School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Republic of Singapore.
J Bioenerg Biomembr. 2012 Jun;44(3):341-50. doi: 10.1007/s10863-012-9442-3. Epub 2012 May 5.
The 95 kDa subunit a of eukaryotic V-ATPases consists of a C-terminal, ion-translocating part and an N-terminal cytosolic domain. The latter's N-terminal domain (~40 kDa) is described to bind in an acidification-dependent manner with cytohesin-2 (ARNO), giving the V-ATPase the putative function as pH-sensing receptor. Recently, the solution structure of the very N-terminal segment of the cytosolic N-terminal domain has been solved. Here we produced the N-terminal truncated form SCa₁₀₄₋₃₆₃ of the N-terminal domain (SCa₁₋₃₆₃) of the Saccharomyces cerevisiae V-ATPase and determined its low resolution solution structure, derived from SAXS data. SCa₁₀₄₋₃₆₃ shows an extended S-like conformation with a width of about 3.88 nm and a length of 11.4 nm. The structure has been superimposed into the 3D reconstruction of the related A₁A₀ ATP synthase from Pyrococcus furiosus, revealing that the SCa₁₀₄₋₃₆₃ fits well into the density of the collar structure of the enzyme complex. To understand the importance of the C-terminus of the protein SCa₁₋₃₆₃, and to determine the localization of the N- and C-termini in SCa₁₀₄₋₃₆₃, the C-terminal truncated form SCa₁₀₆₋₃₂₄ was produced and analyzed by SAXS. Comparison of the SCa₁₀₄₋₃₆₃ and SCa₁₀₆₋₃₂₄ shapes showed that the additional loop region in SCa₁₀₄₋₃₆₃ consists of the C-terminal residues. Whereas SCa₁₀₄₋₃₆₃ is monomeric in solution, SCa₁₀₆₋₃₂₄ forms a dimer, indicating the importance of the very C-terminus in structure formation. Finally, the solution structure of SCa₁₀₄₋₃₆₃ and SCa₁₀₆₋₃₂₄ will be discussed in terms of the topological arrangement of subunit a and cytoheisn-2 in V-ATPases.
真核生物 V-ATP 酶的 95 kDa 亚基 a 由一个 C 端、离子转运部分和一个 N 端胞质域组成。后者的 N 端结构域（~40 kDa）被描述为以酸化依赖的方式与细胞溶质蛋白 2（ARNO）结合，赋予 V-ATP 酶作为 pH 感应受体的假定功能。最近，已解决了胞质 N 端结构域的非常 N 端段的溶液结构。在这里，我们产生了酿酒酵母 V-ATP 酶 N 端结构域的 N 端截断形式 SCa₁₀₄₋₃₆₃（SCa₁₋₃₆₃），并根据 SAXS 数据确定了其低分辨率溶液结构。SCa₁₀₄₋₃₆₃ 显示出伸展的 S 样构象，宽度约为 3.88nm，长度为 11.4nm。该结构已被叠加到来自 Pyrococcus furiosus 的相关 A₁A₀ ATP 合酶的 3D 重建中，表明 SCa₁₀₄₋₃₆₃很好地适合于酶复合物的环结构的密度。为了理解蛋白质 SCa₁₋₃₆₃ 的 C 端的重要性，并确定 SCa₁₋₃₆₃ 中 N 和 C 端的定位，产生了 C 端截断形式 SCa₁₀₆₋₃₂₄ 并通过 SAXS 进行了分析。SCa₁₀₄₋₃₆₃ 和 SCa₁₀₆₋₃₂₄ 形状的比较表明，SCa₁₀₄₋₃₆₃ 中的额外环区由 C 端残基组成。虽然 SCa₁₀₄₋₃₆₃ 在溶液中是单体，但是 SCa₁₀₆₋₃₂₄ 形成二聚体，表明非常 C 端在结构形成中的重要性。最后，将根据 V-ATP 酶中亚基 a 和细胞溶质蛋白 2 的拓扑排列讨论 SCa₁₀₄₋₃₆₃ 和 SCa₁₀₆₋₃₂₄ 的溶液结构。
