Cross-linking between helices within subunit a of Escherichia coli ATP synthase defines the transmembrane packing of a four-helix bundle.
Subunit a of F(1)F(0) ATP synthase is required in the H(+) transport driven rotation of the c-ring of F(0), the rotation of which is coupled to ATP synthesis in F(1). The three-dimensional structure of subunit a is unknown. In this study, Cys substitutions were introduced into two different transmembrane helices (TMHs) of subunit a, and the proximity of the thiol side chains was tested via attempted oxidative cross-linking to form the disulfide bond. Pairs of Cys substitutions were made in TMHs 2/3, 2/4, 2/5, 3/4, 3/5, and 4/5. Cu(+2)-catalyzed oxidation led to cross-link formation between Cys pairs L120C(TMH2) and S144C(TMH3), L120C(TMH2) and G218C(TMH4), L120C(TMH2) and H245C(TMH5), L120C(TMH2) and I246C(TMH5), N148C(TMH3) and E219C(TMH4), N148C(TMH3) and H245C(TMH5), and G218C(TMH4) and I248C(TMH5). Iodine, but not Cu(+2), was found to catalyze cross-link formation between D119C(TMH2) and G218C(TMH4). The results suggest that TMHs 2, 3, 4, and 5 form a four-helix bundle with one set of key functional residues in TMH4 (Ser-206, Arg-210, and Asn-214) located at the periphery facing subunit c. Other key residues in TMHs 2, 4, and 5, which were concluded previously to compose a possible aqueous access pathway from the periplasm, were found to locate to the inside of the four-helix bundle.
Schwem
Brian E
BE
Department of Biomolecular Chemistry, School of Medicine and Public Health University of Wisconsin, Madison, Wisconsin 53706, USA.
Fillingame
Robert H
RH
D019943:Amino Acid Substitution; D025243:Bacterial Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics; D003300:Copper; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007455:Iodine; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D021122:Protein Subunits
D003432:Cross-Linking Reagents; D021122:Protein Subunits; D003300:Copper; D007455:Iodine; D025243:Bacterial Proton-Translocating ATPases; D003545:Cysteine