The dimerization domain of the b subunit of the Escherichia coli F(1)F(0)-ATPase.
In this study a series of N- and/or C-terminal truncations of the cytoplasmic domain of the b subunit of the Escherichia coli F(1)F(0) ATP synthase were tested for their ability to form dimers using sedimentation equilibrium ultracentrifugation. The deletion of residues between positions 53 and 122 resulted in a strongly decreased tendency to form dimers, whereas all the polypeptides that included that sequence exhibited high levels of dimer formation. b dimers existed in a reversible monomer-dimer equilibrium and when mixed with other b truncations formed heterodimers efficiently, provided both constructs included the 53-122 sequence. Sedimentation velocity and (15)N NMR relaxation measurements indicated that the dimerization region is highly extended in solution, consistent with an elongated second stalk structure. A cysteine introduced at position 105 was found to readily form intersubunit disulfides, whereas other single cysteines at positions 103-110 failed to form disulfides either with the identical mutant or when mixed with the other 103-110 cysteine mutants. These studies establish that the b subunit dimer depends on interactions that occur between residues in the 53-122 sequence and that the two subunits are oriented in a highly specific manner at the dimer interface.
Revington
M
M
Department of Biochemistry, University of Western Ontario, London, Ontario N6A 5C1, Canada.
McLachlin
D T
DT
Shaw
G S
GS
Dunn
S D
SD
D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents; D019281:Dimerization; D004220:Disulfides / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D019906:Nuclear Magnetic Resonance, Biomolecular; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013388:Succinimides; D014461:Ultracentrifugation
D003432:Cross-Linking Reagents; D004220:Disulfides; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D013388:Succinimides; C035760:bis(sulfosuccinimidyl)suberate; D006180:Proton-Translocating ATPases