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F1F0-ATP Synthase

BINDING OF delta  SUBUNIT TO A 22-RESIDUE PEPTIDE MIMICKING THE N-TERMINAL REGION OF alpha  SUBUNIT*

Joachim WeberDagger, Alma Muharemagic, Susan Wilke-Mounts, and Alan E. Senior§

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

Received for publication, February 10, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The stator in F1F0-ATP synthase resists strain generated by rotor torque. In Escherichia coli the b2delta subunit complex comprises the stator, bound to subunit a in F0 and to alpha 3beta 3 hexagon of F1. Proteolysis and cross-linking had suggested that N-terminal residues of alpha  subunit are involved in binding delta . Here we demonstrate that a synthetic peptide consisting of the first 22 residues of alpha  ("alpha N1-22") binds specifically to isolated wild-type delta  subunit with high affinity (Kd = 130 nM), accounting for a major portion of the binding energy when delta -depleted F1 and isolated delta  bind together (Kd = 1.4 nM). Stoichiometry of binding of alpha N1-22 to delta  at saturation was 1/1, showing that in intact F1F0-ATP synthase only one of the three alpha  subunits is involved in delta  binding. When alpha N1-22 was incubated with delta  subunits containing mutations in helices 1 or 5 on the F1-binding face of delta , peptide binding was impaired as was binding of delta -depleted F1. Residues alpha 6-18 are predicted to be helical, and a potential helix capping box occurs at residues alpha 3-8. Circular dichroism measurements showed that alpha N1-22 had significant helical content. Hypothetically a helical region of residues alpha N1-22 packs with helices 1 and 5 on the F1-binding face of delta , forming the alpha /delta interface.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ATP synthesis by oxidative phosphorylation occurs on the enzyme F1F0-ATP synthase. In Escherichia coli the enzyme consists of eight different subunit types in stoichiometry alpha 3beta 3gamma delta epsilon ab2cn. Proton movement through the membrane sector of the complex, mediated by subunits a and c, is believed to generate rotation of subunits c, epsilon , and gamma , which collectively form the "rotor." In turn this rotation is believed to act on the catalytic sites, three in number, at alpha /beta interfaces of the alternating alpha 3beta 3 hexagon to generate ATP. A "stator," consisting of subunits b2delta , is necessary to resist the rotor strain. In the reverse direction, ATP hydrolysis in the catalytic sites drives rotation of the rotor, which then generates uphill transport of protons across the bacterial plasma membrane to form the electrochemical gradient essential for nutrient uptake, locomotion, and other functions. Again rotor strain must be resisted by the stator for efficient function. The mechanisms by which catalysis, proton gradient formation, and subunit rotation are functionally integrated are subjects of active investigation (1-4).

This report is concerned with the structure and function of the stator, a topic also of much current interest, which has been reviewed recently in Refs. 5 and 6. The stator (b2delta ) interacts with the alpha 3beta 3 catalytic unit via delta /F1 interactions and with the proton-translocating machinery via b2/a interactions. delta  and b2 interact together via their C-terminal regions. There may also be interaction between b2 and alpha  or beta  subunits. In two recent reports we have studied the binding of the delta  subunit to F1 (7, 8). Using novel tryptophan fluorescence assays, our work established quantitative parameters for delta  binding to F1, demonstrated that helices 1 and 5 of the N-terminal domain of the delta  subunit form the F1-binding surface on delta , and showed that the cytoplasmic domain of the b subunit has a very large effect on the affinity of delta  binding to F1. In this report we move to study the delta -binding surface on F1. delta  subunit (and its mitochondrial homolog oligomycin sensitivity conferral protein) is known from electron microscopy studies to bind at the "top" of F1 (9, 10). Proteolysis (11) and cross-linking (12) experiments have suggested that the extreme N-terminal residues of alpha  subunit could be involved in binding of delta . Removal of the first 15 residues of alpha  by trypsin or of the first 19 residues by chymotrypsin was sufficient to greatly reduce delta  binding to F1 (11). X-ray crystallography studies have not yet been able to determine the structure of these alpha  subunit residues (4, 13, 14).

In this work we took a direct approach and tested the binding of a synthetic peptide, consisting of alpha  subunit residues 1-22, to the isolated wild-type delta  subunit. We found that the peptide did bind to wild-type delta  with high affinity and stoichiometry of 1 mol/mol, producing the same change in fluorescence signal of natural residue delta -Trp-28 in wild-type delta  as was produced by binding of delta -depleted F1 to delta  subunit. Moreover fluorescence responses upon binding of the peptide to isolated mutant delta  subunits were similar to those produced upon binding of delta -depleted F1. The peptide showed alpha -helical structure by circular dichroism, indicating that a predicted helix at residues 6-18 of alpha  subunit may be important for binding of delta .

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Purification of delta  Subunit-- Purification of wild-type, mutant, and proteolytically cleaved delta  subunit (delta ') was as in Refs. 7 and 8.

Synthetic Peptides-- Peptide alpha N1-221 was purchased from United States Biological (immunological grade) (mass = 2562.0 Da). Fresh batches of peptide were dissolved daily in 0.3% NH3 solution in water and used for 1 day only. Experiments showed that, over longer time periods, precipitation occurred. Peptide concentration was determined by Lowry protein assay and agreed closely with that calculated from peptide weight.

Fluorescence Binding Assays-- Tryptophan fluorescence titrations were carried out as described in Refs. 7 and 8 with individual conditions given in the figure legends. Unless noted otherwise the buffer was 50 mM HEPES/NaOH, 5 mM MgSO4, pH 7.0. Binding-induced changes in Trp fluorescence were plotted versus peptide concentration, and from the resulting curves, Kd values were calculated by nonlinear regression. In all cases the fluorescence signal due to the peptide alone was negligible.

Circular Dichroism Measurements-- Measurements were made at 23 °C on a Jasco CD spectropolarimeter Model J710. Peptide was dissolved at 250 µM in 0.3% NH3 solution in water.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Binding of a Synthetic Peptide Comprised of Residues alpha 1-22 to Isolated Wild-type delta  Subunit-- As noted in the Introduction, there is evidence suggesting that the N-terminal region of alpha  subunit of F1 is involved in binding delta  subunit. In E. coli F1, the N-terminal amino acid of alpha  is Met with a free amino group (11). A synthetic peptide comprising residues alpha 1-22 (called "alpha N1-22") with free N and C termini was synthesized by a commercial source. The sequence is MQLNSTEISELIKQRIAQFNVV. When the peptide was mixed with isolated delta  subunit it was seen (Fig. 1A) that the fluorescence signal of the single Trp in delta  (delta -Trp-28) was considerably enhanced, by 50%, and blue-shifted by 4 nm. Exactly the same fluorescence changes were seen on addition of delta -depleted F1 (alpha 3beta 3gamma epsilon ) to isolated delta  subunit (Fig. 1A). Therefore the synthetic peptide mimicked delta -depleted F1 in binding to isolated delta  subunit. Using this fluorescence signal, we had established previously by titration that delta -depleted F1 and wild-type delta  interacted with Kd of 1.4 nM (7). In Fig. 1B we carried out a titration of alpha N1-22 peptide with fixed concentration of wild-type delta . It is seen that binding saturated at a maximal stoichiometry of 1 mol peptide/mol delta  subunit, and the Kd was 130 nM (mean of five experiments). Together these data demonstrate an unexpectedly tight and specific association between the peptide and delta .


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Fig. 1.   Enhancement of fluorescence of wild-type delta  subunit upon addition of alpha N1-22 peptide and determination of Kd of binding. A, fluorescence enhancement of signal of delta -Trp-28 in wild-type delta  subunit on addition of saturating amount of alpha N1-22 peptide compared with addition of delta -depleted F1. Dotted line, fluorescence of delta  subunit alone; open circles, delta  plus alpha N1-22 peptide; solid line, delta  plus delta -depleted F1 (fluorescence of F1 subtracted). B, titration of wild-type delta  subunit and delta ' with alpha N1-22. delta ' is the proteolytic fragment of delta  containing residues 1-135 only. lambda exc = 295 nm; lambda em = 325 nm. Filled circles, wild-type delta ; open circles, delta '.

A further experiment in Fig. 1B showed that binding of alpha N1-22 to delta ', a proteolytic fragment of delta  consisting of residues 1-135 only, which contains the N-terminal helical domain of delta  (15), occurred with the same binding stoichiometry and affinity. Therefore the peptide binds to the N-terminal domain of delta  just as F1 does (7).

Two other peptides were synthesized. One was "alpha N1-22Cam" in which the C-terminal Val residue of alpha N1-22 was amidated. This peptide proved very difficult to dissolve in aqueous buffer and was not further investigated. A second peptide was the 11-mer sequence GTQLSGGQKQR with free N and C termini. This peptide, from the ATP-binding cassette signature sequence of P-glycoprotein, with no resemblance to alpha N1-22, dissolved readily in water but produced no change whatsoever in fluorescence signal of isolated wild-type delta  subunit.

Effect of pH and Mg2+ Ions on Binding of Peptide alpha N1-22 to Wild-type delta  Subunit-- The experiments in Fig. 1 were performed at pH 7.0 to mimic physiological conditions. In previous work we had shown that binding of delta -depleted F1 to wild-type delta  subunit was both Mg2+- and pH-sensitive (7). We repeated these assays with alpha N1-22 peptide and wild-type delta , and the results are shown in Table I alongside the previous data for comparison. It is evident that binding of alpha N1-22 to delta  was not strongly Mg2+-sensitive in contrast to the situation with F1. There was a small pH sensitivity in absence of Mg2+ but much lower than with F1. Interestingly, however, at high pH (9.4) the two sets of data converged, and particularly in absence of Mg2+ we can conclude that, at pH 9.4, the N-terminal residues 1-22 of alpha  provide all of the binding energy for delta  binding. At lower pH and in presence of Mg2+ other interactions come into play with F1 present.


                              
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Table I
Effect of pH and Mg2+ ions on Kd of binding of alpha N1-22 peptide to wild-type delta  subunit
Kd values are given in µM. All values shown are means of at least duplicate assays. Buffers were 50 mM MES/NaOH, pH 6.0; 50 mM HEPES/NaOH, pH 7.0; 50 mM Tris/H2SO4, pH 8.0; and 50 mM glycine/NaOH, pH 9.0 and 9.4. Values for binding delta -depleted F1 to wild-type delta  (taken from Ref. 7) are shown for comparison. MES, 4-morpholine ethane sulfonic acid.

Binding of the Synthetic Peptide alpha N1-22 to Isolated Mutant delta  Subunits-- In Ref. 8 we introduced eight mutations on the F1-binding face of the delta  subunit. Two new Trp residues were introduced at positions delta -11 and delta -79. These proved valuable for monitoring binding of delta  to delta -depleted F1. Most of the mutations impaired binding affinity between delta -depleted F1 and delta . We used these mutants for further characterization of binding of peptide alpha N1-22 to delta . The titration curves are shown in Fig. 2, and the calculated Kd values are summarized in Table II. We have previously reported titration curves and Kd values for binding of delta -depleted F1 to each of the mutant delta  subunits; these values are also shown in Table II.


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Fig. 2.   Titration of mutant delta  subunits with alpha N1-22 peptide. A, delta Y11A (inverted triangles) and delta Y11W/W28L (upright triangles). B, delta A14D (squares) and delta A14L (diamonds). C, delta N75A (inverted triangles) and delta N75E (upright triangles). D, delta V79A (squares) and delta V79W/W28L (diamonds). Concentration of the delta  subunits was 2 µM. lambda exc = 295 nm; lambda em = 325 nm except for delta Y11W/W28L and delta V79W/W28L where lambda em = 360 nm.


                              
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Table II
Comparison of binding of alpha N1-22 peptide versus delta -depleted F1 to isolated wild-type and mutant delta  subunits
Kd values for delta -depleted F1 are taken from Refs. 7 and 8. Values for alpha N1-22 peptide were calculated from titrations of the type shown in Figs. 1 and 2. All values are means of duplicate or triplicate experiments.

In Fig. 2A we show the titrations of alpha N1-22 with delta Y11A and delta Y11W/delta W28L mutant subunits. In the former the fluorescence signal is that of the native delta -Trp-28; in the latter the signal is that of introduced delta -Trp-11 with the natural delta -Trp-28 mutated away (8). With delta Y11A, the fluorescence was enhanced as it is when delta -depleted F1 is added to delta Y11A (8); however, with delta Y11W/W28L the fluorescence was quenched, which is different to the substantial enhancement of fluorescence seen when delta -depleted F1 is added to delta Y11W/W28L (8). Due to limited solubility of the peptide in assay buffer, titration curves could not be extended beyond 30 µM peptide; however, we feel that the calculated Kd values in Table II are reasonably reliable. Fig. 2B shows titration of alpha N1-22 with delta A14L and delta A14D mutant delta  subunits. It is seen that there is no signal change, and therefore no Kd values can be assigned. This could indicate that no binding occurred under these conditions, but it may be noted that no signal change is seen when delta -depleted F1 is added to delta A14L and delta A14D mutants even under conditions where binding does occur (8). Fig. 2C shows titration of alpha N1-22 with the delta N75A and delta N75E mutants. Enhancement of the fluorescence signal, similar to when delta -depleted F1 is titrated with these mutant delta  subunits (8), was seen. Fig. 2D shows titration of alpha N1-22 with the delta V79A and delta V79W/W28L mutant delta  subunits. In the former case substantial enhancement of fluorescence is seen; in the latter case a quench of fluorescence occurs. Both situations mimic what is seen when delta -depleted F1 is added to these mutant subunits. It should also be noted that all of the fluorescence responses were accompanied by blue shifts on addition of alpha N1-22 as was seen on addition of delta -depleted F1. Therefore, in seven of eight mutants, addition of alpha N1-22 peptide elicited a fluorescence response similar to that for delta -depleted F1, the only exception being mutant delta Y11W/W28L where fluorescence was quenched rather than enhanced.

With wild-type and six of the mutant delta  subunits the Kd value for binding of peptide alpha N1-22 was higher than that for delta -depleted F1 (Table II, columns 2 and 3). (With the remaining two mutants, delta A14L and delta A14D, Kd could not be calculated due to lack of signal.) From the ratio of the Kd values (Table II, column 4) the Delta Delta G0 values (Table II, column 5) were calculated to be 10-20 kJ/mol.

Predicted and Measured Structure of the N-terminal Residues 1-22 of alpha  Subunit-- As noted in the Introduction the structure of the N-terminal residues 1-22 of the alpha  subunits in F1F0-ATP synthase is unknown. Several secondary structure prediction algorithms2 indicate that residues alpha 6-18 form an alpha -helix. Residues alpha 3-8 conform to the sequence of a typical helix capping box (17) with both H-bonding and hydrophobic elements. We analyzed the helicity of alpha N1-22 peptide by circular dichroism measurement (18). The results (Fig. 3) indicated helix content of 25%. Since short peptides in aqueous medium often do not form ordered structure (19, 20), this is a significant result.


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Fig. 3.   Circular dichroism spectrum of alpha N1-22 peptide. alpha N1-22 peptide was dissolved in 0.3% NH3 solution in water at 250 µM concentration.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This work establishes that the N-terminal 22 residues of alpha  subunit of F1 provide an important part of the binding surface for the delta  subunit on F1F0-ATP synthase and contribute substantial binding interactions to maintain stator function. Our conclusion is based on the following evidence. 1) The peptide alpha N1-22 consisting of the N-terminal 22 residues of alpha  subunit binds to wild-type delta  subunit with high affinity (Kd = 130 nM), implying specific interaction. 2) Upon binding alpha N1-22 produces the same changes in fluorescence signal of residue delta -Trp-28 (enhancement of signal and blue shift) as does binding of delta -depleted F1 to delta  subunit, showing that alpha N1-22 brings about the same environmental changes of delta -Trp-28. 3) An unrelated peptide produced no effect. 4) alpha N1-22 bound to six mutant delta  subunits with reduced affinity just as delta -depleted F1 does. These mutations were shown previously to be located on the F1-binding surface of delta . 5) The fluorescence response of inserted delta -Trp-79 upon binding of alpha N1-22 (quench of fluorescence and blue shift) was very similar to that seen with delta -depleted F1. With inserted delta -Trp-11, the fluorescence response was different (quench with peptide versus enhancement with delta -depleted F1); however, the decrease in binding affinity was of similar order of magnitude as in other mutants and wild type. Together the data establish that the alpha N1-22 peptide mimics F1 in its binding to delta  subunit.

The peptide alpha N1-22 bound to isolated delta  subunit with 1/1 stoichiometry at saturation. Since the stoichiometry of binding of delta  to F1 is 1/1 (7), this indicates that in intact F1F0-ATP synthase only one of the three alpha  subunits is involved in delta  subunit binding. Our work suggests that the N-terminal region of alpha  forms an alpha -helix. Previous work has shown that the F1-binding surface on delta  is composed of helices 1 and 5 of the N-terminal domain of delta  (8). Therefore we propose that in intact enzyme the alpha -helical N-terminal residues of one of the three alpha  subunits packs on this binding surface of delta , forming the alpha /delta interface. Within the proposed helical region of alpha  are several conserved residues, notably alpha -Glu-7, alpha -Ile-8, alpha -Leu-11, alpha -Phe-19, that might make specific interactions with delta . In this model, two of the alpha  subunit N termini would not be involved in delta  binding and would be available for other functions. The 20 S proteasome structure provides a possible analogy in which protruding helical N termini of the heptameric alpha  subunits adopt different spatial conformations and perform disparate functions (16).

The facts that the Kd of binding of alpha N1-22 to delta  was lower than that for delta -depleted F1 by 100-fold (Kd of 130 versus 1.4 nM) and also that the pH and Mg2+ sensitivity of binding was different in the case of the peptide indicate that other interactions between F1 and delta  also contribute to binding. However, it is clear from the surprisingly high affinity of binding seen with the peptide that the N-terminal 22 residues of alpha  contribute a major component (>= 75%) of the binding energy.

The ability to use synthetic peptides to study the stator function at the top of ATP synthase should greatly expedite future experimentation. One can, for example, readily study the effects of mutations or deletions of residues in the N-terminal region of alpha , a project that we have found difficult to approach by conventional procedures because genetic manipulations that disrupt stator function also interrupt assembly of the enzyme in cells. It should also prove facile to extend these studies to understanding the cooperative effect of the b subunit on binding of delta  subunit to F1 (8).

    ACKNOWLEDGEMENTS

We thank Christina DeVries for excellent technical assistance and Kirti Patel for assistance with CD measurements.

    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 Present address: Dept. of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430.

§ To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, University of Rochester Medical Center, Box 712, Rochester, NY 14642. Tel.: 585-275-2777; Fax: 585-271-2683; E-mail: alan_senior@urmc.rochester.edu.

Published, JBC Papers in Press, February 20, 2003, DOI 10.1074/jbc.C300061200

2 V. A. Eyrich and B. Rost, the META-PredictProtein server (cubic.bioc.columbia.edu/predictprotein/submit_meta.html).

    ABBREVIATIONS

The abbreviation used is: alpha N1-22, synthetic peptide consisting of residues 1-22 of F1F0-ATP synthase alpha  subunit.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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