The Structure of the Chloroplast F1-ATPase at 3.2 Å Resolution*

Georg GrothDagger § and Ehmke Pohl

From the Dagger  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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 alpha 3beta 3gamma epsilon . Subunit delta  was removed before crystallization to improve the diffraction of the crystals. The overall structure of the noncatalytic alpha -subunits and the catalytic beta -subunits is highly similar to those of the mitochondrial and thermophilic subunits. However, in the crystal structure of the chloroplast enzyme, all alpha - and beta -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 gamma - and epsilon -subunits in the complex. However, clear electron density was present at the core of the alpha 3beta 3-subcomplex, which probably represents the C-terminal domain of the gamma -subunit. The structure of the spinach chloroplast F1 has a potential binding site for the phytotoxin, tentoxin, at the alpha beta -interface near beta Asp83 and an insertion from beta Gly56-Asn60 in the N-terminal beta -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

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 alpha 3beta 3gamma delta epsilon . In contrast to the homologous bacterial and mitochondrial enzymes, isolated CF1 is a latent ATPase that requires activation.

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 alpha  and beta  subunits. The central cavity of the alpha 3beta 3 core complex is partially filled with subunit gamma . Previous studies on subunit deficient chloroplast F1 demonstrated that the epsilon -subunit is necessary to preserve the arrangement of the central gamma -subunit. Removal of subunit delta  showed no effect on the integrity of the central mass in the alpha 3beta 3 hexagon (4). Interaction of the catalytic F1 domain and the membrane embedded F0 complex, however, was affected in CF1-deficient in subunit delta  (5).

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 alpha  and beta  subunits. Binding sites are located on each of the six alpha beta -interfaces.

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 alpha 3beta 3gamma complex (9), the alpha 3beta 3 core complex from the thermophilic bacterium PS3 (10), and the alpha 3beta 3gamma epsilon 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 gamma Cys199 and gamma Cys205. In addition to these mechanisms, isolated CF1 is activated by heat, proteolytic cleavage in the vicinity of the regulatory disulfide in the gamma -subunit, alcohol, and mild detergents (summarized in Ref. 1). Removal of the delta -subunit shows no effect on the catalytic activity. In contrast CF1-epsilon is always activated (12).

The alpha  and beta  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 gamma , which contains a regulatory insert of about 20 amino acids (gamma 183-206) including two redox-active cysteine residues in the chloroplast enzymes of higher plants and green algae. The important role of the gamma -subunit in the catalytic cycle was demonstrated in sophisticated experiments which showed that subunit gamma  rotates in the alpha 3beta 3 core and is probably related to sequential conformational changes of the alpha beta -pairs (13-15). Three different conformations of the beta -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 beta DP, beta TP, and beta E. The conformation of the three noncatalytic alpha -subunits was very similar in the bovine MF1 structure because all alpha -subunits were filled with the nonhydrolyzable nucleotide analogue AMPPNP (7). Deletion of the 20 C-terminal residues of the gamma -subunit resulted in an active chloroplast enzyme and questioned the significance of the gamma -rotation for the catalytic process in the chloroplast ATPase (16). Hence, rotation of subunit gamma  was recently also visualized for isolated CF1 (17).

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 alpha -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 gamma . 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 gamma -subunit.

In this paper we present the first high resolution structure of a chloroplast alpha 3beta 3gamma epsilon complex. The structure was obtained in the absence of magnesium and shows 3-fold symmetry. Thus, subunits gamma  and epsilon , which are present in single copies in the complex, are not clearly resolved. Despite the different nucleotide content the chloroplast alpha  and beta  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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 delta  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, alpha  = 90°, beta  = 90°, and gamma  = 120°. Further crystallographic parameters are given in Table I.

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 alpha beta -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 alpha Ebeta DP-dimer and the closely related alpha DPbeta TP conformer. In these dimers the nucleotide binding sites on the alpha -subunits are filled with the nonhydrolyzable nucleotide analogue AMPPNP, whereas the beta -subunits contain either ADP or ATP. Calculations using the beta E conformation of the beta -subunit where no nucleotides are bound in the catalytic site resulted in a correlation coefficient of 0.63. These results suggested that a alpha beta -dimer in the chloroplast alpha 3beta 3gamma epsilon complex resembles the dimer formed by nucleotide-filled alpha  and beta -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 alpha  and beta  subunits were defined in the rigid body minimization corresponding to residues alpha 19-95, alpha 96-379, and alpha 380-510, and beta 9-82, beta 83-363, and beta 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).

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).


    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Overall Structure of the Chloroplast alpha beta -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 delta  was degraded, whereas all other subunits remained intact (data not shown). Thus, in subsequent preparations subunit delta  was removed from the CF1 complex and the remaining alpha 3beta 3gamma epsilon complex was used in crystallization experiments (Fig. 1). No trace of subunit delta  could be detected in this complex either by silver staining or by immunoblotting. The alpha 3beta 3gamma epsilon complex crystallized in the space group R32 having one single alpha beta -pair and 0.33 gamma -subunit and 0.33 epsilon -subunit in the asymmetric unit of the crystal. In contrast to the structure of the rat liver enzyme, unambiguous determination of the gamma  structure was not possible because of the 3-fold crystallographic symmetry in the complex. The single alpha  and beta  subunits in the asymmetric unit represent a superposition of the three copies present in the chloroplast alpha 3beta 3gamma epsilon complex. With the exception of the C termini the electron density is well defined, suggesting that all alpha  and beta  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 alpha 3beta 3gamma epsilon complex. The purified CF1-delta 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 alpha - and beta -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 sigma .

The final model of the chloroplast alpha beta -dimer includes 942 residues (alpha 25-501 and beta 19-485). The final parameter of refinement and model stereochemistry are summarized in Table I. Residues alpha Leu345, alpha Gly353, alpha Leu387, alpha Gln389, alpha Phe399, alpha Ser401, alpha Thr407, alpha Asn409-Arg413, alpha Tyr464, alpha Thr481-Phe482, alpha Glu499, beta Leu159, beta Ala331, beta Gly355, beta Gly381, beta Ala406, beta Ile407, beta Gly409, beta Glu416, beta Asp417, beta Gly446, beta Ile455, beta Leu462, and beta 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 beta Arg122 have main chain dihedral angles that fall within the allowed regions of the Ramachandran diagram as defined by the program PROCHECK (37).


                              
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Table I
Crystallographic data and refinement statistics

The overall structure of the alpha  and beta  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 beta -barrel (alpha 25-96 and beta 19-93), a central domain of beta -strands with associated alpha -helices that contains the nucleotide binding site (alpha 97-371 and beta 94-381), and a C-terminal alpha -helical bundle (alpha 372-501 and beta 382-485). In comparison with the mitochondrial enzyme, the chloroplast beta -subunit contains two insertions in the N-terminal beta -barrel domain (beta Lys39 and beta Gly56-Asn60), a single residue inserted in the central domain of the beta -subunit (beta Glu224), and a deletion of 8 residues between helix B and strand 6 in the central nucleotide binding domain of the alpha -subunit (alpha Lys187-Asp194). The insertions beta Lys39 and beta Glu224 and the deletion in subunit alpha  show only minor effects on the structure. However, the insertion beta 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 beta -barrel domain of the adjacent alpha -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 alpha - and beta -subunits. A, schematic representation of the alpha -subunit structure (blue). B, schematic representation of the chloroplast beta -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 alpha 3beta 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 beta Gly56-Asn60 in the N-terminal domain of the beta -subunit that partially cover the adjacent alpha -subunit are indicated by the arrows.

The conformation of the chloroplast alpha -subunit is similar to the three conformations found in the bovine mitochondrial structure. The Calpha atoms superimpose with rms deviations of 1.31, 1.31, and 0.95 Å with the mitochondrial alpha TP, alpha DP, and alpha E conformation. In contrast the chloroplast beta -subunit superimposes well only with the beta TP or beta DP conformation of the mitochondrial enzyme. The Calpha atoms show rms deviations of 1.24 and 1.19 Å, respectively. Comparison with the nucleotide-free beta E conformation of the mitochondrial structure reveals a rms deviation of 3.89 Å. Thus the chloroplast alpha  and beta -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 alpha - and beta -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 beta -subunit. To discriminate these different conformations and to reduce the effects of model bias, residues beta 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 beta DP and beta 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 beta -subunit. Calculations of a composite omit, cross-validated, sigma -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 beta E and beta DP conformation superimposed. A, stereo image of a sigma A-weighted 2Fo - Fc electron density map of the chloroplast beta -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 beta E is shown is red, and beta DP is drawn in black.

As an additional test, whether the catalytic beta -subunits in the crystal is present as a mixture of closed and open conformations, crystallographic refinement was carried out with a model containing the beta E and beta DP conformation at various occupancies. In comparison with a model having the beta -subunit exclusively in the closed conformation (1.00 beta DP), refinement of a model with 0.66 beta DP conformation and 0.33 beta 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 beta -subunit in the crystal, although local disorder and structural distinct conformations cannot be completely excluded at the present resolution.

Comparison of the Crown Region-- The overall structure of the N-terminal six-stranded beta -barrel domain is highly similar in the mitochondrial (7), thermophilic (10), and chloroplast enzymes (Fig. 5). However, the chloroplast and the thermophilic beta -subunit contain an insert of five and seven residues, respectively. In the chloroplast alpha 3beta 3gamma epsilon 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 beta -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 beta Asn40 and alpha Arg90 and van der Waal's interaction of residues beta Asn38-alpha Ser21 and beta Glu41-alpha Met48 are proposed to cause this increased stability (10). The high resolution structure of the chloroplast enzyme suggests that a salt bridge beta Asp83-alpha Arg297 enhances the stability of the enzyme. Both residues are within 3.4-3.5 Å, whereas the corresponding residues in the thermophilic (beta Asp68-alpha -Arg296) and the mitochondrial (beta Glu67-alpha 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 beta -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 beta -barrel domain of the beta -subunit in the mitochondrial (A), chloroplast (B), and thermophilic ATPase (C).

Structure of the gamma -Subunit-- In contrast to the rat liver mitochondrial F1 complex that also crystallized in the space group R32 (9), unambiguous tracing of the gamma -subunit in the chloroplast alpha 3beta 3gamma epsilon 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 alpha 3beta 3 core complex, which probably corresponds to the 3-fold superimposed C terminus of the chloroplast gamma -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 alpha 3beta 3 domain suggests that the extreme C-terminal end of the chloroplast gamma -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 gamma -subunits remains to be solved.



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Fig. 6.   Electron density in the central cavity of the chloroplast alpha 3beta 3 core. Calculated electron density map at 1 sigma  attributed to the chloroplast gamma -subunit. Parts of the density map show the superimposed 3-fold crystallographic symmetry on the gamma -subunit in the center of the alpha 3beta 3 complex. The structure is shown with the crystallographic axis in the vertical position.

A less ordered, unfolded structure rather than a more buried well ordered alpha -helical conformation might account for the observed fast modification of gamma Cys322 located at the extreme C-terminal end of the gamma -subunit and might also explain why this portion of the gamma -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 gamma -subunit in the near future.

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 alpha - and beta -subunits (7-10). Noncatalytic sites are predominately formed by the alpha -subunits, and catalytic sites are mainly located on the beta -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 alpha 3beta 3gamma epsilon complex revealed that no nucleotides were bound to the alpha -subunit, even though the conformation of the alpha -subunit resembles the closed, AMPPNP-filled conformation of the bovine mitochondrial enzyme. The same closed conformation of the alpha -subunits lacking bound nucleotides was also found in the thermophilic alpha 3beta 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 alpha 3beta 3gamma epsilon 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 beta -subunits. Again, no bound nucleotides were detected in the crystal structure. However, some additional density was detected in the P-loop region (alpha Gly170-Thr177 and beta 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 alpha 3beta 3gamma epsilon 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 beta -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.

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 gamma -disulfide or displacement of the epsilon -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.

The structure of the chloroplast alpha 3beta 3gamma epsilon 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 gamma -subunit or in the catalytic beta -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 gamma -subunit caused by reduction of the disulfide or by altered interaction with the epsilon -subunit. These conformational changes involving the transformation of the nucleotide binding sites might be related to a displacement of the central axis of subunit gamma  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).

Potential Tentoxin-binding Site at the alpha beta -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 alpha beta -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 beta -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 beta -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 alpha beta -interface. Residues alpha 49-52, alpha 65-69, alpha 95-96, alpha 131-133, alpha 297, beta 26-29, beta 51-54, beta 78, beta 80-85, and beta 242, which are located within 10.0 Å of beta Asp83 in the structure of the chloroplast alpha 3beta 3gamma epsilon 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 beta Asp83-alpha Arg297 controls the overall conformation of the binding pocket and adjusts the alpha beta -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 alpha 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 alpha Arg128, alpha Lys132, and alpha 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 beta Asp53 and beta Thr54, which are located at the top of the cleft. Adjacent to these residues a hydrophobic surface is formed by residues alpha Leu129, alpha Ser132, and alpha Pro133, which might provide essential contacts for the inhibitor. Further analysis of the CF1 structure suggests that residues alpha Met274 and alpha 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 beta Glu67 is blocked by alpha Arg128 and beta Glu42. In the thermophilic structure the corresponding beta 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 alpha Glu130 and alpha 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.



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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 beta Asp83 in CF1 and the corresponding residues beta Asp67 in MF1 and beta 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 alpha 3beta 3 complex.

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 alpha Ala96 and alpha 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.


    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.


    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.


    ABBREVIATIONS

The abbreviations used are: CF1, chloroplast F1; AMPPNP, adenosine 5'-(beta ,gamma -imino)triphosphate; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; rms, root mean square.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES


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