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Abstract:
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 withDCCDas well as Na+-DCCD competition experiments revealed only one binding site for DCCD and Na+, indicating that the mature c subunit of this A1AO 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 V1VO 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 A1AO ATP synthases.
