From the Heinrich-Heine-Universität, Biochemie
der Pflanzen, Universitätsstrasse 1, D-40225 Düsseldorf,
Germany and ¶ EMBL Hamburg, Notkestrasse 85, D-22603 Hamburg, Germany
Received for publication, September 1, 2000, and in revised form, October 10, 2000
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ABSTRACT |
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The structure of the F1-ATPase
from spinach chloroplasts was determined to 3.2 Å resolution by
molecular replacement based on the homologous structure of the bovine
mitochondrial enzyme. The crystallized complex contains four different
subunits in a stoichiometry of The membrane-bound proton translocating ATP synthase of
chloroplasts,
CF1F0,1
catalyzes ATP synthesis and ATP hydrolysis coupled to proton translocation across the thylakoid membrane (for a recent review see
Ref. 1). The enzyme is comprised of nine different polypeptides organized in two separate sectors. The membrane-integrated
F0 complex, composed of subunits I, II, III, and IV,
mediates proton transport and provides specific sites for the
attachment of the catalytic F1 complex. The membrane
extrinsic F1 domain is an assembly of five different
polypeptides in a stoichiometry of
The overall shape, dimension, and mass distribution of the chloroplast
ATPase were revealed by electron microscopy and image analysis (2, 3).
The catalytic F1 complex resembles a pseudo-hexagonal, symmetrical ring of alternating The location and the orientation of the catalytic and the regulatory
sites in the chloroplast F1 domain were identified in photo-labeling and fluorescence resonance energy transfer studies (6),
which demonstrated that nucleotide binding is associated with the At present, most detailed structural information about the
F1 complex comes from the high resolution structure of the
bovine mitochondrial ATPase (7, 8). Additional structural information was obtained from the rat liver The Another striking difference that is expected to be reflected in the
structure of the chloroplast ATPase is the specific interaction of the
extrinsic CF1 complex of certain plant species with the fungal phytotoxin, tentoxin. Binding of a single molecule of tentoxin that has no effect on mitochondrial or bacterial ATPases is
sufficient to inhibit CF1, whereas binding of a second and
third molecule results in its reactivation (18, 19).
Differences in nucleotide binding of the chloroplast ATPase were
reported depending on whether isolated or membrane-bound CF1 were studied (20-22). In addition magnesium was shown
to play a crucial role in nucleotide binding (23, 24). Depending on their binding characteristics sites were designated 1-5 (21) or A-C
(25, 26). Isolated CF1 contains one or two endogenous bound
nucleotides probably located on the noncatalytic In this paper we present the first high resolution structure of a
chloroplast Crystallization and Data Collection--
Purification and
crystallization of the membrane extrinsic F1 domain of the
chloroplast ATP synthase have been described previously (28). Briefly,
CF1 was released from thylakoid membranes of spinach plants
in a medium containing 300 mM sucrose, 2 mM
Tricine, pH 7.8, 2 mM dithiothreitol, 0.875 mM
EDTA, and 0.002% (w/v) phenylmethylsulfonyl fluoride. Membrane
fragments were removed by centrifugation, and the supernatant was
applied to anion exchange chromatography on POROS HQ (Applied
Biosystems). The protein was eluted from the anion exchange column by
applying a linear gradient of 225-1500 mM NaCl in 25 mM Bis-trispropane/Tris borate, pH 7.5. In contrast to the
former protocol subunit Structure Determination and Refinement--
The structure was
solved by molecular replacement using the program AMoRe (34). The
atomic coordinates of a poly-alanine model derived from the refined
structure of bovine mitochondrial F1 (7) were used as a
search model in these calculations. All conformations of SDS-Polyacrylamide Gel Electrophoresis--
Proteins were
analyzed by SDS-polyacrylamide gel electrophoresis on 12%
polyacrylamide gels according to Laemmli (41). After electrophoresis,
subunit composition of the purified complex was detected by silver
staining (42).
Overall Structure of the Chloroplast
The final model of the chloroplast
The overall structure of the
The conformation of the chloroplast
As an additional test, whether the catalytic Comparison of the Crown Region--
The overall structure of the
N-terminal six-stranded Structure of the
A less ordered, unfolded structure rather than a more buried well
ordered Catalytic and Noncatalytic Nucleotide-binding Sites--
The high
resolution structures of the mitochondrial and thermophilic ATPase
revealed that the nucleotide binding sites are located at the
interfaces between the Mechanistic Implications of the Chloroplast Structure--
In
contrast to the bacterial and mitochondrial enzymes CF1,
either in isolated or membrane-bound form, is a latent ATPase. Nucleotides cannot exchange effectively with medium nucleotides until
the enzyme is activated by reduction of the
The structure of the chloroplast Potential Tentoxin-binding Site at the
On the basis of the present structural information obtained with the
chloroplast F1 complex, a sequence alignment of sensitive and resistant species suggests that residues 3
3
.
Subunit
was removed before crystallization to improve the
diffraction of the crystals. The overall structure of the noncatalytic
-subunits and the catalytic
-subunits is highly similar to those
of the mitochondrial and thermophilic subunits. However, in the crystal
structure of the chloroplast enzyme, all
- and
-subunits adopt a
closed conformation and appear to contain no bound adenine
nucleotides. The superimposed crystallographic symmetry in the space
group R32 impaired an exact tracing of the
- and
-subunits in the complex. However, clear electron density was
present at the core of the
3
3-subcomplex, which probably represents the C-terminal domain of the
-subunit. The
structure of the spinach chloroplast F1 has a potential
binding site for the phytotoxin, tentoxin, at the
-interface near
Asp83 and an insertion from
Gly56-Asn60 in the N-terminal
-barrel
domain probably increases the thermal stability of the complex. The
structure probably represents an inactive latent state of the ATPase,
which is unique to chloroplast and cyanobacterial enzymes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
3
3
. In contrast to the
homologous bacterial and mitochondrial enzymes, isolated
CF1 is a latent ATPase that requires activation.
and
subunits. The central cavity of the
3
3 core complex is
partially filled with subunit
. Previous studies on subunit
deficient chloroplast F1 demonstrated that the
-subunit
is necessary to preserve the arrangement of the central
-subunit.
Removal of subunit
showed no effect on the integrity of the central
mass in the
3
3 hexagon (4). Interaction
of the catalytic F1 domain and the membrane embedded F0 complex, however, was affected in
CF1-deficient in subunit
(5).
and
subunits. Binding sites are located on each of the six
-interfaces.
3
3
complex (9), the
3
3 core complex from the
thermophilic bacterium PS3 (10), and the
3
3
complex from Escherichia
coli (11). For the chloroplast enzyme, however, we still rely on
the limited structural information from electron microscopy,
cross-linking, and fluorescence resonance energy transfer studies
because no high resolution structure was available until now. Even
though the chloroplast enzyme shares many structural and functional
characteristics with the mitochondrial and bacterial enzymes,
CF1 is unique in several aspects of enzyme activation and
sensitivity toward specific energy transfer inhibitors. Control of the
catalytic activity in membrane-bound CF1 is achieved by the
transmembrane proton gradient, the inhibitory tight binding of ADP to a
catalytic site, and the redox modulation of
Cys199 and
Cys205. In addition to these mechanisms, isolated
CF1 is activated by heat, proteolytic cleavage in the
vicinity of the regulatory disulfide in the
-subunit, alcohol, and
mild detergents (summarized in Ref. 1). Removal of the
-subunit
shows no effect on the catalytic activity. In contrast
CF1-
is always activated (12).
and
subunits of CF1 share 58.5 and 67.5%
sequence identity, respectively, with their mitochondrial counterparts,
suggesting that they have similar overall structures. In contrast, the
smaller subunits are not well conserved. Particular interest is
focussed on subunit
, which contains a regulatory insert of about 20 amino acids (
183-206) including two redox-active cysteine residues in the chloroplast enzymes of higher plants and green algae. The important role of the
-subunit in the catalytic cycle was
demonstrated in sophisticated experiments which showed that subunit
rotates in the
3
3 core and is probably
related to sequential conformational changes of the
-pairs
(13-15). Three different conformations of the
-subunit, which
contains most of the catalytic binding site, were found in the high
resolution structure of the bovine MF1 complex (7).
Depending on the nucleotide bound in the catalytic site the subunits
were named
DP,
TP, and
E.
The conformation of the three noncatalytic
-subunits was very
similar in the bovine MF1 structure because all
-subunits were filled with the nonhydrolyzable nucleotide analogue
AMPPNP (7). Deletion of the 20 C-terminal residues of the
-subunit
resulted in an active chloroplast enzyme and questioned the
significance of the
-rotation for the catalytic process in the
chloroplast ATPase (16). Hence, rotation of subunit
was recently
also visualized for isolated CF1 (17).
-subunit (26-28).
The presence of adenine nucleotides at the noncatalytic sites, however,
is not a prerequisite for catalytic turnover but promotes the release
of inhibitory tightly bound ADP from a catalytic site (29). Tight
binding of adenine nucleotides to noncatalytic sites is stimulated by
the presence of magnesium, which also stabilizes the inhibitory
enzyme-bound ADP located on a catalytic site (25). Fluorescence
resonance energy transfer studies demonstrated that the asymmetry of
the nucleotide binding sites of CF1 is not a permanent
feature and that site switching in the catalytic cycle is controlled by
magnesium (30, 31). The molecular structures of the rat liver (9) and
the thermophilic ATPase (10) suggest that the asymmetry of the
catalytic sites in the F1 complex resolved in the bovine
F1 (7) is controlled by magnesium and/or subunit
. Both
structures show 3-fold symmetry and were obtained in the absence of
magnesium and for the thermophilic structure also in the absence of the
-subunit.
3
3
complex. The
structure was obtained in the absence of magnesium and shows 3-fold
symmetry. Thus, subunits
and
, which are present in single
copies in the complex, are not clearly resolved. Despite the different
nucleotide content the chloroplast
and
subunits adopt a
conformation that resembles the conformations of the bovine
MF1 complex containing bound nucleotides. This difference
might be related to the unique latent ATPase activity of the
chloroplast enzyme.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
was removed from the catalytic F1 domain by incubation with 20% (v/v) ethanol/14% (v/v)
glycerol in 25 mM Bis-trispropane/Tris borate, pH 7.5, when
CF1 was bound to the anion exchange column. Rhombohedral
crystals, space group R32, were grown in 25 mM
HEPES, pH 7.5, 1 mM dithiothreitol, 0.002% (w/v)
phenylmethylsulfonyl fluoride, 0.01% (w/v) NaN3 using 1.5 M ammonium sulfate as a precipitant at 20 °C by the
micro batch technique. Adenine nucleotides, ADP (0.02 mM),
and AMPPNP (1 mM) were added to the crystallization buffer
to stabilize the purified protein. A first diffraction set was
collected at 3.5 Å resolution from flash-frozen crystals on beam line
X11 (EMBL/DESY Hamburg). Subsequently a second data set at 3.2 Å resolution was measured on beam line BW7B at EMBL/DESY. Diffraction
data were processed with the programs DENZO (32) and MOSFLM (33),
revealing unit cell parameters of a = 147.70 Å,
b = 147.70 Å, and c = 385.05 Å,
= 90°,
= 90°, and
= 120°. Further
crystallographic parameters are given in Table I.
-dimers
found in the mitochondrial F1 structure (7) were tested.
Data from 15-5 Å resolution were included in the molecular
replacement calculations. A clear solution with a correlation
coefficient of 0.71 was obtained with the
E
DP-dimer and the closely related
DP
TP conformer. In these dimers the nucleotide binding sites on the
-subunits are filled with the nonhydrolyzable nucleotide analogue AMPPNP, whereas the
-subunits contain either ADP or ATP. Calculations using the
E
conformation of the
-subunit where no nucleotides are bound in the
catalytic site resulted in a correlation coefficient of 0.63. These
results suggested that a
-dimer in the chloroplast
3
3
complex resembles the dimer
formed by nucleotide-filled
and
-subunits in the mitochondrial
enzyme. The model was mutated to the chloroplast sequence in O (35).
Initial rigid body refinement was carried out by the program CNS (36)
for all reflections from 15 to 5 Å. According to the structure of the
mitochondrial F1 complex, three domains in the
and
subunits were defined in the rigid body minimization corresponding to
residues
19-95,
96-379, and
380-510, and
9-82,
83-363, and
364-474, respectively. Further crystallographic
refinement was accomplished by Cartesian coordinate molecular dynamics
and restrained B-factor minimization in iterative cycles.
The model was revised at each cycle of the refinement by inspection of
the FoFc and
2FoFc maps and manual
rebuilding in O (35). A bulk solvent correction was applied in the
final refinement cycle. The model converged to a final crystallographic R-factor of 31.9% (Rfree = 35.0%) for reflections from 6-3.2 Å. Model geometry was verified
with PROCHECK (37). Figures were produced using the programs Bobscript
(38), Molscript (39), and Swiss-PdbViewer (40). The structure has been
submitted to the Protein Data Bank (PDB accession code 1FX0).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-Dimer--
Previously
we have described purification and crystallization of the chloroplast
F1 domain (28). However, these crystals diffracted only up
to 6-8 Å resolution. Analysis of the crystals by SDS-polyacrylamide
gel electrophoresis revealed that subunit
was degraded, whereas all
other subunits remained intact (data not shown). Thus, in subsequent
preparations subunit
was removed from the CF1 complex
and the remaining
3
3
complex was
used in crystallization experiments (Fig.
1). No trace of subunit
could be
detected in this complex either by silver staining or by
immunoblotting. The
3
3
complex
crystallized in the space group R32 having one single
-pair and 0.33
-subunit and 0.33
-subunit in the asymmetric
unit of the crystal. In contrast to the structure of the rat liver
enzyme, unambiguous determination of the
structure was not possible
because of the 3-fold crystallographic symmetry in the complex. The
single
and
subunits in the asymmetric unit represent a
superposition of the three copies present in the chloroplast
3
3
complex. With the exception of
the C termini the electron density is well defined, suggesting that all
and
subunits are in similar conformations even though, because
of local disorder in parts of the C-terminal domain, alternative conformations in the individual subunits cannot be completely excluded.
A representative plot of part of the 3.2 Å resolution electron density
map with the refined model coordinates superimposed is shown in Fig.
2.
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Fig. 1.
Subunit composition of purified
chloroplast
3
3
complex. The purified CF1-
was denatured in
SDS and separated on a 12% polyacrylamide gel that was silver stained
for the detection of the individual subunits. Positions of
CF1-subunits are indicated on the left.
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Fig. 2.
Electron density map corresponding to the
chloroplast - and
-subunit. View of the 3.2 Å resolution
electron density map used for the model building of the chloroplast
F1 structure with the refined coordinates
superimposed. The contour level of the
2Fo
Fc map is at 1
.
-dimer includes 942 residues
(
25-501 and
19-485). The final parameter of
refinement and model stereochemistry are summarized in
Table I. Residues
Leu345,
Gly353,
Leu387,
Gln389,
Phe399,
Ser401,
Thr407,
Asn409-Arg413,
Tyr464,
Thr481-Phe482,
Glu499,
Leu159,
Ala331,
Gly355,
Gly381,
Ala406,
Ile407,
Gly409,
Glu416,
Asp417,
Gly446,
Ile455,
Leu462, and
Asp467-Ser468
were not clearly visible in the electron density map; several other
surface exposed residues in the C-terminal domain display high
temperature factors for their side chain atoms. All residues except
Arg122 have main chain dihedral angles that fall within
the allowed regions of the Ramachandran diagram as defined by the
program PROCHECK (37).
Crystallographic data and refinement statistics
and
subunits (Fig.
3, A and B) is
highly similar to those of the mitochondrial (7, 8) and the
thermophilic subunits (10). As in the MF1 and
TF1 structures (7-10), both subunits have almost identical
folds and are arranged alternately about a central axis (Fig.
3C). Three different domains can be distinguished in each
subunit: a N-terminal six-stranded
-barrel (
25-96 and
19-93), a central domain of
-strands with associated
-helices
that contains the nucleotide binding site (
97-371 and
94-381),
and a C-terminal
-helical bundle (
372-501 and
382-485). In
comparison with the mitochondrial enzyme, the chloroplast
-subunit
contains two insertions in the N-terminal
-barrel domain
(
Lys39 and
Gly56-Asn60), a
single residue inserted in the central domain of the
-subunit (
Glu224), and a deletion of 8 residues between helix B
and strand 6 in the central nucleotide binding domain of the
-subunit (
Lys187-Asp194). The insertions
Lys39 and
Glu224 and the deletion in
subunit
show only minor effects on the structure. However, the
insertion
Gly56-Asn60, which is located in
an extended loop between strands 3 and 4, seems to affect the structure
of the chloroplast F1 complex. As shown in Fig.
3C the additional residues wrap up part of the
-barrel domain of the adjacent
-subunit and appear to provide additional contacts, thereby increasing the stability of the CF1 crown
region (Fig. 3C).
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Fig. 3.
Crystal structure of the chloroplast
- and
-subunits.
A, schematic representation of the
-subunit structure
(blue). B, schematic representation of the
chloroplast
-subunit (red). The overall fold of both
subunits is similar. Three different structural domains are apparent in
each subunit. Both subunits are shown with their 3-fold
crystallographic axis in the vertical orientation. C,
schematic representation of the molecular structure of the chloroplast
3
3 core complex. The structure is shown
from the top as it would appear on the membrane; the 3-fold axis points
toward the viewer. The inserted residues
Gly56-Asn60 in the N-terminal domain of the
-subunit that partially cover the adjacent
-subunit are indicated
by the arrows.
-subunit is similar to the three
conformations found in the bovine mitochondrial structure. The C
atoms superimpose with rms deviations of 1.31, 1.31, and 0.95 Å with
the mitochondrial
TP,
DP, and
E conformation. In contrast the chloroplast
-subunit
superimposes well only with the
TP or
DP
conformation of the mitochondrial enzyme. The C
atoms show rms
deviations of 1.24 and 1.19 Å, respectively. Comparison with the
nucleotide-free
E conformation of the mitochondrial structure reveals a rms deviation of 3.89 Å. Thus the chloroplast
and
-subunit seem to reflect the closed nucleotide-filled
conformation of the mitochondrial enzyme rather than the
open nucleotide-free conformation. However, the high
temperature factors of the C-terminal domains of the
- and
-subunits raise the possibility that more than one single, discrete
conformation exists in the crystal structure. Different conformations
might exist in particular of the catalytic
-subunit. To discriminate
these different conformations and to reduce the effects of model bias,
residues
Glu383-Phe430 which are involved
in the conformational changes associated with nucleotide binding and
catalytic turnover, were omitted in the structure refinement. Fig.
4 shows the calculated electron density map and the protein backbone corresponding to the
DP and
E conformation of the bovine mitochondrial ATPase (7).
Although the corresponding calculated electron density is somewhat
weaker than when all residues in the C-terminal region were included in
the refinement, the density clearly indicate a closed
conformation of the
-subunit. Calculations of a composite omit,
cross-validated,
-A weighted map, where small regions of the
model (10%) are systematically excluded in the refinement, support
this conclusion (Fig. 4B).
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Fig. 4.
Calculated electron density omit map of the
chloroplast F1 complex with the peptide backbone
corresponding to the mitochondrial
E and
DP conformation superimposed.
A, stereo image of a
A-weighted
2Fo
Fc electron
density map of the chloroplast
-subunit where residues
Glu383-Phe430 of the model were excluded in
the refinement. B, composite omit, cross-validated map where
10% of the model were systematically excluded (36). The peptide
backbone of
E is shown is red, and
DP is drawn in black.
-subunits in the
crystal is present as a mixture of closed and
open conformations, crystallographic refinement was carried
out with a model containing the
E and
DP
conformation at various occupancies. In comparison with a model having
the
-subunit exclusively in the closed conformation (1.00
DP), refinement of a model with 0.66
DP
conformation and 0.33
E conformation increased the free
R-factor by 3.5%. These calculations further support the
suggestion that the closed conformation is the predominant
form of the catalytic
-subunit in the crystal, although local
disorder and structural distinct conformations cannot be completely
excluded at the present resolution.
-barrel domain is highly similar in the
mitochondrial (7), thermophilic (10), and chloroplast enzymes (Fig.
5). However, the chloroplast and the
thermophilic
-subunit contain an insert of five and seven residues,
respectively. In the chloroplast
3
3
complex, these residues form an extended loop located between strands 3 and 4 (Fig. 5B), whereas in the thermophilic enzyme a more
rigid structure of an extended
-strand is formed by the additional
residues (Fig. 5C). Both structures should promote
additional contacts in the N-terminal crown-region of the
F1 complex, which might increase the thermal stability of
the enzyme. In the thermophilic enzyme a hydrogen bond between
Asn40 and
Arg90 and van der Waal's
interaction of residues
Asn38-
Ser21 and
Glu41-
Met48 are proposed to cause this
increased stability (10). The high resolution structure of the
chloroplast enzyme suggests that a salt bridge
Asp83-
Arg297 enhances the stability of
the enzyme. Both residues are within 3.4-3.5 Å, whereas the
corresponding residues in the thermophilic (
Asp68-
-Arg296) and the mitochondrial
(
Glu67-
Arg304) enzyme are 7.7-9.9 and
5.7-11.6 Å apart. The proposed increased stability of the chloroplast
F1 complex caused by the additional contacts in the
-barrel domain is reflected by the fact that the isolated enzyme
tolerates incubation at 60 °C for heat activation of the latent
ATPase activity (43).
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Fig. 5.
Schematic representation of the N-terminal
six-stranded -barrel domain of the
-subunit in the mitochondrial (A),
chloroplast (B), and thermophilic ATPase
(C).
-Subunit--
In contrast to the rat liver
mitochondrial F1 complex that also crystallized in the
space group R32 (9), unambiguous tracing of the
-subunit
in the chloroplast
3
3
complex was
not possible because of the superimposed 3-fold crystallographic
symmetry. The calculated electron density maps of the chloroplast
ATPase, however, showed significant, additional electron density in the central cavity of the chloroplast
3
3 core
complex, which probably corresponds to the 3-fold superimposed C
terminus of the chloroplast
-subunit (Fig.
6). Nevertheless, a clear interpretation
of this density was not possible at the present resolution of the
diffraction data. Inspection of the additional density in the center of
the
3
3 domain suggests that the extreme
C-terminal end of the chloroplast
-subunit is less ordered than the
corresponding residues in the mitochondrial enzyme. Whether this
disorder is due to the superimposed 3-fold crystallographic symmetry or
is an intrinsic characteristic of the chloroplast
-subunits remains
to be solved.
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Fig. 6.
Electron density in the central cavity of the
chloroplast
3
3
core. Calculated electron density map at 1
attributed to the
chloroplast
-subunit. Parts of the density map show the superimposed
3-fold crystallographic symmetry on the
-subunit in the center of
the
3
3 complex. The structure is shown
with the crystallographic axis in the vertical position.
-helical conformation might account for the observed fast
modification of
Cys322 located at the extreme C-terminal
end of the
-subunit and might also explain why this portion of the
-subunit is not crucial for rotational catalysis in the chloroplast
enzyme as suggested by Sokolov et al. (16). These
conclusions drawn from our present data, however, are preliminary and
have to be considered with caution. Data at higher resolution and the
addition of cross-linking agents might allow a clear interpretation of
the complete density attributed to the
-subunit in the near future.
- and
-subunits (7-10). Noncatalytic
sites are predominately formed by the
-subunits, and catalytic sites
are mainly located on the
-subunits. Folds and structural motifs in
the different ATPases are highly similar. Clear differences, however,
were reported in the occupancy of the nucleotide binding sites, which
might reflect different physiological states or an artificial state of
the enzyme caused by the crystallization conditions. Inspection of the
noncatalytic nucleotide binding sites in our crystal structure of the
chloroplast
3
3
complex revealed
that no nucleotides were bound to the
-subunit, even though the
conformation of the
-subunit resembles the closed, AMPPNP-filled
conformation of the bovine mitochondrial enzyme. The same closed
conformation of the
-subunits lacking bound nucleotides was also
found in the thermophilic
3
3 core complex
(10). This complex, however, was assembled from individual subunits
overexpressed in E. coli (44), and the significance of
the nucleotide occupancy in this complex might be questioned. The
chloroplast
3
3
complex crystallized
in this study, however, was isolated from its natural source and
retained catalytic activity (12, 28). Thus, the closed conformation
might also exist in the absence of nucleotides in the native
F1 complex. The situation is similar for the catalytic binding sites on the chloroplast
-subunits. Again, no bound
nucleotides were detected in the crystal structure. However, some
additional density was detected in the P-loop region
(
Gly170-Thr177 and
Gly172-Thr179) of the chloroplast catalytic
and noncatalytic binding sites that might represent bound phosphate or
sulfate. The refinement showed no significant changes in model geometry
or free R-factor when sulfate was included in the catalytic
and noncatalytic site. Thus, at the present resolution we cannot
discriminate whether the P-loop contributes to the additional density
or whether it indicates a phosphate ion bound to the P-loop. Because of
the 3-fold crystallographic symmetry, we also have to be cautious about
the occupancy of the nucleotide binding sites in the chloroplast
3
3
complex in general because
substoichiometric binding of adenine nucleotides to 1 or 2 sites in the
crystal cannot be completely excluded on the basis of the present data.
Substoichiometric binding of 1 ADP was determined in the purified
chloroplast ATPase by luciferin/luciferase (28). Although crystals were
grown in the presence of 1 mM AMPPNP and 0.02 mM ADP, rebinding of adenine nucleotides to the catalytic
and noncatalytic sites is limited because magnesium, which was shown to
stimulate the tight binding of nucleotides (25), was excluded in the
crystallization trials. Thus, the substoichiometric nucleotide content
found in the purified CF1 complex might also reflect the
situation in the crystallized enzyme. To resolve any substoichiometric
binding in the CF1 structure, ADP at an occupancy of 0, 0.33, and 0.66 was included in the crystallographic refinement. The
adenine nucleotide was located in the
-subunit according to the
structure of the bovine mitochondrial F1 complex (7) with
B-factors set to the P-loop region of the chloroplast structure. The
different calculations showed no significant change in the free
R-factor. In addition, no clear negative peak was visible at
the position of the nucleotide in the electron density difference maps.
Thus, on the basis of the present data, we cannot completely rule out
the possibility that at least a single site in the chloroplast
F1 complex is occupied, even though the electron density
map in the nucleotide binding domain shows no clear density corresponding to a bound adenine nucleotide.
-disulfide or
displacement of the
-subunit (12). Activation always precedes catalysis and controls the catalytic cycle of the enzyme to prevent the
futile hydrolysis of ATP in the dark under physiological conditions.
3
3
complex described in this work might reflect the inactive, latent state
of the enzyme that is not found in the bacterial or mitochondrial
enzymes because those F1 complexes exhibit high rates of
ATP hydrolysis without previous activation. Catalytic and noncatalytic
binding sites in the structure are in a closed conformation and contain
no or substoichiometric amounts of adenine nucleotides. Therefore, any catalytic turnover, which might result in conformational changes in the
-subunit or in the catalytic
-subunit, is blocked efficiently in
the inactive oxidized complex. Transformation of this inactive, transient state of the chloroplast F1 complex into an
active state might be related to conformational changes in the
-subunit caused by reduction of the disulfide or by altered
interaction with the
-subunit. These conformational changes
involving the transformation of the nucleotide binding sites might be
related to a displacement of the central axis of subunit
as
discussed for the symmetrical rat liver F1 complex (9) to
convert the symmetrical inactive form into the asymmetrical form
described for the bovine mitochondrial ATPase (7, 8) that reflects the
binding change mechanism (45).
-Interface--
The
isolated and membrane-bound chloroplast ATPase of certain sensitive
species of plants is inhibited by the cyclic tetrapeptide, tentoxin,
which is produced by several phytopathogenic fungi (46). The precise
binding site and the mechanism of the phytotoxin are not identified
yet. Labeling studies suggest a high affinity inhibitory binding site,
probably located on the
-interface and one or two low affinity
binding sites that cause reactivation of the enzyme (18-19).
Mutational studies suggested that residue Asp83 in the
N-terminal domain of the
-subunit controls the sensitivity to the
inhibitor (47, 48). However, tentoxin-resistant enzymes of the
thermophilic bacterium PS3 and E. coli also
contain aspartate in the corresponding position of their
-subunit.
Thus, the binding motif seems to be more complex and can probably not
be reduced to single residues but rather to a certain structural
conformation formed at the catalytic
-interface. Residues
49-52,
65-69,
95-96,
131-133,
297,
26-29,
51-54,
78,
80-85, and
242, which are located within 10.0 Å of
Asp83 in the structure of the chloroplast
3
3
complex, might be involved in
the binding of the phytotoxin. Comparison with the corresponding domain
in the mitochondrial and thermophilic enzymes (Fig.
7) suggests that the potential salt
bridge
Asp83-
Arg297 controls the overall
conformation of the binding pocket and adjusts the
-interface to
the inhibitor. Neither the mitochondrial nor the thermophilic ATPase
show contacts between the corresponding residues in their structures.
The F1 structures of the different species, however,
suggest that the compensation of the positively charged
Arg297 in the chloroplast enzyme by the salt bridge
might be essential to promote the binding of the toxin as in the
thermophilic and the mitochondrial F1 complex positively
charged residues
Arg128,
Lys132, and
Arg132 are exposed at the surface of the potential
binding cleft (Fig. 7, B and C). Additional
negative surface charge at the binding site is promoted in the
chloroplast F1 complex by
Asp53 and
Thr54, which are located at the top of the cleft.
Adjacent to these residues a hydrophobic surface is formed by residues
Leu129,
Ser132, and
Pro133, which might provide essential contacts for the
inhibitor. Further analysis of the CF1 structure suggests
that residues
Met274 and
Leu277 located
at the inner face of the cleft might also contribute to this
hydrophobic cluster. In the mitochondrial structure access to the
potential binding site located around
Glu67 is blocked
by
Arg128 and
Glu42. In the thermophilic
structure the corresponding
Asp68 is accessible and
located almost in the center of the binding cleft. However, the surface
charge distribution at the entrance site of the binding pocket clearly
differs from those in the CF1 structure. In the
thermophilic enzyme, charged residues
Glu130 and
Arg132 are located at the position corresponding to the
hydrophobic cluster in the chloroplast structure, and a hydrophobic
surface instead of a negatively charged entrance site is found at the top of the binding cleft.
View larger version (80K):
[in a new window]
Fig. 7.
Suggested potential Tentoxin binding site of
the chloroplast ATPase (A) and corresponding domains
in the mitochondrial (B) and thermophilic
F1 complex (C). Surface
representation of residues within 20.0 Å of the essential
Asp83 in CF1 and the corresponding residues
Asp67 in MF1 and
Asp68 in
TF1. Structures are shown in stereo view with the
crystallographic axis in the vertical position as they would appear
from the outside of the
3
3 complex.
Ala96 and
Pro133 are involved in the binding and might confer the
sensitivity of the F1 complex to the phytotoxin. In
resistant species alanine is substituted by hydrophobic residues with
bulky side chains, and proline is replaced by basic or hydrophobic
residues with extended side chains. The mechanism by which tentoxin
inhibits and activates the chloroplast ATPase remains still open and
additional structural information and studies of protein dynamics seem
necessary. Recent progress in co-crystallization of CF1 and
tentoxin2 promises more
detailed structural information on the inhibitory complex and might
also identify the reason for the reactivation of the chloroplast ATPase
caused by tentoxin.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. John Walker for critical reading of the manuscript and helpful discussions. We gratefully acknowledge his support, continuous encouragement, and permanent interest in the project. We are grateful to Prof. H. Strotmann, Dr. Andrew Leslie, and Dr. Daniela Stock for helpful comments on the manuscript and valuable suggestions. We also thank Daniela for guidance at the beam line and valuable help with data collection. For support during beam time at EMBL beam lines X11 and BW7B, we thank the EMBL staff.
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FOOTNOTES |
---|
* This work was supported by Deutsche Forschungsgemeinschaft Grant Gr1616/4-1 and by funds from the Bennigsen-Foerder-Preis of Nordrhein-Westfalen (to G. G.).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.
The atomic coordinates and the structure factors (code 1FX0) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
This article is dedicated to Wolfgang Junge (Osnabrück) on the occasion of his 60th birthday.
§ To whom correspondence should be addressed. Tel.: 49-211-8112822; Fax: 49-211-8113706; E-mail: georg.groth@uni- duesseldorf.de.
Published, JBC Papers in Press, October 13, 2000, DOI 10.1074/jbc.M008015200
2 G. Groth, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
CF1, chloroplast F1;
AMPPNP, adenosine
5'-(,
-imino)triphosphate;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
rms, root mean square.
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