title: Individual interactions of the b subunits within the stator of the Escherichia coli ATP synthase.
abstract: FOF1 ATP synthases are rotary nanomotors that couple proton translocation across biological membranes to the synthesis/hydrolysis of ATP. During catalysis, the peripheral stalk, composed of two b subunits and subunit δ in Escherichia coli, counteracts the torque generated by the rotation of the central stalk. Here we characterize individual interactions of the b subunits within the stator by use of monoclonal antibodies and nearest neighbor analyses via intersubunit disulfide bond formation. Antibody binding studies revealed that the C-terminal region of one of the two b subunits is principally involved in the binding of subunit δ, whereas the other one is accessible to antibody binding without impact on the function of FOF1. Individually substituted cysteine pairs suitable for disulfide cross-linking between the b subunits and the other stator subunits (b-α, b-β, b-δ, and b-a) were screened and combined with each other to discriminate between the two b subunits (i.e. bI and bII). The results show the b dimer to be located at a non-catalytic α/β cleft, with bI close to subunit α, whereas bII is proximal to subunit β. Furthermore, bI can be linked to subunit δ as well as to subunit a. Among the subcomplexes formed were a-bI-α, bII-β, α-bI-bII-β, and a-bI-δ. Taken together, the data obtained define the different positions of the two b subunits at a non-catalytic interface and imply that each b subunit has a different role in generating stability within the stator. We suggest that bI is functionally related to the single b subunit present in mitochondrial ATP synthase.
authors.affiliation: Department of Microbiology, University of Osnabrück, 49069 Osnabrück, Germany.
authors.forename: Karsten
authors.initials: K
authors.lastname: Brandt
authors.forename: Sarah
authors.initials: S
authors.lastname: Maiwald
authors.forename: Brigitte
authors.initials: B
authors.lastname: Herkenhoff-Hesselmann
authors.forename: Kerstin
authors.initials: K
authors.lastname: Gnirß
authors.forename: Jörg-Christian
authors.initials: JC
authors.lastname: Greie
authors.forename: Stanley D
authors.initials: SD
authors.lastname: Dunn
authors.forename: Gabriele
authors.initials: G
authors.lastname: Deckers-Hebestreit
mesh_terms: D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D021122:Protein Subunits; D006180:Proton-Translocating ATPases; C101709:AtpH protein, E coli
keywords: ATP Synthase; Disulfide; Escherichia coli; F-ATPase; Membrane Proteins; Peripheral Stator Stalk; Protein Cross-linking