Crystal structures of the ATP-binding and ADP-release dwells of the V1 rotary motor

V 1 -ATPases are highly conserved ATP-driven rotary molecular motors found in various membrane systems. We recently reported the crystal structures for the Enterococcus hirae A 3 B 3 DF (V 1 ) complex, corresponding to the catalytic dwell state waiting for ATP hydrolysis. Here we present the crystal structures for two other dwell states obtained by soaking nucleotide-free V 1 crystals in ADP. In the presence of 20 μM ADP, two ADP molecules bind to two of three binding sites and cooperatively induce conformational changes of the third site to an ATP-binding mode, corresponding to the ATP-binding dwell. In the presence of 2 mM ADP, all nucleotide-binding sites are occupied by ADP to induce conformational changes corresponding to the ADP-release dwell. Based on these and previous findings, we propose a V 1 -ATPase rotational mechanism model. V 1 -ATPases are rotary molecular motors that are powered by ATP hydrolysis. Here, the authors report two of the missing rotary states of this protein complex, and perform biochemical analysis to investigate the binding mode of the nucleotides.