Site-directed cross-linking of b to the alpha, beta, and a subunits of the Escherichia coli ATP synthase.
The b subunit dimer of the Escherichia coli ATP synthase, along with the delta subunit, is thought to act as a stator to hold the alpha(3)beta(3) hexamer stationary relative to the a subunit as the gammaepsilonc(9-12) complex rotates. Despite their essential nature, the contacts between b and the alpha, beta, 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 b(24-156), 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 F(1) sector or to complete F(1)F(0) was attempted. Cross-linking in both the full-length and truncated forms of b was obtained at positions 92 (to alpha and beta), and 109 and 110 (to alpha only). Mass spectrometric analysis of peptide fragments derived from the b(24-156)A92C cross-link revealed that cross-linking took place within the region of alpha between Ile-464 and Met-483. This result indicates that the b dimer interacts with the alpha subunit near a non-catalytic alpha/beta 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 F(0) 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 b(24-156)A92C and beta as well as b(24-156)I109C and alpha 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.
McLachlin
D T
DT
Department of Biochemistry, University of Western Ontario, London, Ontario, Canada N6A 5C1.
Coveny
A M
AM
Clark
S M
SM
Dunn
S D
SD
D019943:Amino Acid Substitution; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D003432:Cross-Linking Reagents; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011994:Recombinant Proteins; D006180:Proton-Translocating ATPases; D003545:Cysteine