Site-directed cross-linking of b to the α, β, and a subunits of the Escherichia coli ATP synthase

Abstract

The b subunit dimer of the Escherichia coli ATP synthase, along with the δ subunit, is thought to act as a stator to hold the α3β3 hexamer stationary relative to the a subunit as the γεc9-12 complex rotates. Despite their essential nature, the contacts between b and the α, β, and a subunits remain largely undefined. We have introduced cysteine residues individually at various positions within the wild type membrane-bound b subunit, or within b24-156, a truncated, soluble version consisting only of the hydrophilic C-terminal domain. The introduced cysteine residues were modified with a photoactivatable cross-linking agent, and cross-linking to subunits of the F1 sector or to complete F1F0 was attempted. Cross- linking in both the full-length and truncated forms of b was obtained at positions 92 (to α and β), and 109 and 110 (to α only). Mass spectrometric analysis of peptide fragments derived from the b24-156A92C crosslink revealed that cross-linking took place within the region of a between Ile-464 and Met-483. This result indicates that the b dimer interacts with the a subunit near a non-catalytic α/β interface. A cysteine residue introduced in place of the highly conserved arginine at position 36 of the b subunit could be cross-linked to the a subunit of F0 in membrane-bound ATP synthase, implying that at least 10 residues of the polar domain of b are adjacent to residues of a. Sites of cross-linking between b24-156A92C and β as well as b24-156I109C and α are proposed based on the mass spectrometric data, and these sites are discussed in terms of the structure of b and its interactions with the rest of the complex.