Dimerization interactions of the b subunit of the Escherichia coli F1F0-ATPase.

Site-directed mutagenesis and N-terminal truncations were used to examine dimerization interactions in the b subunit of Escherichia coli F1F0-ATPase. Individual cysteine residues were incorporated into bsyn, a soluble form of the protein lacking the membrane-spanning N-terminal domain, in two main areas: the heptad repeat region and the hydrophobic region which begins at residue Val-124. The tendencies of these cysteine residues to form disulfide bonds with the corresponding cysteine in the bsyn dimer were tested using disulfide exchange by glutathione and air oxidation catalyzed by Cu2+. Within the heptad repeat region, only cysteines at residues 59 and 60, which occupy the b and c positions of the heptad repeat, showed significant tendencies to form disulfides, a result inconsistent with a coiled-coil model for bsyn. Mixed disulfide formation most readily occurred with the S60C + L65C and A61C + L65C pairs. Cysteines at positions 124, 128, 132, and 139 showed strong tendencies to form disulfides with their mates in the dimer, suggesting a parallel alpha-helical interaction between the subunits in this region. Deletion of residues N-terminal to either Glu-34 or Asp-53 had no apparent effect on dimerization as determined by sedimentation equilibrium, while deletion of all residues N-terminal to Lys-67 produced a monomeric form. These results imply that residues 53-66 but not 24-52 are essential for bsyn dimerization. Taken together the results are consistent with a model in which the two b subunits interact in more than one region, including a parallel alignment of helices containing residues 124-139.