Mechanical Modulation of ATP-binding Affinity of V1-ATPase

V₁-ATPase is a rotary motor protein that rotates the central shaft in a counterclockwise direction hydrolyzing ATP. Although the ATP-binding process is suggested to be the most critical reaction step for torque generation in F₁-ATPase (the closest relative of V₁-ATPase evolutionarily), the role of ATP binding for V₁-ATPase in torque generation has remained unclear. In the present study, we performed single-molecule manipulation experiments on V₁-ATPase from Thermus thermophilus to investigate how the ATP-binding process is modulated upon rotation of the rotary shaft. When V₁-ATPase showed an ATP-waiting pause, it was stalled at a target angle and then released. Based on the response of the V₁-ATPase released, the ATP-binding probability was determined at individual stall angles. It was observed that the rate constant of ATP binding (k_on) was exponentially accelerated with forward rotation, whereas the rate constant of ATP release (k_off) was exponentially reduced. The angle dependence of the k_off of V₁-ATPase was significantly smaller than that of F₁-ATPase, suggesting that the ATP-binding process is not the major torque-generating step in V₁-ATPase. When V₁-ATPase was stalled at the mean binding angle to restrict rotary Brownian motion, k_on was evidently slower than that determined from free rotation, showing the reaction rate enhancement by conformational fluctuation. It was also suggested that shaft of V₁-ATPase should be rotated at least 277° in a clockwise direction for efficient release of ATP under ATP-synthesis conditions.