Open AccessInteracting helical faces of subunits a and c in the F 1 F o ATP synthase of Escherichia coli defined by disulfide cross-linking
Author(s) -
Weiping Jiang,
Robert H. Fillingame
Publication year - 1998
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.95.12.6607
Subject(s) - transmembrane domain , protein subunit , atp synthase , dimer , atp synthase gamma subunit , transmembrane protein , helix (gastropod) , stereochemistry , mutant , chemistry , escherichia coli , biochemistry , crystallography , biology , membrane , enzyme , atp hydrolysis , atpase , ecology , receptor , organic chemistry , snail , gene
Subunitsa andc of Fo are thought to cooperatively catalyze proton translocation during ATP synthesis by theEscherichia coli F1 Fo ATP synthase. Optimizing mutations in subunita at residues A217, I221, and L224 improves the partial function of thec A24D/c D61G double mutant and, on this basis, these three residues were proposed to lie on one face of a transmembrane helix of subunita , which then interacted with the transmembrane helix of subunitc anchoring the essential aspartyl group. To test this model, in the present work Cys residues were introduced into the second transmembrane helix of subunitc and the predicted fourth transmembrane helix of subunita . After treating the membrane vesicles of these mutants with Cu(1,10-phenanthroline)2 SO4 at 0°, 10°, or 20°C, stronga–c dimer formation was observed at all three temperatures in membranes of 7 of the 65 double mutants constructed, i.e., in thea S207C/c I55C,a N214C/c A62C,a N214C/c M65C,a I221C/c G69C,a I223C/c L72C,a L224C/c Y73C, anda I225C/c Y73C double mutant proteins. The pattern of cross-linking aligns the helices in a parallel fashion over a span of 19 residues with thea N214C residue lying close to thec A62C andc M65C residues in the middle of the membrane. Lessera–c dimer formation was observed in nine other double mutants after treatment at 20°C in a pattern generally supporting that indicated by the seven landmark residues cited above. Cross-link formation was not observed between helix-1 of subunitc and helix-4 of subunita in 19 additional combinations of doubly Cys-substituted proteins. These results provide direct chemical evidence that helix-2 of subunitc and helix-4 of subunita pack close enough to each other in the membrane to interact during function. The proximity of helices supports the possibility of an interaction between Arg210 in helix-4 of subunita and Asp61 in helix-2 of subunitc during proton translocation, as has been suggested previously.