A New Type of Na(+)-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif

The anaerobic bacterium Fusobacterium nucleatum uses glutamate decarboxylation to generate a transmembrane gradient of Na(+). Here, we demonstrate that this ion-motive force is directly coupled to ATP synthesis, via an F(1)F(o)-ATP synthase with a novel Na(+) recognition motif, shared by other human pathogens. Molecular modeling and free-energy simulations of the rotary element of the enzyme, the c-ring, indicate Na(+) specificity in physiological settings. Consistently, activity measurements showed Na(+) stimulation of the enzyme, either membrane-embedded or isolated, and ATP synthesis was sensitive to the Na(+) ionophore monensin. Furthermore, Na(+) has a protective effect against inhibitors targeting the ion-binding sites, both in the complete ATP synthase and the isolated c-ring. Definitive evidence of Na(+) coupling is provided by two identical crystal structures of the c(11) ring, solved by X-ray crystallography at 2.2 and 2.6 Å resolution, at pH 5.3 and 8.7, respectively. Na(+) ions occupy all binding sites, each coordinated by four amino acids and a water molecule. Intriguingly, two carboxylates instead of one mediate ion binding. Simulations and experiments demonstrate that this motif implies that a proton is concurrently bound to all sites, although Na(+) alone drives the rotary mechanism. The structure thus reveals a new mode of ion coupling in ATP synthases and provides a basis for drug-design efforts against this opportunistic pathogen.