Structure of the membrane domain of subunit b of the Escherichia coli F0F1ATP synthase

The structure of the N-terminal transmembrane domain (residues 1-34) of sub unit b of the Escherichia coli F0F1-ATP synthase has been solved by two-dim ensional H-1 NMR in a membrane mimetic solvent mixture of chloroform/methan ol/H2O (4:4:1), Residues 4-22 form an cu-helix, which is likely to span the hydrophobic domain of the lipid bilayer to anchor the largely hydrophilic subunit b in the membrane. The helical structure is interrupted by a rigid bend in the region of residues 23-26 with cu-helical structure resuming at Pro-27 at an angle offset by 20 degrees from the transmembrane helix. In na tive subunit b, the hinge region and C-terminal alpha-helical segment would connect the transmembrane helix to the cytoplasmic domain. The transmembra ne domains of the two subunit b in F-0 were shown to be close to each other by cross-linking experiments in which single Cys were substituted for resi dues 2-21 of the native subunit and b-b dimer formation tested after oxidat ion with Cu(II) (phenanthroline)(2). Cys residues that formed disulfide cro ss-links were found with a periodicity indicative of one face of an alpha-h elix, over the span of residues 2-18, where Cys at positions 2, 6, and 10 f ormed dimers in highest yield. A model for the dimer is presented based upo n the NMR structure and distance constraints from the cross-linking data. T he transmembrane Lu-helices are positioned at a 23 degrees angle to each ot her with the side chains of Thr 6, Gln-10, Phe-14, and Phe-17 at the interf ace between subunits, The change in direction of helical packing at the hin ge region may be important in the functional interaction of the cytoplasmic domains.