Structure of the gamma-epsilon complex of cyanobacterial F-1-ATPase reveals a suppression mechanism of the gamma subunit on ATP hydrolysis in phototrophs

F-1-ATPase forms the membrane-associated segment of F0F1-ATP synthase - the fundamental enzyme complex in cellular bioenergetics for ATP hydrolysis and synthesis. Here, we report a crystal structure of the central F-1 subcomplex, consisting of the rotary shaft gamma subunit and the inhibitory epsilon subunit, from the photosynthetic cyanobacterium Thermosynechococcus elongatus BP-1, at 1.98 angstrom resolution. In contrast with their homologous bacterial and mitochondrial counterparts, the gamma subunits of photosynthetic organisms harbour a unique insertion of 35-40 amino acids. Our structural data reveal that this region forms a beta-hairpin structure along the central stalk. We identified numerous critical hydrogen bonds and electrostatic interactions between residues in the hairpin and the rest of the gamma subunit. To elaborate the critical function of this beta-hairpin in inhibiting ATP hydrolysis, the corresponding domain was deleted in the cyanobacterial F-1 subcomplex. Biochemical analyses of the corresponding alpha(3)beta(3)gamma complex confirm that the clinch of the hairpin structure plays a critical role and accounts for a significant interaction in the alpha(3)beta(3) complex to induce ADP inhibition during ATP hydrolysis. In addition, we found that truncating the beta-hairpin insertion structure resulted in a marked impairment of the interaction with the epsilon subunit, which binds to the opposite side of the gamma subunit from the beta-hairpin structure. Combined with structural analyses, our work provides experimental evidence supporting the molecular principle of how the insertion region of the gamma subunit suppresses F-1 rotation during ATP hydrolysis.