; open
triangles, unlabeled
S10C; open circles, wild-type
subunit as a control.
Figure 2:
Cross-linking between the and
subunits in ECF
from the mutant
M138C. ECF
from the mutant
M138C was labeled with photoreactive
maleimide TFPAM3 at room temperature for 1 h. After removal of the
excess label by one centrifuge column, ATP (5 mM) or ATP (5
mM) + Mg
(5.5 mM) was added to
the sample and incubated for 30 min. Samples were analyzed by
10-18% SDS-polyacrylamide gel electrophoresis. Lane 1,
ATP + EDTA (dark); lane 2, ATP +
Mg
(dark); lane 3, ATP
(photolyzed); lane 4, ATP + Mg
(photolyzed).
The
location of residues 43 and 65 were examined by reacting subunit
with TFPAM3 in the dark, removing excess reagent and then binding to
ECF
, treated to remove endogenous
subunit with
trypsin, followed by photolysis to activate the TFPA group. A
cross-linked product between the
and
subunits was observed
in ECF
containing the T43C mutation (see below). No
cross-link was observed with the S65C mutant.
Figure 3:
Cross-linking and trypsin digestion of
ECF from the mutant
S10C. ECF
from the
mutant
S10C was cross-linked by TFPAM3 and treated with trypsin
for 1 h. After stopping the trypsin digestion with 4 mM freshly prepared phenylmethylsulfonyl fluoride, samples were
analyzed by 10-18% SDS-polyacrylamide gel electrophoresis (panel A), and the presence of a truncated
-
cross-linked product was confirmed by immunoblotting (panel
B). Panel A, lane 1, photolyzed; lane
2, photolyzed + trypsin; lane 3, unphotolyzed +
trypsin. Panel B, Western blot of the sample of photolyzed
+ trypsin was incubated with anti-
III mAb (lane 1)
and anti-
I mAb (lane 2).
The approximate M 19,000 band was excised from polyacrylamide
gels, protein was collected by electroelution, purified by HPLC, and
then digested further with trypsin. The small fragments, so generated,
were separated by high pressure liquid chromatography, and the peaks
were resolved and then analyzed by N-terminal amino acid sequencing.
One peak contained a peptide of
containing Cys-10, present in
equimolar amounts with a fragment of the
subunit identified by
its sequence as including residues 222-242.
As shown in Fig. 4, the sequence of the fragment continued beyond
Cys-10, as expected, because the cross-link involves the side chain of
the Cys, and Edman degradation is not prevented. In contrast,
sequencing of the
fragment stopped abruptly before tyrosine 228,
consistent with cross-linking via the tetrafluorophenylazide into the
backbone of the polypeptide between residues 227 and 228. We have seen
a similar insertion of TFPAM into the backbone of the
subunit
next to a tryptophan residue in experiments to identify the interaction
site between
and
(Aggeler et al., 1993). It
appears that the TFPA moiety tends to stack against aromatic residues,
such as tyrosines and tryptophans, in an orientation that causes
insertion of the reactive nitrene into the backbone.
Figure 4:
Sequencing to determine the site of
cross-linking from S10C to the
subunit. Complete trypsin
digestion of the truncated
-
cross-linked product generated a
tryptic fragment containing the N-terminal sequence of the
subunit, i.e. AMTY. Further Edman degradation of this tryptic
fragment allowed identification of two sequences, one the N-terminal
amino acid sequence of
(upper plot) and the second
identified as the sequence of the
subunit beginning at residue
222.
An experiment involving the mutant H38C is shown in Fig. 5. Isolated
subunit containing the mutation H38C was
reacted with TFPAM3 in the dark, and then the modified
subunit
reacted with ECF
that had been treated with trypsin to
remove endogenous
and
subunits and at the same time cleave
to
and
. Two forms of
ECF
were used, one containing the mutation
S8C, the
other
V286C (Aggeler and Capaldi, 1992). These sites were labeled
with CM to identify the N-terminal
fragment or
C-terminal
fragment on gels. As shown in Fig. 5, cross-linking from the Cys at 38 to the
subunit
with TFPAM3 gave a product of approximate M
22,000, which contained Cys-286 but not Cys-8 of the
subunit and is, therefore, a covalent product of the
subunit at
position 38 with the C-terminal region 202-286 of
.
Figure 5:
Localization of the site of TFPAM3
cross-linking between H38C and the
subunit. Pure
H38C
subunit, labeled with TFPAM3 in the dark, was added to trypsin-treated
and CM-labeled ECF
from the mutants
S8C and
V286C. After photolysis, the samples were analyzed by 10-18%
SDS-polyacrylamide gel electrophoresis. The gel was later visualized on
a UV light box. Lanes 1 and 2, cross-linking using
ECF
from the mutant
V286C; lanes 3 and 4, cross-linking using ECF
from the mutant
S8C. Lanes 1 and 3, unphotolyzed; lanes 2 and 4, photolyzed.
Similar experiments were then used to localize the site of
interaction of the subunit from residue 43 with the
subunit. These show cross-linking of the
fragment to the same
C-terminal part of the
subunit (results not shown).
Figure 6:
Sucrose gradient centrifugation to measure
the binding of subunit to trypsin-treated ECF
.
ECF
that had been treated with trypsin at pH 7.0 or 8.0 was
incubated with a 10-fold molar excess of pure wild-type
subunit,
and the mixtures were applied on a 10-40% sucrose step gradient.
Samples from the gradient were concentrated and then loaded onto a
10-18% SDS-polyacrylamide gel for electrophoresis followed by
electroblotting onto a polyvinylidene difluoride membrane. The Western
blot was later analyzed with anti-
I mAb. Lane 1, pure
subunit (applied on the gel as control); lane 2,
ECF
treated with trypsin at pH 8.0; lanes 3 and 4, ECF
(pH 8.0) +
; lane 5,
ECF
, treated with trypsin at pH 7.0 +
.
Figure 7:
Effects of chemical modification of a Cys
at position 106 of the subunit on
subunit inhibition.
ECF
from the mutant
T106C modified with NEM or CM was
digested with trypsin to remove the
subunit. Purified
subunit (wild type) was added at different molar ratios with respect to
ECF
, and the ATPase activity was measured. The filled
circles show
T106C labeled with CM; the open squares are for
T106C labeled with NEM; the open circles are
for unmodified
T106C, while the open triangles show
wild-type ECF
, with all three experiments fitted to a
single rate (dashed line).
Studies presented here focus on the interaction faces of the
and the
subunits for each other. The results are summarized
in Fig. 8, which shows the recently obtained structure of the
N-terminal domain of the
subunit involving residues 1-86 in
part A (Wilkens et al., 1995) and the predicted folding
pattern of the
subunit in part B. Genetic studies have predicted
that the
subunit is a 2-domain protein (Kuki et al.,
1988), and this is evident in the NMR structure determination of the
isolated
subunit in solution. Residues 1-86 form a
10-stranded
barrel, or sandwich, while the C-terminal 48 residues
are arranged as an
-helix-loop-
-helix structure. In solution,
the helix-loop-helix domain binds back on the
barrel at one end.
Figure 8:
Structural models of the and the
subunits from ECF
. A, a recently refined
structure of the N-terminal
barrel domain of the
subunit
shown down the axis through the
sheet sandwich, kindly provided
by Dr. Stephan Wilkens. The open labeled spheres are the
carbons of the residues changed to Cys in the experiments described
here. The shaded spheres are side chains of the residues 15,
9, 79, 77, 68, and 42 that provide a hydrophobic patch on the side of
that binds the
subunit. B, hypothetical secondary
structure of the
subunit based on 1) the secondary structure
prediction methods of Chou and Fasman(1978) and Garnier et
al.(1978), 2) the crystal structure of bovine mitochondrial
F
-ATPase; and 3) our previous trypsin digestion studies
(Tang et al., 1994) along with chemical modification and
cross-linking studies reported here. Column,
helix; arrow,
sheet; line,
turn or undefined
region. The residue number at the ends of the
helix/
sheet
are labeled, and the trypsin cleavage sites are identified along with
residue Thr-106.
The subunit interacts with the
and
subunits of the
F
part through the C-terminal domain, as indicated by
cross-linking from Ser-108 to Glu-381 in the DELSEED region of the
subunit by
1-ethyl-3-[3-dimethylamino)propyl]carbodiimide (Dallmann et al., 1992), by disulfide bond formation from a Cys at
position 108 of
to a Cys in place of Glu-381 in
(Aggeler et al., 1995b), and by disulfide bond formation between Cys at
108 of
and a Cys at position 411 in the
subunit (this is
the equivalent residue to Glu-381 of
). (
)Finally, as
shown here, there is cross-linking of the
subunit to a
subunit by TFPAM3 from a Cys replacing Met-138 at the C terminus of
.
In addition to interacting with and
subunits, the
subunit is now known to interact with the c subunits of the
F
(Zhang et al., 1994, 1995; Watts et
al., 1995), and this linkage is via the opposite end of the
sheet sandwich from that which binds the helix-loop-helix domain (Fig. 8A).
The subunit also binds to the
subunit in ECF
(Dunn, 1986; Aggeler et al., 1992;
Skakoon and Dunn, 1993). The homologous subunit in chloroplasts (
)
also binds to the
subunit in CF
(Suss, 1986;
Soteropoulos et al., 1994). The results presented here
identify Ser-10, His-38, and Thr-43 as close to, or involved in, this
reaction based on mutagenesis to Cys and then cross-linking from these
sites and/or by chemical modification of the introduced Cys residue and
consequent steric effects on
binding. These three residues are on
one face of the
barrel and, as shown in Fig. 8, are close
to a patch of hydrophobic residues, which may play a key role in the
binding to the
subunit.
The cross-linking results presented
here indicate a role of residues between 202 and around 240 in
subunit binding. TFPAM cross-linking from the Cys-10 of
is with a
tyrosine at residue 228 of the
subunit. Cross-linking from Cys-38
or Cys-43 in the mutants
H38C and
T43C is within the region
of
from 202 to 286. The C terminus from residues 222-286 is
organized as a long
helix that, from around residue 240 to the
very C terminus, is intercalated within the cavity formed by the
hexagonally arranged
and
subunits (Abrahams et
al., 1994). Our data indicate that the
subunit binds at, or
close to, this
helix as it extends from the
barrel through the stalk region and
makes contact with the c subunits of the F
part somewhere
between residues 202 and 229 (Watts et al., 1995).
The
protease digestion data focus attention on the region of around
residue 70, as well as on the region between residues 202 and 212 in
binding the
subunit. Residue 70 is in the epitope for a
monoclonal antibody to the
subunit, described by Dunn and
colleagues, which reacts with ECF
only when the
subunit is first removed (Skakoon and Dunn, 1993). Residues
202-212 are close to the region of the
subunit in which
there is an insertion in the
subunit of chloroplasts that
contains two Cys residues, which regulate CF
activity by
reduction and oxidation reactions involving the protein thioredoxin
(reviewed by Soteropoulos et al. (1994)). The
subunit
has been shown to affect the oxidation and reduction reaction of these
Cys residues and protect this region from proteolytic digestion in
CF
(Schumann et al., 1985).
In addition to the
region around residue 70 and the C-terminal part from residues 202 to
around 240, our chemical modification studies also place Thr-106 of
in the
subunit binding site. The recent x-ray structure
determination shows that the
subunit has three segments in
contact with
and
subunits, an N-terminal
-helix of
residues 1-50, a short central
-helical residue region
83-99 in the numbering system of E. coli, as well as the
C-terminal
-helix (Abrahams et al., 1994). It is
noteworthy that the remainder of this subunit, including several of the
sites identified here as in, or close to, the
binding site, is
predicted to be in the
sheet and turn structure. It seems likely,
then, that the
barrel of the
subunit binds in part to an
equivalent structure formed by much of the region of the
subunit,
as well as binding to the extension of the C-terminal helix region.