Identification of the F1-binding Surface on the delta -Subunit of ATP Synthase*

Joachim Weber, Susan Wilke-Mounts, and Alan E. SeniorDagger

From the Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642

Received for publication, November 26, 2002, and in revised form, January 16, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The stator function in ATP synthase was studied by a combined mutagenesis and fluorescence approach. Specifically, binding of delta -subunit to delta -depleted F1 was studied. A plausible binding surface on delta -subunit was identified from conservation of amino acid sequence and the high resolution NMR structure. Specific mutations aimed at modulating binding were introduced onto this surface. Affinity of binding of wild-type and mutant delta -subunits to delta -depleted F1 was determined quantitatively using the fluorescence signals of natural delta -Trp-28, inserted delta -Trp-11, or inserted delta -Trp-79. The results demonstrate that helices 1 and 5 in the N-terminal domain of the delta -subunit provide the F1-binding surface of delta . Unexpectedly, mutations that impaired binding between F1 and delta  were found to not necessarily impair ATP synthase activity. Further investigation revealed that inclusion of the soluble cytoplasmic domain of the b subunit substantially enhanced affinity of binding of delta -subunit to F1. The new data show that the stator is "overengineered" to resist rotor torque during catalysis.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ATP synthase is the membrane enzyme responsible for ATP synthesis in oxidative and photophosphorylation of prokaryotes and eukaryotes, and also for ATP-driven proton pumping to generate the transmembrane proton gradient in bacterial membranes. In Escherichia coli, ATP synthase consists of a complex of eight subunits, alpha 3beta 3gamma delta epsilon ab2cn. It was defined in earlier times in terms of a membrane-peripheral F1 sector (alpha 3beta 3gamma delta epsilon ) containing three catalytic sites, and a membrane-embedded F0 sector (ab2cn), which carries out transmembrane proton transport. Recent work has demonstrated that the enzyme functions as a rotary motor. The centrally located "rotor" consists of subunits gamma epsilon cn. At the top, it rotates inside the alpha 3beta 3 hexagon, and thus modifies the activities of the catalytic sites; at the base, it rotates against subunit a, thereby facilitating proton movement. In this way, the energy of the proton gradient is transduced into the energy of ATP synthesis/hydrolysis. Understanding the mechanism by which this occurs is currently of major interest. To ensure that subunits a and alpha 3beta 3 remain firmly fixed in relation to each other they are connected by a peripheral structure, the "stator" stalk, consisting of subunits b2 and delta . For recent reviews of the structure and function of ATP synthase, see Refs. 1-3.

The stator must be able to resist strain resulting from rotor torque, thus its construction is of considerable importance. The dimer of b-subunits forms an elongated helical connection between subunit a and the C-terminal domain of the delta -subunit, it lies at one side of the alpha 3beta 3 hexagon, and its functional domains have been well characterized (4-6). There may exist functional interactions between b2 and the alpha 3beta 3 hexagon (7). Currently only partial high-resolution structure has been reported for the b subunit (8, 9). The delta -subunit has been shown by electron microscopy to bind to the very top ("crown") of the alpha 3beta 3 hexagon (10), and the homologous mitochondrial OSCP1 subunit also binds at the top of the molecule, with its C-terminal domain at the side (11). The N-terminal region of alpha -subunit (residues alpha 1-15 or alpha 1-19) was shown to be necessary for delta  binding by proteolysis experiments (12), and cross-linking between an inserted Cys at residue alpha 2 and natural Cys residue(s) in delta  (13) provided further evidence that the N terminus of alpha , which is not seen in high-resolution x-ray structures, binds to delta . In our laboratory, quantitative determination of the affinity of binding between delta -subunit and the alpha 3beta 3gamma epsilon complex (also called "delta -depleted F1"), using a novel fluorescence assay dependent upon the fluorescence of the single natural Trp in delta , revealed a Kd of 1.4 nM in the E. coli enzyme (14). This is equivalent to a binding energy of ~50 kJ/mol, approximately equivalent to rotor torque (15, 16). A similar Kd value was reported for the chloroplast enzyme (17) using a fluorescent probe attached to delta . We were able to show that a fragment of delta  containing only N-terminal residues 1-1342 (called delta ') bound with the same affinity as intact delta , demonstrating that all the determinants of binding lie in the N-terminal region of delta  (14). The structure of the N-terminal domain of delta  (residues 2-106 in the fragment delta ') has been determined to high-resolution by NMR (18). Examination of this structure in combination with homology searches of the sequences of other species of delta  and OSCP suggested possible residues that might be involved in binding interaction at the F1/delta interface. Using the quantitative binding assay that we had developed, in combination with mutagenesis, we were able here to test these ideas. From the results, this paper presents the identification of the surface on the delta -subunit that provides interactions with F1. In addition we show that inclusion of the soluble cytoplasmic domain of the b subunit substantially enhanced the affinity of binding of delta -subunit to F1.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Purification of F1, Purification of delta -Subunit, Preparation of delta -depleted F1, Fluorescence Binding Assays, Assay of ATP-driven Proton Pumping in Reconstituted Membrane Vesicles, Routine Procedures-- These were all as described previously (14). For measurement of binding of purified delta -subunit to delta -depleted F1, the delta -depleted F1 was the "beta W107 F1" from strain pSWM86/DK8 as previously described (14). beta W107 F1 contains only the Trp at residue beta -107, it has normal functional properties, and has a low Trp background signal in the delta -binding assay. For the ATP-driven proton pumping assay, the delta -depleted F1 was from strain pSWM92/DK8 (delta W28L), which is easier to completely deplete of delta -subunit than wild-type F1 (14). Growth yields on limiting glucose were measured as in Ref. 19.

E. coli Strains and delta -Mutagenesis-- Strains pSWM86/DK8 and pSWM92/DK8 were described previously (14). Strain AN2015 has the genotype uncH241, argH, pyrE, entA, recA. It contains the mutation delta W28stop, and was a kind gift of G. B. Cox and F. Gibson. For mutagenesis of delta , the oligonucleotide-directed mutagenesis procedure in which an EcoRI-SmaI fragment from plasmid pJC1 (20) was moved into the M13mp19 template was used (14). After mutagenesis the same fragment was moved back to pJC1, creating a new mutant plasmid that was transformed into strain AH3 for expression of mutant delta  (14). For the delta Y11W/delta W28L and delta V79W/delta W28L mutants the initial EcoRI-SmaI fragment in M13mp19 was taken from pSWM101, containing the delta W28L mutation. New mutations, oligonucleotides and resultant mutant plasmids were as follows: delta Y11A, GTAGCTCGCCCCGCGGCCAAAGCAGC, plasmid pSWM104, introduces SacII site; delta Y11W, GGTAGCTCGCCCATGGGCCAAAGCAGC, plasmid pSWM105, introduces NcoI site; delta A14L, CCCTACGCCAAGCTAGCTTTTGACTTTGC, plasmid pSWM107, introduces NheI site; delta A14D, CCTACGCCAAAGATGCATTTGACTTTGC, plasmid pSWM106, introduces NsiI site; delta N75E, CGAAAACGGTCAGGAGCTCATTCGGGTTATGGC, plasmid pSWM113, introduces SacI site; delta N75A, CGAAAACGGTCAAGCGCTGATTCGGGTTATG, plasmid pSWM112, introduces Eco47III site; delta V79A, GAACCTGATTCGGGCCATGGCTGAAAATGG, plasmid pSWM114, introduces NcoI site; delta V79W, GAACCTGATTCGGTGGATGGCGGAAAATGGTCG, plasmid pSWM115, introduces EciI site; and delta G150D, GTCTGTAATGGCGGACGTTATCATCCG, plasmid pSWM119, introduces EciI site.

Expression and Purification of the Cytoplasmic Domain of b Subunit in Soluble Form-- The soluble form of the cytoplasmic domain of the b subunit named bST34-156 was expressed from plasmid pJB3 (20) after transformation into strain DK8. The procedure followed essentially that described in Ref. 20. Briefly, six 1-liter cultures of strain pJB3/DK8 were grown and harvested, cells were broken by a French press, and the supernatant fraction was collected after centrifugation. Ammonium sulfate was added to 60% saturation, the pellet was resuspended and dialyzed, the protein was then purified by sequential chromatography on Whatman DE-52 anion exchange resin and by gel filtration on Sephacryl S-100. Buffers were described as in Ref. 20. Yield of bST34-156 was 23 mg of protein/liter of culture.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A Potential F1-binding Surface on the delta -Subunit-- Fig. 1 shows the NMR structure of the N-terminal domain of the delta -subunit of E. coli ATP synthase (18) as determined using the proteolytic fragment (delta ') consisting of residues 1-134 (intact delta  has 177 residues). As noted in the Introduction, because proteolytic removal of the 43 C-terminal residues has no effect on the affinity for binding to delta -depleted F1 (14), it is highly likely that the domain shown in Fig. 1 contains most, if not all, of the amino acid residues involved in binding of delta  to F1. Comparison of the sequences of delta  or OSCP from 90 species by BLAST search (21) showed that the highly conserved residues are clustered on a relatively small portion of the delta  surface, as demonstrated in Fig. 2. Because delta  sequences are in general not highly conserved, for example, in comparison with those of alpha - or beta -subunits (22), it appeared probable that this conserved part of delta , formed by helices 1 and 5 (colored dark blue and yellow, respectively, in Fig. 1), constituted the F1-binding site. We decided to test this hypothesis by mutagenesis of four conserved residues, delta -Tyr-11, delta -Ala-14, delta -Asn-75, and delta -Val-79. From the BLAST search (above) we found the following degrees of conservation of these residues. At position delta -11, Tyr occurred in 95% of species and Phe in 5%; at delta -14, Ala occurred in 90%; at delta -75 Asn occurred in 78% and Asp in 6%; and at delta -79 a hydrophobic residue (Val, Leu, or Ile) occurred in 92% of species. Fig. 2A shows the location of these residues on the hypothesized binding surface.


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Fig. 1.   High resolution (NMR) structure of the N-terminal domain of delta -subunit. The protein backbone from residue delta -Ser-2 (helix 1, dark blue) to residue delta -Thr-106 (helix 6, red) is shown. Helices are numbered 1-6. Residue delta -Trp-28, located in helix 2 (light blue), is depicted in "spacefill" representation. Adapted from Ref. 18 and displayed using PyMOL (31).


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Fig. 2.   Sequence conservation on a proposed binding surface of delta -subunit. A, the surface of the N-terminal domain of delta  is shown in the same orientation as in Fig. 1. Strongly conserved or conservatively replaced residues are colored in red; partially conserved residues are colored in pink. The remaining residues of delta  (gray) show a high degree of variability. Residues 11 and 14 in helix 1 and 75 and 79 in helix 5 were mutated in this work. Residue 85 was mutated in previous work and is discussed in the text. B, series of views obtained by rotating the molecule in 90° steps. The central depiction shows the molecule from the same angle as in A above and in Fig. 1.

Mutagenesis and Purification of the delta -Subunit-- Two mutations were introduced in each of the four conserved positions, namely: delta Y11A,delta Y11W; delta A14D,delta A14L; delta N75A,delta N75E; delta V79A,delta V79W. The Ala mutations were designed to give an estimate of the contribution of the natural residue to subunit-subunit binding energy. The introduced Trp residues could give new probes for assaying the interaction of delta  with F1 and in both cases they were combined with the delta W28L mutation to remove the only naturally occurring Trp in delta . The remaining mutations were designed to perturb binding of delta  to F1. The mutation delta G150D was included as a control. This mutation is known to completely impair ATP synthase function (23), but because it occurs in the C-terminal domain of the delta -subunit it is expected to interfere with binding of delta  to the b subunit (24) and not with binding to F1.

For each mutant, we purified delta -subunit following previously published procedures (14, 20), except in the cases of delta A14D and delta G150D, where the concentration of saturated ammonium sulfate necessary to precipitate delta  was 50% rather than 32%. Yields ranged from 0.5 to 3 mg/liter culture. Purity of the mutant delta  was checked by SDS gels, and in each case was the same as with wild-type as previously shown in Ref. 14. Each purified mutant delta  was assayed to determine the affinity of binding to F1 using the fluorescence assay developed previously (14). This assay depends on the fact that binding of delta  to F1 that has been depleted of delta  produces a large enhancement of fluorescence of the natural delta -Trp-28 residue. As noted above, for the delta Y11W and delta V79W mutants, we removed the natural delta -Trp-28 (by combining with delta W28L) and used the new Trp as the probe. For each mutation we also assayed the effect on function in vivo, by measuring growth of mutant cells on succinate plates or in limiting glucose medium, and we measured effects on ATP-driven proton pumping in vitro by reconstitution with membrane vesicles and delta -depleted F1 using purified mutant delta .

Fluorescence Spectra of Purified Mutant delta -- Fig. 3, A-E, shows the Trp fluorescence spectra of the purified mutant delta -subunits in the absence of F1. Except for the delta Y11W/W28L and delta V79W/W28L mutants (dashed curves in Fig. 3, A and D, respectively), the fluorophor is the natural Trp in position delta -28. Several of the spectra of mutant delta  (delta A14L, delta V79A, and delta G150D) resemble closely that of wild-type delta  (dotted lines in Fig. 3, A-E) with a maximum at 326 nm, indicating a relatively unpolar environment for the tryptophan side chain. The wavelength position of the spectra for delta N75A and delta N75E (Fig. 3C) is also very similar; however, the fluorescence intensity is 20-30% higher, suggesting that the mutations caused minor changes in the environment of delta -Trp-28. In the delta A14D mutant, the spectrum of delta -Trp-28 was red-shifted by 2 nm, in the delta Y11A mutant by 5 nm, indicative of an increased polarity experienced by the fluorophor. In general, however, the mutations appeared not to perturb the tertiary structure to any large extent, by this criterion.


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Fig. 3.   Tryptophan fluorescence spectra of purified mutant delta -subunits. Spectra (lambda exc = 295 nm) were measured in 50 mM HEPES, 5 mM MgSO4, pH 7.0, and corrected. In all panels the wild-type spectrum is shown as the dotted line. A, solid line, delta Y11A; dashed line, delta Y11W/W28L. B, solid line, delta A14D; dashed line, delta A14L. C, solid line, delta N75A; dashed line, delta N75E. D, solid line, delta V79A; dashed line, delta V79W/W28L. E, solid line, delta G150D.

A tryptophan inserted in position delta -11, in the delta Y11W/W28L mutant, has an emission maximum of 337 nm (Fig. 3A), suggesting clearly a more polar environment than for delta -Trp-28 in wild-type. A tryptophan in position delta -79 (Fig. 3D) has nearly aqueous surroundings, as indicated by fluorescence maximum at 350 nm of the delta V79W/W28L mutant. These data are consistent with the predicted location from the structure (Figs. 1 and 2).

Effect of Addition of delta -depleted F1 on the Fluorescence Spectra of Trp in delta -Subunits-- Wild-type and most of the mutant delta  preparations contained delta -Trp-28 as the sole Trp. The response of the delta -Trp-28 fluorescence upon binding of delta -depleted F1 to wild-type delta  is shown in Fig. 4A. As described previously (14), there was an increase in fluorescence intensity of about 50%, combined with a slight blue-shift (~4 nm), indicative of a change to a more unpolar environment upon binding. The same change was seen in the delta V79A and delta G150D mutants (data not shown). In the delta N75A and delta N75E mutants the fluorescence signal of unbound delta  was higher to begin with (Fig. 3C), but the final signal after binding to delta -depleted F1 was very similar to wild-type delta , as in Fig. 4A (data not shown). In the delta Y11A mutant, which had a lower intensity and a more red-shifted spectrum before addition of F1, the fluorescence increase upon F1 binding was about 40%, and the blue-shift was by 6 nm. Mutants delta A14D and delta A14L did not show any change in fluorescence under the experimental conditions used (discussed below).


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Fig. 4.   Tryptophan fluorescence spectra of purified wild-type and mutant delta -subunits after addition of delta -depleted F1. Spectra are shown before (dotted lines) and after (solid lines) addition of 2-3-fold excess of delta -depleted F1. All spectra are corrected for the contribution by F1 and unbound delta . A, wild-type delta . B, delta Y11W/W28L. C, delta V79W/W28L.

The engineered delta -Trp-11, in the delta Y11W/W28L mutant, experiences a very pronounced fluorescence increase upon F1 binding, by 80-90% at 330 nm, combined with a blue-shift of 4 nm (Fig. 4B). In contrast, the fluorescence of the engineered delta -Trp-79, in the delta V79W/W28L mutant, is quenched upon F1 binding, by about 50% at 350 nm, accompanied by a blue-shift of 14 nm (Fig. 4C). The blue-shifts of all three Trp residues (delta -Trp-11, delta -Trp-28, and delta -Trp-79) reflect a more unpolar environment of the tryptophan side chains upon F1 binding, suggesting that the fluorophors are better shielded from the medium in this state. This, in turn, provides good evidence that all three fluorophors are actually located at, or close to the F1-binding surface on the delta -subunit. These data indicated that for all except the delta A14D and delta A14L mutants a direct fluorescence assay was available for binding of delta -subunit to delta -depleted F1, and that in the cases of delta A14D and delta A14L a competition assay would be required.

Fluorescence Titration of Binding of delta -Subunit to delta -depleted F1 and Determination of Binding Affinities of Mutant delta -Subunits-- Binding of mutant delta  to delta -depleted F1 was measured by monitoring the tryptophan fluorescence of delta , either because of the natural delta -Trp-28 or because of the engineered delta -Trp-11 or delta -Trp-79. For each mutant, initially an F1 concentration of 0.05 µM was chosen. If this concentration was too high to obtain a reliable Kd value (because the resulting titration curve was "stoichiometric," for example, the curve for wild-type delta  in Fig. 5A), the titrations were repeated using 0.01 µM F1. If the initial F1 concentration was too low (i.e. the titration curves did not reach saturation), the experiments were repeated using 0.5 µM F1. Typical titration curves are shown in Figs. 5 and 6 and the resultant calculated Kd values are given in Table I. In all cases where a binding stoichiometry could be determined reliably (i.e. with stoichiometric or close-to-stoichiometric binding curves), it was very close to 1 (0.9 to 1.2) mol of delta  per mol of F1.


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Fig. 5.   Fluorescence titrations to assess binding of delta -Tyr-11 and delta -Ala-14 mutant delta  to delta -depleted F1. Pure delta -subunit was mixed with delta -depleted F1, and the resulting fluorescence enhancement at 325 nm (after subtraction of the contribution of delta  alone and of F1 alone) was plotted against the concentration of delta . F1 concentrations are given in the figure. A, open circles/dotted line, wild-type delta ; inverted triangles/solid line, delta Y11A; triangles/dashed line, delta Y11W/W28L. B, inverted triangles/solid line, delta Y11A; triangles/dashed line, delta Y11W/W28L; squares, delta A14D; diamonds, delta A14L.


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Fig. 6.   Fluorescence titrations to assess binding of delta -Asn-75 and delta -Val-79 mutant delta  to delta -depleted F1. Pure delta -subunit was mixed with delta -depleted F1, and the resulting fluorescence changes at 325 nm (A and B) or 360 nm (C) were plotted (after subtraction of the contribution of delta  alone and of F1 alone) against the concentration of delta . F1 concentrations are given in the figure. A, open circles/dotted line, wild-type delta ; inverted triangles/solid line, delta N75A; triangles/dashed line, delta N75E. B, delta V79A. C, delta V79W/W28L.


                              
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Table I
Effect of mutations in delta -subunit on affinity of binding of delta  to delta -depleted F1
Binding affinities (Kd) were calculated from the titration curves in Figs. 5 and 6.

Both mutations in position delta -11 resulted in a significantly decreased binding affinity. Based on the results obtained with the delta Y11A mutant, the delta -Tyr-11 side chain in wild-type delta  contributes about 12 kJ/mol of binding energy (Table I). In this case, tryptophan is not a good substitute for tyrosine, because the delta Y11W mutation also causes a loss of close to 12 kJ/mol in binding energy (calculated by comparing delta Y11W/W28L to delta W28L mutant).

Titrations with the delta A14D and delta A14L mutants, even using 0.5 µM F1 and up to 2.5 µM delta , did not result in significant changes in the delta -Trp-28 fluorescence (Fig. 5B). One possible explanation is that the binding affinity is so low that even at the highest concentrations used, binding is negligible. In this case, Kd would be >5 µM (and the loss in binding energy >20 kJ/mol). Alternatively, it is possible that binding occurs, but that the mutations affect the interaction between delta  and F1 in such a way that the fluorescence of delta -Trp-28 does not respond to binding. To address this question, we performed competition titration experiments, which showed that a 6-10-fold excess of delta A14D or delta A14L mutant delta -subunit did not significantly reduce binding of wild-type delta . On the basis of these results, we can calculate that the Kd for delta A14D or delta A14L mutant is at least 0.1 µM. Thus, from either calculation, both mutations at residue delta -14 perturb interaction between delta  and F1 considerably.

In position delta -75, neither mutation delta N75A nor delta N75E has a significant effect on the F1 binding affinity (Fig. 6A and Table I). Specifically the results obtained with the delta N75A mutant suggest that the natural asparagine side chain does not contribute binding energy. In contrast, the valine side chain of residue delta -Val-79 makes a moderate contribution of about 6 kJ/mol (Table I). A tryptophan in this position strongly interferes with binding to F1, with a loss of binding energy of more than 11 kJ/mol (comparing delta V79W/W28L to delta W28L). As expected, the mutation delta G150D in the C-terminal region of delta , used as a control here, had no effect on F1 binding affinity.

Effect of the delta  Mutations on ATP Synthesis in Vivo-- Plasmids containing mutant uncH (delta -subunit) genes were transformed into strain AN2015, which contains the chromosomal mutation delta -Trp-28 right-arrow stop and is therefore unable to grow by oxidative phosphorylation. All plasmids carrying mutations in positions 11, 14, 75, and 79 of delta  were able to restore oxidative phosphorylation, as demonstrated by their growth yields on limiting glucose (Table II) and their ability to grow on plates containing succinate as the sole carbon source (not shown). In contrast, the mutation delta G150D prevented ATP synthesis by the ATP synthase in vivo (Table II).


                              
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Table II
Effect of mutations in delta -subunit on growth of cells in vivo and ATP-driven proton pumping in vitro
Growth yields in limiting glucose medium and ATP-driven proton pumping in membrane vesicles were measured as described under "Experimental Procedures." For the former, plasmid containing wild-type or mutant delta  (uncH) gene was transformed into strain AN2015, which contains a nonsense mutation delta W28stop. For the latter, 500 µg of KSCN-stripped membranes were reconstituted with 100 µg of delta -depleted F1 plus 10 µg of wild-type or mutant delta -subunit, and ATP-induced quench of acridine orange fluorescence was measured.

Effect of delta  Mutations on ATP-driven H+ Pumping in Vitro-- KSCN-stripped membranes were reconstituted with wild-type or mutant delta  together with delta -depleted F1 and proton pumping was initiated by addition of ATP. It was found that none of the mutants at positions 11, 14, 75, or 79 of delta  caused significant impairment of ATP-driven H+ pumping (Table II). These data indicate that, in the presence of intact F0, functional binding of the mutant delta -subunits to F1 did occur. From the considerations presented in the Introduction, one likely mechanism for such an effect would be through involvement of the b subunit dimer, and specifically the cytoplasmic domain of b that interacts with delta . In contrast, the delta G150D mutant prevented formation of a proton gradient upon ATP hydrolysis (Table II) as expected from previous work (23). As already noted, this mutation occurs in the C-terminal region of delta  and is expected to interrupt interaction of delta  with b.

Effect of the Soluble Cytoplasmic Domain of the b Subunit on Binding of delta  to delta -depleted F1-- For these experiments the soluble cytoplasmic domain of the b subunit that we used was purified bST34-156 (20). It consists of residues 34 through 156 (C terminus) of the b subunit with an additional Ser-Thr- sequence at the N terminus. The purified protein showed a single major band on SDS gels with the expected mobility (20). Functional integrity of bST34-156 was evaluated using the method of Dunn (25). In this assay, inhibition of reconstitution of ATP-dependent proton pumping by F1 in stripped membranes is measured. Inhibition comes about as a result of competition between F0 and added b subunit cytoplasmic domain for a limited number of F1 molecules. We found that reconstitution was inhibited by bST34-156 in a dose-dependent manner. The ratio of bST34-156/F1 giving 50% inhibition of reconstitution was 2 µg/µg, the same as in Ref. 25. Theoretically bST34-156 should contain no Trp residue. Calculation of the Trp content of our preparation, from the fluorescence spectrum in 6 M guanidine hydrochloride, revealed a Trp content of 0.1 mol/mol. The fluorescence because of Trp contamination in bST34-156 was corrected routinely.

We demonstrated that addition of bST34-156 to wild-type or any of the mutant delta -subunits in the absence of F1 had no effect on the Trp fluorescence of the delta -subunit. In initial experiments with F1 present, we chose to use a mutant delta  with a high Trp signal and a relatively low affinity for binding to F1, namely delta Y11W/W28L. Fig. 7A demonstrates an experiment in which bST34-156 and delta -depleted F1 were added at a constant concentration and the amount of added delta Y11W/W28L subunit was varied. This fluorescence titration showed that addition of bST34-156 to the binding assay had a very large effect, reducing Kd for binding of delta Y11W/W28L to F1 from 0.5 µM to <5 nM. In Fig. 7B we determined the apparent Kd for bST34-156. Here, with constant concentrations of delta -depleted F1 and delta Y11W/W28L subunit present, the concentration of bST34-156 was varied. The apparent Kd for the bST34-156 dimer (the physiological form, Refs. 4 and 5) was 150 nM, with two independent experiments showing excellent agreement.


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Fig. 7.   Effect of bST34-156 on binding of delta Y11W/W28L mutant delta  to delta -depleted F1. The signal used was the fluorescence increase of residue delta -Trp-11 upon binding of delta Y11W/W28L mutant delta  to delta -depleted F1. Contributions of mutant delta  alone, delta -depleted F1, and bST34-156 were subtracted. A, titration of delta -depleted F1 with delta Y11W/W28L mutant delta  in the presence of 10 µM bST34-156. B, titration of equimolar (100 nM) concentrations of delta -depleted F1 and delta Y11W/W28L mutant delta  with bST34-156 (plotted as bST34-156 dimer).

Fig. 8 shows the effect of inclusion of bST34-156 on the binding of wild-type and mutant delta -subunits to delta -depleted F1. We used a concentration of 4 µM bST34-156 in these experiments to be sure of saturation while reducing the impact of the Trp fluorescence because of contaminants in bST34-156 to a minimum. Presence of this contamination restricted the concentration range accessible for titration experiments to >= 100 nM F1 for all delta  mutants except delta Y11W/W28L, with its larger signal, where 20 nM F1 could be used. Nevertheless, it is clear from Fig. 8, A-C, that in mutants delta Y11W/W28L, delta Y11A, delta V79A, and delta V79W/W28L, inclusion of bST34-156 in the binding assay brought about a substantial increase in binding affinity between the delta -subunit and F1. The titration curves (Fig. 8) are all fully or close to "stoichiometric," thus we can only stipulate that in these cases that the Kd was reduced to <5 nM (except for delta Y11W/W28L where we can say Kd was reduced to <1 nM). Still, by comparison with the Kd values obtained in the absence of bST34-156 (shown in Table I), it is clear that the changes are large, indeed >= 500-fold in the case of delta Y11W/W28L mutant.


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Fig. 8.   Effect of bST34-156 on binding of mutant delta -subunits to delta -depleted F1. Mutant delta -subunit was added to delta -depleted F1 (concentration given in the figure) in the presence of 4 µM bST34-156. Trp fluorescence intensity changes at 325 nm (A-C, squares) or 360 nm (C, diamonds) were plotted against the concentration of delta . A, open circles/dotted line, wild-type delta ; inverted triangles/solid line, delta Y11A. B, delta Y11W/W28L. C, squares/solid line, delta V79A; diamonds/dashed line, delta V79W/W28L.

With regard to wild-type delta , the above mentioned technical constraints prevented us from determining whether the binding affinity for F1 was increased by inclusion of bST34-156 (the Kd in the absence of bST34-156 was already 1.4 nM, Table I). If we assume that a 500-fold increase in affinity occurs for wild-type, as it did for delta Y11W/W28L, this would give a Kd for wild-type delta  in the presence of bST34-156 of <= 3 pM.

In contrast, with mutants delta A14D and delta A14L the titration curves (not shown) looked the same as those in Fig. 5B (squares and diamonds) even when bST34-156 was present. From this we can conclude that either these mutant delta -subunits do not bind significantly to F1 even in the presence of bST34-156, or that if they do bind, the binding does not engender a change in the fluorescence signal of delta -Trp-28.

Titration of Mutant delta -Subunits with KSCN-stripped Membranes and delta -depleted F1 in the Reconstituted Proton-pumping Assay-- Table I demonstrated that the mutant delta A14D and delta A14L subunits did reconstitute ATP-driven proton pumping in membrane vesicles when added back under saturating conditions with delta -depleted F1 to KSCN-stripped membranes. To investigate the binding properties of these mutant delta -subunits in the presence of intact F0 further we titrated them in this assay with constant amounts of stripped membranes and delta -depleted F1. Whereas we had shown before (14) that such a titration cannot be used to determine absolute values of Kd for binding of delta  to F1, nevertheless, we expected that by comparison of the mutants with wild-type we could get qualitatitve information regarding the binding of the delta A14D and delta A14L proteins. Fig. 9 indicates that the titration curves obtained with these (and other) mutants are not very dissimilar from wild-type. We therefore conclude that the failure to see enhancement of fluorescence when these mutant subunits are added to delta -depleted F1 in the presence of bST34-156 (above) is most likely because of the fact that the mutations affect the environment of residue delta -Trp-28 and prevent enhancement of its fluorescence signal upon binding.


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Fig. 9.   ATP-driven proton pumping in stripped membranes reconstituted with mutant delta -subunit and delta -depleted F1: titration with mutant delta . Membrane vesicles were stripped of F1 by KSCN treatment, then reconstituted with delta -depleted F1 and varying amounts of mutant or wild-type delta -subunit. ATP-driven proton pumping was monitored by quenching of acridine orange fluorescence, and the maximal percent quench was plotted as a function of delta  concentration in the cuvette. Open circles/dotted line, wild-type delta ; inverted triangles/solid line, delta Y11A; open squares/solid line, delta A14D; open diamonds/dashed line, delta A14L; filled diamonds/dashed line, delta V79W/W28L.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The goal of this study was to investigate in detail the interaction between the delta -subunit and F1 in ATP synthase, and specifically to identify the F1-binding surface on the delta -subunit. Based on the location of conserved or conservatively replaced residues in one particular region of the high resolution NMR structure of delta , we hypothesized that the F1-binding surface might be formed by helices 1 and 5 (see Figs. 1 and 2). Our results confirmed the hypothesis. However, several mutations in delta  that clearly disrupted binding of delta  to F1 did not lead to impaired function, an unexpected finding. Additional studies showed that inclusion of the soluble cytoplasmic domain of the b subunit substantially enhanced the binding affinity between delta  and F1 and compensated for loss of binding affinity caused by delta  mutations.

As target residues for mutational analysis we selected delta -Tyr-11 and delta -Ala-14 in helix 1 and delta -Asn-75 and delta -Val-79 in helix 5. The first evidence that these residues are at or close to the F1-binding site came from tryptophan substitutions in positions 11 and 79, whose fluorescence signals responded strongly to binding of F1 (Fig. 4, B and C). Further evidence came from the titrations in Figs. 5 and 6, and from competition binding experiments. Calculated binding affinity measurements (Kd values, see Table I) indicated that three of the four residues are directly involved in binding. The tyrosine side chain of residue delta -Tyr-11 contributes about 12 kJ/mol binding energy, possibly because of pi -pi or pi -cation interactions. Increasing the size of the side chain in position delta -Ala-14, either by itself (delta A14L mutant) or in combination with introduction of a negative charge (delta A14D mutant) reduces the affinity for F1 significantly, probably by affecting interprotein surface complementarity. The valine side chain in position delta -Val-79 contributes about 6 kJ/mol binding energy, very likely because of hydrophobic interactions. The lack of binding energy contribution of the side chain of residue delta -Asn-75 suggests this residue is not directly involved in binding. However, it might not necessarily be taken as an argument against its location at the F1-binding surface; in an analysis of the interaction between the human growth hormone and its receptor (26) it was found that only one-quarter of the residues at the protein-protein interface had a significant impact on the binding energy. Whereas this might be an extreme case, at many protein-protein interface amino acid side chains can be found that should have the potential to participate in binding, but in fact play no or only a very minor role (see examples summarized in Ref. 27).

Surprisingly, despite losses in F1-binding energy of up to 15 kJ/mol, all mutations of residues in helices 1 and 5 were still fully or nearly fully functional in vivo and in vitro. In a previous study (14) we determined that the alpha G29D mutation reduces the binding affinity between delta  and F1 moderately, corresponding to a loss in binding energy of about 7 kJ/mol. We ascribed the strong functional impairment of this mutant ATP synthase to the interruption of binding of delta  to F1. This led us to conclude that the stator resistance function was finely balanced. However, the results of the present study showed that much larger losses of binding energy between delta  and F1 were well tolerated. Thus, we must now conclude that not all of the binding energy necessary to affix the stator stalk to F1, to resist the elastic strain generated by rotational catalysis, must necessarily be derived from delta /F1 interactions. In all likelihood, interactions between the b subunits and delta  and/or F1 also contribute.

To explore this possibility we included the soluble cytoplasmic domain (bST34-156, Ref. 20) in delta  binding assays and found that it substantially decreased the Kd of binding of delta  to F1. Due to technical limitations of the fluorescence assays, absolute values for this Kd in presence of bST34-156 could not be obtained, but an enhancement of >= 500-fold in affinity was evident from the results, equivalent to an additional binding energy of >= 15 kJ/mol. The additional binding energy could come from interactions between b subunit and alpha  or beta , and/or from b-induced conformational changes in delta . Interestingly, the Kd for interaction between bST34-156 and isolated delta  (in the absence of F1) was 5-10 µM (20) but the Kd measured here for binding of bST34-156 in the presence of isolated delta  and delta -depleted F1 was 150 nM, indicating a considerable cooperativity between the stator subunits. Overall, the new data indicate that the wild-type stator stalk is "overengineered," i.e. it is equipped with excess binding energy. This might explain why only very few impairing point mutations in delta  have been found (23, 28).

The experiments presented here supplement and extend earlier studies on OSCP. A study using deletion mutants of bovine OSCP indicated that removal of the N-terminal 28 residues, corresponding approximately to all of helix 1 in E. coli delta , impaired binding of OSCP to F1 (29). Also a study of rat OSCP showed that the strongly conserved residue delta -Arg-85 (Arg or Lys in 95% of sequences, residue Arg-94 in OSCP) contributed significantly to F1-binding energy (30). This residue occurs in the loop following helix 5 (see Fig. 2). Interestingly, despite the loss of binding energy, the delta R85A and delta R85Q mutants were still functional in vitro (30), and the authors concluded, as we do here, that other interactions provide binding energy. In E. coli, the mutation delta R85Q reduced the membrane-bound ATPase activity, by 50%, but had little effect on growth characteristics (28).

The quantitative binding assays described here for wild-type and mutant delta  binding to delta -depleted F1 in the presence or absence of the soluble cytoplasmic domain of the b subunit will allow us in future work to assess further aspects of stator subunit interactions. The influence of specific residues of the b subunits on delta  binding affinity can readily be studied by mutagenesis, for example. In addition, we can now investigate, by mutagenesis and other techniques, the binding site for delta  on F1, which appears to consist to a significant extent of one or more of the extreme N termini of the three alpha  subunits, for which a high resolution structure is not yet available.

    ACKNOWLEDGEMENTS

We thank Alma Muharemagic and Christina DeVries for excellent technical assistance. We also thank Dr. Stanley D. Dunn (University of Western Ontario) for plasmid pJB3 and for helpful discussions.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM25349 (to A. E. S.).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.

Dagger To whom correspondence should be addressed. Tel.: 585-275-2777; Fax: 585-271-2683; E-mail: alan_senior@urmc.rochester.edu.

Published, JBC Papers in Press, January 29, 2003, DOI 10.1074/jbc.M212037200

2 E. coli residue numbers are used throughout.

    ABBREVIATIONS

The abbreviations used are: OSCP, oligomycin-sensitivity conferral protein (the mitochondrial homolog of E. coli delta ); bST34-156, soluble cytoplasmic domain of b subunit (20).

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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