THE SUBUNIT-DELTA SUBUNIT-B DOMAIN OF THE ESCHERICHIA-COLI F(1)F(0)ATPASE - THE B-SUBUNITS INTERACT WITH F1 AS A DIMER AND THROUGH THE DELTA-SUBUNIT

The delta and b subunits are both involved in binding the F-1, to the F-0, part in the Escherichia coli ATP synthase (ECF1F0,). The interact ion of the purified delta subunit and the isolated hydrophilic domain of the b subunit (b(sol),,,) has been studied here. Purified delta bin ds to b(sol),,, weakly in solution, as indicated by NMR studies and pr otease protection experiments. On F-1,, i.e. in the presence of ECF1-d elta,, delta, and b(sol),,, interact strongly, and a complex of ECF1 . b(sol), b(sol),,, can be isolated by native gel electrophoresis. Both delta subunit and b(sol),,, are protected from trypsin cleavage in th is complex. In contrast, the delta subunit is rapidly degraded by the protease when bound to ECF1, when b(sol),,, is absent. The interaction of b(sol),,, with ECF1, involves the C-terminal domain of delta as de lta((1-134)), cannot replace intact delta in the binding experiments. As purified, b(sol),,, is a stable dimer with 80% alpha helix. A monom eric form of b(sol),,, can be obtained by introducing the mutation A12 8D (Howitt, S. M., Rodgers, A. J.,W., Jeffrey, P. D., and Cox, G., B. (1996) J. Biol. Chem. 271, 7038-7042). Monomeric b(sol),,, has less cu helix, i.e. only 58%, is much more sensitive to trypsin cleavage than dimer, and unfolds at much lower temperatures than the dimer in circu lar dichroism melting studies, indicating a less stable structure. The b(sol),,, dimer, but not monomer, binds to delta in ECF1,. To examine whether subunit b is a monomor or dimer in intact ECF1F0,, CuCl2, was used to induce cross-link formation in the mutants bS60C, bQ104C, bA1 28C, bG131C, and bS146C. With the exception of bS60C, CuCl2, treatment resulted in formation of b subunit dimers in all mutants. Cross-linki ng yield was independent of nucleotide conditions and did not affect A TPase activity. These results show the b subunit to be dimeric for a l arge portion of the C terminus, with residues 124-131 likely forming a pair of parallel alpha helices.