Site-directed Cross-linking of b to the α, β, anda Subunits of the Escherichia coli ATP Synthase

• Published in: The Journal of biological chemistry Vol. 275; no. 23; pp. 17571 - 17577
• Main Authors: McLachlin, Derek T., Coveny, Angela M., Clark, Sonya M., Dunn, Stanley D.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 09.06.2000
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M000375200

The b subunit dimer of theEscherichia coli ATP synthase, along with the δ subunit, is thought to act as a stator to hold the α3β3 hexamer stationary relative to thea subunit as the γεc9–12complex rotates. Despite their essential nature, the contacts betweenb 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 bsubunit, 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 ofb 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 cross-link revealed that cross-linking took place within the region of α between Ile-464 and Met-483. This result indicates that the b dimer interacts with the α 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 ofb 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.