Abstract
The stator in F(1)F(o)-ATP synthase resists strain generated by rotor torque. In Escherichia coli, the b(2)delta subunit complex comprises the stator, bound to subunit a in F(o) and to the alpha(3)beta(3) hexagon of F(1). Previous work has shown that N-terminal residues of alpha subunit are involved in binding delta. A synthetic peptide consisting of the first 22 residues of alpha (alphaN1-22) binds specifically to isolated wild-type delta subunit with 1:1 stoichiometry and high affinity, accounting for a major portion of the binding energy between delta and F(1). Residues alpha6-18 are predicted by secondary structure algorithms and helical wheels to be alpha-helical and amphipathic, and a potential helix capping box occurs at residues alpha3-8. We introduced truncations, deletions, and mutations into alphaN1-22 peptide and examined their effects on binding to the delta subunit. The deletions and mutations were introduced also into the N-terminal region of the uncA (alpha subunit) gene to determine effects on cell growth in vivo and membrane ATP synthase activity in vitro. Effects seen in the peptides were well correlated with those seen in the uncA gene. The results show that, with the possible exception of residues close to the initial Met, all of the alphaN1-22 sequence is required for binding of delta to alpha. Within this sequence, an amphipathic helix seems important. Hydrophobic residues on the predicted nonpolar surface are important for delta binding, namely alphaIle-8, alphaLeu-11, alphaIle-12, alphaIle-16, and alphaPhe-19. Several or all of these residues probably make direct interaction with helices 1 and 5 of delta. The potential capping box sequence per se appeared less important. Impairment of alpha/delta binding brings about functional impairment due to reduced level of assembly of ATP synthase in cells.
Highlights
The structure and function of the stator have been reviewed recently [8, 9]
An earlier report had used NMR to establish the structure of the N-terminal domain of ␦ subunit, which is composed of a six-helix bundle, and had shown that helices 1 and 5 form a hydrophobic groove [16]
In Ref. 17, we showed that a 22-residue synthetic peptide corresponding in sequence to the N-terminal residues of ␣ subunit with free N and C termini (␣N1–22)1 was able to bind to wild-type ␦ subunit with high affinity and specificity (Kd ϭ 130 nM) and with 1:1 stoichiometry, effectively mimicking the binding of intact F1 to ␦
Summary
Purification of ␦ Subunit, Purification of F1, Preparation of Membrane Vesicles, Assay of ATPase Activity, Assay of ATP-driven Proton Pumping in Membrane Vesicles, and Growth Yield Assays—These were as described previously [14, 15]. Point mutations in the uncA gene were generated by oligonucleotide-directed mutagenesis following previous methods [14] except that the template was M13mp containing the SphI-SalI fragment from pBWU13.4. (In intact ATP synthase, the N terminus of ␣ subunit is free Met.) For fluorescence binding assays, the peptides were dissolved at 10 mg/ml in dry Me2SO and used for 1 day only. Binding-induced changes in Trp fluorescence were plotted versus peptide concentration, and from the resulting curves, Kd values were calculated by nonlinear regression following the methods of Eftink [28], using the equation,. Where F is the measured fluorescence change at fixed ␦ concentration Eo and added peptide concentration Ltotal, n is the stoichiometry of binding, and m is a factor that converts fluorescence data to ligand bound (equals F at saturation divided by [Eo]). Values of n (stoichiometry of binding of peptide to ␦ subunit) were consistent with one peptide molecule binding per ␦ subunit molecule in all cases
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