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Abstract

We have structurally characterized the c-ring from the thermoalkaliphilic Bacillus sp. strain TA2.A1 F₁F_o-ATP synthase. Atomic force microscopy imaging and cryo-electron microscopy analyses confirm previous mass spectrometric data indicating that this c-ring contains 13 c-subunits. The cryo-electron microscopy map obtained from two-dimensional crystals shows less closely packed helices in the inner ring compared to those of Na⁺-binding c₁₁ rings. The inner ring of α-helices in c₁₁ rings harbors a conserved GxGxGxGxG motif, with glycines located at the interface between c-subunits, which is responsible for the close packing of these helices. This glycine motif is altered in the c₁₃ ring of Bacillus sp. strain TA2.A1 to AxGxSxGxS, leading to a change in c–c subunit contacts and thereby enlarging the c-ring diameter to host a greater number of c-subunits. An altered glycine motif is a typical feature of c-subunit sequences in alkaliphilic Bacillus species. We propose that enlarged c-rings in proton-dependent F-ATP synthases may represent an adaptation to facilitate ATP synthesis at low overall proton-motive force, as occurs in bacteria that grow at alkaline pH.