Structure of the membrane domain of subunit b of the Escherichia coli F0F1 ATP synthase.
The structure of the N-terminal transmembrane domain (residues 1-34) of subunit b of the Escherichia coli F0F1-ATP synthase has been solved by two-dimensional 1H NMR in a membrane mimetic solvent mixture of chloroform/methanol/H2O (4:4:1). Residues 4-22 form an alpha-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 alpha-helical structure resuming at Pro-27 at an angle offset by 20 degrees from the transmembrane helix. In native subunit b, the hinge region and C-terminal alpha-helical segment would connect the transmembrane helix to the cytoplasmic domain. The transmembrane domains of the two subunit b in F0 were shown to be close to each other by cross-linking experiments in which single Cys were substituted for residues 2-21 of the native subunit and b-b dimer formation tested after oxidation with Cu(II)(phenanthroline)2. Cys residues that formed disulfide cross-links were found with a periodicity indicative of one face of an alpha-helix, over the span of residues 2-18, where Cys at positions 2, 6, and 10 formed dimers in highest yield. A model for the dimer is presented based upon the NMR structure and distance constraints from the cross-linking data. The transmembrane alpha-helices are positioned at a 23 degrees angle to each other with the side chains of Thr-6, Gln-10, Phe-14, and Phe-17 at the interface between subunits. The change in direction of helical packing at the hinge region may be important in the functional interaction of the cytoplasmic domains.
Dmitriev
O
O
Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.
Jones
P C
PC
Jiang
W
W
Fillingame
R H
RH
D000595:Amino Acid Sequence; D003545:Cysteine / Q000235:genetics; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry; D017433:Protein Structure, Secondary; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics; D012997:Solvents / Q000737:chemistry
D008565:Membrane Proteins; D010446:Peptide Fragments; D012997:Solvents; D006180:Proton-Translocating ATPases; D003545:Cysteine