Xiong H, Zhang D, Vik S B
Department of Biological Sciences, Southern Methodist University, Dallas, Texas 75275, USA.
Biochemistry. 1998 Nov 17;37(46):16423-9. doi: 10.1021/bi981522i.
Structural models of subunit epsilon of the ATP synthase from Escherichia coli have been determined recently by NMR [Wilkens et al. (1995) Nat. Struct. Biol. 2, 961-967] and by X-ray crystallography [Uhlin et al. (1997) Structure 5, 1219-1230], revealing a two-domain protein. In this study, six new epsilon mutants were constructed and analyzed: Y63A, D81A, T82A, and three truncated mutants, tr80(S), tr94(LAS), and tr117(AS). Seven mutants constructed previously were also analyzed: E31A, E59A, S65A, E70A, T77A, R58A, and D81A/R85A. Subunits were purified by isoelectric focusing from extracts of cells that overproduced these 13 mutants. F1 was prepared lacking subunit epsilon by immobilized-Ni affinity chromatography. Three mutants, E70A, S65A, and E31A, showed somewhat higher affinities and extents of inhibition than the wild type. Three mutants, T82A, R85A, and tr94(LAS), showed both lower affinities and extents of inhibition, over the concentration range tested. Two showed no inhibition, D81A and tr80(S). The others, T77A, Y63A, E59A, and tr117(AS), showed lower affinities than wild type, but the extents of inhibition were nearly normal. Results indicate that the C-terminal domain of subunit epsilon contributes to inhibition of ATP hydrolysis, but it is not necessary for ATP-driven proton translocation. Interactions with subunit gamma are likely to involve a surface containing residues S65, E70, T77, D81, and T82, while residues R85 and Y63 are likely to be important in the conformation of subunit epsilon.
最近通过核磁共振[威尔肯斯等人（1995年），《自然结构生物学》2，961 - 967]和X射线晶体学[乌林等人（1997年），《结构》5，1219 - 1230]确定了来自大肠杆菌的ATP合酶ε亚基的结构模型，揭示了一种双结构域蛋白。在本研究中，构建并分析了六个新的ε突变体：Y63A、D81A、T82A，以及三个截短突变体，tr80(S)、tr94(LAS)和tr117(AS)。还分析了之前构建的七个突变体：E31A、E59A、S65A、E70A、T77A、R58A和D81A/R85A。通过等电聚焦从过量表达这13个突变体的细胞提取物中纯化亚基。通过固定化镍亲和色谱法制备不含ε亚基的F1。在测试的浓度范围内，三个突变体E70A、S65A和E31A显示出比野生型略高的亲和力和抑制程度。三个突变体T82A、R85A和tr94(LAS)显示出较低的亲和力和抑制程度。两个突变体没有抑制作用，即D81A和tr80(S)。其他突变体T77A、Y63A、E59A和tr117(AS)显示出比野生型低的亲和力，但抑制程度接近正常。结果表明，ε亚基的C末端结构域有助于抑制ATP水解，但对于ATP驱动的质子转运不是必需的。与γ亚基的相互作用可能涉及一个包含残基S65、E70、T77、D81和T82的表面，而残基R85和Y63可能在ε亚基的构象中起重要作用。
