F1F0-ATP synthase. Binding of delta subunit to a 22-residue peptide mimicking the N-terminal region of alpha subunit.
The stator in F(1)F(0)-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(0) and to alpha(3)beta(3) hexagon of F(1). 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 (K(d) = 130 nm), accounting for a major portion of the binding energy when delta-depleted F(1) and isolated delta bind together (K(d) = 1.4 nm). Stoichiometry of binding of alpha N1-22 to delta at saturation was 1/1, showing that in intact F(1)F(0)-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 F(1)-binding face of delta, peptide binding was impaired as was binding of delta-depleted F(1). 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 F(1)-binding face of delta, forming the alpha/delta interface.
Weber
Joachim
J
Department of Biochemistry and Biophysics, University of Rochester Medical Center, New York 14642, USA.
Muharemagic
Alma
A
Wilke-Mounts
Susan
S
Senior
Alan E
AE
D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D007477:Ions; D010455:Peptides; D006180:Proton-Translocating ATPases; D008274:Magnesium