Laboratory for Chemistry and Life Science, Tokyo Institute of Technology, Nagatsuta 4259-R1-8, Midori-Ku, Yokohama 226-8503, Japan; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Tokyo 102-0075, Japan.
Laboratory for Chemistry and Life Science, Tokyo Institute of Technology, Nagatsuta 4259-R1-8, Midori-Ku, Yokohama 226-8503, Japan.
Biochim Biophys Acta Bioenerg. 2018 May;1859(5):319-325. doi: 10.1016/j.bbabio.2018.02.004. Epub 2018 Feb 19.
F is a soluble part of FF-ATP synthase and performs a catalytic process of ATP hydrolysis and synthesis. The γ subunit, which is the rotary shaft of F motor, is composed of N-terminal and C-terminal helices domains, and a protruding Rossman-fold domain located between the two major helices parts. The N-terminal and C-terminal helices domains of γ assemble into an antiparallel coiled-coil structure, and are almost embedded into the stator ring composed of αβ hexamer of the F molecule. Cyanobacterial and chloroplast γ subunits harbor an inserted sequence of 30 or 39 amino acids length within the Rossman-fold domain in comparison with bacterial or mitochondrial γ. To understand the structure-function relationship of the γ subunit, we prepared a mutant F-ATP synthase of a thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1, in which the γ subunit is split into N-terminal α-helix along with the inserted sequence and the remaining C-terminal part. The obtained mutant showed higher ATP-hydrolysis activities than those containing the wild-type γ. Contrary to our expectation, the complexes containing the split γ subunits were mostly devoid of the C-terminal helix. We further investigated the effect of post-assembly cleavage of the γ subunit. We demonstrate that insertion of the nick between two helices of the γ subunit imparts resistance to ADP inhibition, and the C-terminal α-helix is dispensable for ATP-hydrolysis activity and plays a crucial role in the assembly of F-ATP synthase.
F 是 FF-ATP 合酶的可溶性部分，执行 ATP 水解和合成的催化过程。γ 亚基是 F 马达的旋转轴，由 N 端和 C 端螺旋结构域以及位于两个主要螺旋结构域之间的突出的 Rossman 折叠结构域组成。γ 的 N 端和 C 端螺旋结构域组装成反平行的螺旋卷曲结构，并几乎嵌入由 F 分子的αβ六聚体组成的定子环。与细菌或线粒体的γ 相比，蓝细菌和叶绿体的γ 亚基在 Rossman 折叠结构域内具有 30 或 39 个氨基酸长度的插入序列。为了了解 γ 亚基的结构-功能关系，我们制备了一种嗜热蓝细菌 Thermosynechococcus elongatus BP-1 的突变型 F-ATP 合酶，其中 γ 亚基沿着插入序列分裂成 N 端的α-螺旋和剩余的 C 端部分。获得的突变体显示出比含有野生型 γ 更高的 ATP 水解活性。与我们的预期相反，含有分裂 γ 亚基的复合物大多缺乏 C 端螺旋。我们进一步研究了 γ 亚基组装后的切割的影响。我们证明，在 γ 亚基的两个螺旋之间插入切口赋予对 ADP 抑制的抗性，并且 C 端的α-螺旋对于 ATP 水解活性是可有可无的，并在 F-ATP 合酶的组装中发挥关键作用。
