title: Analysis of sequence determinants of F1Fo-ATP synthase in the N-terminal region of alpha subunit for binding of delta subunit.
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.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642, USA.
authors.forename: Joachim
authors.initials: J
authors.lastname: Weber
authors.forename: Alma
authors.initials: A
authors.lastname: Muharemagic
authors.forename: Susan
authors.initials: S
authors.lastname: Wilke-Mounts
authors.forename: Alan E
authors.initials: AE
authors.lastname: Senior
mesh_terms: D000595:Amino Acid Sequence; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D006180:Proton-Translocating ATPases / Q000737:chemistry*
chemical_list: D021122:Protein Subunits; D006180:Proton-Translocating ATPases