Structure analysis of membrane-reconstituted subunit c-ring of E. coli H+-ATP synthase by solid-state NMR.

The subunit c-ring of H(+)-ATP synthase (F(o) c-ring) plays an essential role in the proton translocation across a membrane driven by the electrochemical potential. To understand its structure and function, we have carried out solid-state NMR analysis under magic-angle sample spinning. The uniformly [(13)C, (15)N]-labeled F(o) c from E. coli (EF(o) c) was reconstituted into lipid membranes as oligomers. Its high resolution two- and three-dimensional spectra were obtained, and the (13)C and (15)N signals were assigned. The obtained chemical shifts suggested that EF(o) c takes on a hairpin-type helix-loop-helix structure in membranes as in an organic solution. The results on the magnetization transfer between the EF(o) c and deuterated lipids indicated that Ile55, Ala62, Gly69 and F76 were lined up on the outer surface of the oligomer. This is in good agreement with the cross-linking results previously reported by Fillingame and his colleagues. This agreement reveals that the reconstituted EF(o) c oligomer takes on a ring structure similar to the intact one in vivo. On the other hand, analysis of the (13)C nuclei distance of [3-(13)C]Ala24 and [4-(13)C]Asp61 in the F(o) c-ring did not agree with the model structures proposed for the EF(o) c-decamer and dodecamer. Interestingly, the carboxyl group of the essential Asp61 in the membrane-embedded EF(o) c-ring turned out to be protonated as COOH even at neutral pH. The hydrophobic surface of the EF(o) c-ring carries relatively short side chains in its central region, which may allow soft and smooth interactions with the hydrocarbon chains of lipids in the liquid-crystalline state.