Structural basis of ATP hydrolysis mechanism of V1-ATPase motor

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Vacuolar ATPases (V-ATPases) function as proton pumps in various membrane systems within the cells, which are involved in a number of processes such as bone resorption and cancer metastasis. The hydrophilic V1 portion is known as a rotary motor, in which a central axis DF complex rotates inside a hexagonally arranged catalytic A3B3 complex using ATP hydrolysis energy, although the molecular mechanism is not well defined due to a lack of high-resolution structural information.We have established the in vitro expression, purification, and reconstitution of Enterococcus hirae V1-ATPase from the A3B3 and DF complexes. In this study, we solved the crystal structures of AMP-PNP-bound and Pi-bound V1-ATPase, and also characterized the several site-directed mutants. On the basis of these findings, we proposed the molecular mechanism of the rotary motor.