title: Second-site revertants of an arginine-210 to lysine mutation in the a subunit of the F0F1-ATPase from Escherichia coli: implications for structure.
abstract: Arg-210 of the a subunit of the Escherichia coli F0F1-ATPase has been proposed previously as a component of the proton pore. A mutant in which lysine was substituted for Arg-210 was generated and was found to be unable to translocate protons. A plasmid carrying this mutation, along with wild-type genes encoding the c and b subunits, was unusual in that it failed to complement a chromosomal c-subunit mutation on succinate minimal medium. Three revertants on succinate minimal medium contained plasmids that showed complementation with chromosomal c-subunit but not with a-subunit mutations. One of these had a deletion in the a subunit. The other two were point mutations, resulting in the substitution of aspartic acid by Gly-53 and of arginine for Leu-211. The Gly-53 to aspartic acid change implied that Gly-53 and Arg-210 are normally in close proximity. To test this idea further, a series of mutants in which aspartic acid was placed in helix I at positions ranging from 42 to 57 was generated. Full complementation was regained only when the aspartic acid residue was present on the same side of a putative helix as Gly-53 over a span of three turns of the alpha-helix. These results and others suggest modifications of a previously proposed model for the transmembrane helices of the F0 portion of the F0F1-ATPase. The implications of these modifications for the mechanism of proton translocation are discussed.
authors.affiliation: Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, Canberra.
authors.forename: S M
authors.initials: SM
authors.lastname: Howitt
authors.forename: G B
authors.initials: GB
authors.lastname: Cox
mesh_terms: D001120:Arginine / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D001693:Biological Transport, Active; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000648:ultrastructure; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D017433:Protein Structure, Secondary; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000648:ultrastructure; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D001120:Arginine; D006180:Proton-Translocating ATPases; D008239:Lysine