The b and delta subunits of the Escherichia coli ATP synthase interact via residues in their C-terminal regions.
An affinity resin for the F1 sector of the Escherichia coli ATP synthase was prepared by coupling the b subunit to a solid support through a unique cysteine residue in the N-terminal leader. b24-156, a form of b lacking the N-terminal transmembrane domain, was able to compete with the affinity resin for binding of F1. Truncated forms of b24-156, in which one or four residues from the C terminus were removed, competed poorly for F1 binding, suggesting that these residues play an important role in b-F1 interactions. Sedimentation velocity analytical ultracentrifugation revealed that removal of these C-terminal residues from b24-156 resulted in a disruption of its association with the purified delta subunit of the enzyme. To determine whether these residues interact directly with delta, cysteine residues were introduced at various C-terminal positions of b and modified with the heterobifunctional cross-linker benzophenone-4-maleimide. Cross-links between b and delta were obtained when the reagent was incorporated at positions 155 and 158 (two residues beyond the normal C terminus) in both the reconstituted b24-156-F1 complex and the membrane-bound F1F0 complex. CNBr digestion followed by peptide sequencing showed the site of cross-linking within the 177-residue delta subunit to be C-terminal to residue 148, possibly at Met-158. These results indicate that the b and delta subunits interact via their C-terminal regions and that this interaction is instrumental in the binding of the F1 sector to the b subunit of F0.
McLachlin
D T
DT
Department of Biochemistry, University of Western Ontario, London, Ontario, Canada N6A 5C1.
Bestard
J A
JA
Dunn
S D
SD
D025181:ATP Synthetase Complexes; D001426:Bacterial Proteins / Q000378:metabolism; D001577:Benzophenones / Q000378:metabolism; D001667:Binding, Competitive; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents / Q000378:metabolism; D003488:Cyanogen Bromide / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D008301:Maleimides / Q000378:metabolism; D009097:Multienzyme Complexes / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000737:chemistry*; D011485:Protein Binding; D017421:Sequence Analysis; D014461:Ultracentrifugation
D001426:Bacterial Proteins; D001577:Benzophenones; D003432:Cross-Linking Reagents; D008301:Maleimides; D009097:Multienzyme Complexes; D010446:Peptide Fragments; C046109:benzophenone-4-maleimide; D025181:ATP Synthetase Complexes; D017856:Phosphotransferases (Phosphate Group Acceptor); D003488:Cyanogen Bromide