Structural basis of ATP hydrolysis mechanism of V1-ATPase motor

空泡ATPase(V-ATPase)在细胞内的各种膜系统中作为质子泵发挥作用，参与骨吸收和癌症转移等一系列过程。V1的亲水性部分被称为旋转马达，其中中心轴的DF复合体利用ATP的水解能在六角排列的催化A3B3复合体中旋转，但由于缺乏高分辨结构信息，其分子机制尚未很好地确定。我们从A3B3和DF复合体中建立了肠球菌Hirae V1-ATPase的体外表达、纯化和重组。在本研究中，我们解决了AMP-PNP结合和PI结合的V1-ATPase的晶体结构，并对几个定点突变体进行了鉴定。在此基础上，提出了旋转马达的分子机制。

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.