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J Biol Chem. 2012 Nov 16;287(47):39327-37. doi: 10.1074/jbc.M112.411223. Epub 2012 Sep 24.

A c subunit with four transmembrane helices and one ion (Na+)-binding site in an archaeal ATP synthase: implications for c ring function and structure.

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Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe University Frankfurt/Main, 60438 Frankfurt, Germany.


The ion-driven membrane rotors of ATP synthases consist of multiple copies of subunit c, forming a closed ring. Subunit c typically comprises two transmembrane helices, and the c ring features an ion-binding site in between each pair of adjacent subunits. Here, we use experimental and computational methods to study the structure and specificity of an archaeal c subunit more akin to those of V-type ATPases, namely that from Pyrococcus furiosus. The c subunit was purified by chloroform/methanol extraction and determined to be 15.8 kDa with four predicted transmembrane helices. However, labeling with DCCD as well as Na(+)-DCCD competition experiments revealed only one binding site for DCCD and Na(+), indicating that the mature c subunit of this A(1)A(O) ATP synthase is indeed of the V-type. A structural model generated computationally revealed one Na(+)-binding site within each of the c subunits, mediated by a conserved glutamate side chain alongside other coordinating groups. An intriguing second glutamate located in-between adjacent c subunits was ruled out as a functional Na(+)-binding site. Molecular dynamics simulations indicate that the c ring of P. furiosus is highly Na(+)-specific under in vivo conditions, comparable with the Na(+)-dependent V(1)V(O) ATPase from Enterococcus hirae. Interestingly, the same holds true for the c ring from the methanogenic archaeon Methanobrevibacter ruminantium, whose c subunits also feature a V-type architecture but carry two Na(+)-binding sites instead. These findings are discussed in light of their physiological relevance and with respect to the mode of ion coupling in A(1)A(O) ATP synthases.

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