Substrate-induced Changes in Domain Interaction of Vacuolar H+-Pyrophosphatase
Substrate-induced Changes in Domain Interaction of Vacuolar H+-Pyrophosphatase
复制标题
DOI：
10.1074/jbc.m114.568139
复制
发表时间：
2015-01-09
影响因子：
4.8
通讯作者：
Pan, Rong-Long
中科院分区：
文献类型：
--
作者：
Hsu, Shen-Hsing;Lo, Yueh-Yu;Pan, Rong-Long
利用单分子原子力显微镜（smAFM）研究了绿豆（Vigna radiata）中一种独特的液泡型H+-焦磷酸酶（EC 3.6.1.1）的跨膜结构域相互作用。H+-焦磷酸酶是一种膜包埋的同源二聚体蛋白，含有单一类型的多肽，并将PPi水解与质子转运联系起来。每个亚基由16个跨膜结构域组成，两端面向管腔侧。在这项调查中，H+-焦磷酸酶重组到脂质双层在相同的方向，有效的捕鱼出膜的smAFM。重组的H+-焦磷酸酶在脂质双层中显示出真正的二聚体结构，并且每个单体的大小在长度上类似于4 nm，在宽度上类似于2nm，并且在突起高度上为± 1 nm。在提取的H+-焦磷酸酶的膜，力-距离曲线包含10个峰，获得并分配到不同的域。在焦磷酸盐，磷酸盐和亚氨二磷酸盐的存在下，相互作用曲线的数量分别改变为7，8和10，伴随着显着的修改力的强度。进一步替换底物结合残基以验证底物结合后这些结构域的变化。因此，提出了一个工作模型来显示在有和没有底物及其类似物的情况下H+-焦磷酸酶跨膜结构域之间的相互作用。
Single molecule atomic force microscopy (smAFM) was employed to unfold transmembrane domain interactions of a unique vacuolar H+-pyrophosphatase (EC 3.6.1.1) from Vigna radiata. H+-Pyrophosphatase is a membrane-embedded homodimeric protein containing a single type of polypeptide and links PPi hydrolysis to proton translocation. Each subunit consists of 16 transmembrane domains with both ends facing the lumen side. In this investigation, H+-pyrophosphatase was reconstituted into the lipid bilayer in the same orientation for efficient fishing out of the membrane by smAFM. The reconstituted H+-pyrophosphatase in the lipid bilayer showed an authentically dimeric structure, and the size of each monomer was similar to 4 nm in length,similar to 2 nm in width, and+1 nm in protrusion height. Upon extracting the H+-pyrophosphatase out of the membrane, force-distance curves containing 10 peaks were obtained and assigned to distinct domains. In the presence of pyrophosphate, phosphate, and imidodiphosphate, the numbers of interaction curves were altered to 7, 8, and 10, respectively, concomitantly with significant modification in force strength. The substrate-binding residues were further replaced to verify these domain changes upon substrate binding. A working model is accordingly proposed to show the interactions between transmembrane domains of H+-pyrophosphatase in the presence and absence of substrate and its analog.