Mutations in the dimerization domain of the b subunit from the Escherichia coli ATP synthase. Deletions disrupt function but not enzyme assembly.
The b subunit dimer of Escherichia coli ATP synthase serves essential roles as an assembly factor for the enzyme and as a stator during rotational catalysis. To investigate the functional importance of its coiled coil dimerization domain, a series of internal deletions including each individual residue between Lys-100 and Ala-105 (b(deltaK100)-b(deltaA105)), b(deltaK100-A103), and b(deltaK100-Q106) as well as a control b(K100A) missense mutation were prepared. All of the mutants supported assembly of ATP synthase, but all single-residue deletions failed to support growth on acetate, indicating a severe defect in oxidative phosphorylation, and b(deltaK100-Q106) displayed moderately reduced growth. The membrane-bound ATPase activities of these strains showed a related reduction in sensitivity to dicyclohexylcarbodiimide, indicative of uncoupling. Analysis of dimerization of the soluble constructs of b(deltaK100) and the multiple-residue deletions by sedimentation equilibrium revealed reduced dimerization compared with wild type for all deletions, with b(deltaK100-Q106) most severely affected. In cross-linking studies it was found that F1-ATPase can mediate the dimerization of some soluble b constructs but did not mediate dimerization of b(deltaK100) and b(deltaK100-Q106); these two forms also were defective in F1 binding analyses. We conclude that defective dimerization of soluble b constructs severely affects F1 binding in vitro, yet allows assembly of ATP synthase in vivo. The highly uncoupled nature of enzymes with single-residue deletions in b indicates that the b subunit serves an active function in energy coupling rather than just holding on to the F1 sector. This function is proposed to depend on proper, specific interactions between the b subunits and F1.
Cipriano
Daniel J
DJ
Department of Biochemistry, University of Western Ontario, London, Ontario N6A 5C1, Canada.
Wood
Kristi S
KS
Bi
Yumin
Y
Dunn
Stanley D
SD
D000595:Amino Acid Sequence; D025243:Bacterial Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D003432:Cross-Linking Reagents; D025243:Bacterial Proton-Translocating ATPases