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

Derek T Mclachlin,Angela M Coveny,Sonya M Clark,Stanley D Dunn

doi:10.1074/jbc.m000375200

Abstract

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.

Highlights

Subunits from each sector contribute to the formation of two stalks that join F1 and F0
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
Cross-linking of A92C to F1—Previously, we expressed the hydrophilic domain of the b subunit, consisting of residues

Summary

Introduction

Subunits from each sector contribute to the formation of two stalks that join F1 and F0. Chemical cross-linking of purified ATP synthase [22] or F0 [23] indicates that a and b are proximal, and analysis of second-site revertants to a bG9D mutation identified residue 240 of the a subunit, within the fifth putative transmembrane helix [24, 25], as a site that may be close to b within the membrane [26]. When cross-linking of the soluble domain of b to a or b was observed, the mutation was inserted into the full-length b subunit for analysis in membrane-bound ATP synthase.

Results

Conclusion