Stam Nicholas J, Wilkens Stephan
From the Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210.
From the Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210.
J Biol Chem. 2017 Feb 3;292(5):1749-1761. doi: 10.1074/jbc.M116.766790. Epub 2016 Dec 13.
Eukaryotic vacuolar H-ATPase (V-ATPase) is a multisubunit enzyme complex that acidifies subcellular organelles and the extracellular space. V-ATPase consists of soluble V-ATPase and membrane-integral V proton channel sectors. To investigate the mechanism of V-ATPase regulation by reversible disassembly, we recently determined a cryo-EM reconstruction of yeast V The structure indicated that, when V is released from V, the N-terminal cytoplasmic domain of subunit a (a) changes conformation to bind rotor subunit d However, insufficient resolution precluded a precise definition of the a-d interface. Here we reconstituted V into lipid nanodiscs for single-particle EM. 3D reconstructions calculated at ∼15-Å resolution revealed two sites of contact between a and d that are mediated by highly conserved charged residues. Alanine mutagenesis of some of these residues disrupted the a-d interaction, as shown by isothermal titration calorimetry and gel filtration of recombinant subunits. A recent cryo-EM study of holo V-ATPase revealed three major conformations corresponding to three rotational states of the central rotor of the enzyme. Comparison of the three V-ATPase conformations with the structure of nanodisc-bound V revealed that V is halted in rotational state 3. Combined with our prior work that showed autoinhibited V-ATPase to be arrested in state 2, we propose a model in which the conformational mismatch between free V and V functions to prevent unintended reassembly of holo V-ATPase when activity is not needed.
真核生物液泡H⁺-ATP酶（V-ATP酶）是一种多亚基酶复合体，可酸化亚细胞器和细胞外空间。V-ATP酶由可溶性V-ATP酶和膜整合V质子通道部分组成。为了研究V-ATP酶通过可逆拆卸进行调节的机制，我们最近确定了酵母V-ATP酶的冷冻电镜重建结构。该结构表明，当V从V-ATP酶释放时，亚基a（a）的N端胞质结构域会改变构象以结合转子亚基d。然而，分辨率不足妨碍了对a-d界面的精确定义。在这里，我们将V-ATP酶重组到脂质纳米盘中用于单颗粒电镜分析。在约15埃分辨率下计算的三维重建揭示了a和d之间由高度保守的带电残基介导的两个接触位点。对其中一些残基进行丙氨酸诱变破坏了a-d相互作用，等温滴定量热法和重组亚基的凝胶过滤分析表明了这一点。最近一项关于全酶V-ATP酶的冷冻电镜研究揭示了三种主要构象，对应于该酶中心转子的三种旋转状态。将三种V-ATP酶构象与纳米盘结合的V-ATP酶结构进行比较，发现V-ATP酶在旋转状态3时停止。结合我们之前的研究结果，即显示自抑制的V-ATP酶在状态2时停滞，我们提出了一个模型，其中游离的V和V-ATP酶之间的构象不匹配起到了防止在不需要活性时全酶V-ATP酶意外重新组装的作用。
