Interaction and stoichiometry of the peripheral stalk subunits NtpE and NtpF and the N-terminal hydrophilic domain of NtpI of Enterococcus hirae V-ATPase
Interaction and stoichiometry of the peripheral stalk subunits NtpE and NtpF and the N-terminal hydrophilic domain of NtpI of Enterococcus hirae V-ATPase
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DOI：
10.1074/jbc.m801772200
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发表时间：
2008-07-11
影响因子：
4.8
通讯作者：
Murata, Takeshi
中科院分区：
文献类型：
--
作者：
Yamamoto, Misaki;Unzai, Satoru;Murata, Takeshi
液泡型ATPase(V-ATPase)由一个可溶性催化区和一个由中心茎和少数外围茎连接的完整膜结构域组成。外周茎秆亚基的数量和排列仍然存在争议。Hirae肠球菌Na+转位V-ATPase的外周茎可能由NtpE和NtpF亚基(对应于真核V-ATPase的G亚基)以及NtpI的N端亲水结构域(对应于真核V-ATPase的a亚基)组成。在这里，我们纯化了NtpE、NtpF和NtpI的N端亲水结构域(NtpI(NTerm))作为单独的重组His标记蛋白，并通过一个标记亚基的下拉实验、CD光谱、表面等离子体共振和分析超速离心法检测了这三个亚基之间的相互作用。NtpI(NTerm)直接与NtpF结合，但不与NtpE结合。NtpE与NtpF结合紧密。NtpI(NTerm)与NtpE-F复合体的结合强度仅强于NtpF，表明NtpE增加了NtpI(NTerm)与NtpF的结合亲和力。纯化的NtpE-F-I-NTerm络合物呈单分散状态，分析超速离心法和小角X射线散射(SAXS)测定的分子质量表明该三元络合物的化学计量比为1：1：1。从小角X射线光谱分析数据得到的低分辨率结构模型显示出一个拉长的“L”形状。
The vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain and an integral membrane domain connected by a central stalk and a few peripheral stalks. The number and arrangement of the peripheral stalk subunits remain controversial. The peripheral stalk of Na+-translocating V-ATPase from Enterococcus hirae is likely to be composed of NtpE and NtpF(corresponding to subunit G of eukaryotic V-ATPase) subunits together with the N-terminal hydrophilic domain of NtpI (corresponding to subunit a of eukaryotic V-ATPase). Here we purified NtpE, NtpF, and the N-terminal hydrophilic domain of NtpI (NtpI(Nterm)) as separate recombinant His-tagged proteins and examined interactions between these three subunits by pulldown assay using one tagged subunit, CD spectroscopy, surface plasmon resonance, and analytical ultracentrifugation. NtpI(Nterm) directly bound NtpF, but not NtpE. NtpE bound NtpF tightly. NtpI(Nterm) bound the NtpE-F complex stronger than NtpF only, suggesting that NtpE increases the binding affinity between NtpI(Nterm) and NtpF. Purified NtpE-F-I-Nterm complex appeared to be monodisperse, and the molecular masses estimated from analytical ultracentrifugation and small-angle x-ray scattering (SAXS) indicated that the ternary complex is formed with a 1:1:1 stoichiometry. A low resolution structure model of the complex produced from the SAXS data showed an elongated "L" shape.