Rishikesan Sankaranarayanan, Manimekalai Malathy S S, Grüber Gerhard
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Republic of Singapore.
Biochim Biophys Acta. 2010 Oct;1798(10):1961-8. doi: 10.1016/j.bbamem.2010.06.012. Epub 2010 Jun 27.
Subunit G is an essential stalk subunit of the eukaryotic proton pump V(1)V(O) ATPase. Previously the structure of the N-terminal region, G(1)(-)(59), of the 13kDa subunit G was solved at higher resolution. Here solution NMR was performed to determine the structure of the recombinant C-terminal region (G(61)(-)(101)) of subunit G of the Saccharomyces cerevisiae V(1)V(O) ATPase. The protein forms an extended alpha-helix between residues 64 and 100, whereby the first five- and the last residues of G(61)(-)(101) are flexible. The surface charge distribution of G(61)(-)(101) reveals an amphiphilic character at the C-terminus due to positive and negative charge distribution at one side and a hydrophobic surface on the opposite side of the structure. The hydrophobic surface pattern is mainly formed by alanine residues. The alanine residues 72, 74 and 81 were exchanged by a single cysteine in the entire subunit G. Cysteines at positions 72 and 81 showed disulfide formation. In contrast, no crosslink could be formed for the mutant Ala74Cys. Together with the recently determined NMR solution structure of G(1)(-)(59), the presented solution structure of G(61)(-)(101) enabled us to present a first structural model of the entire subunit G of the S. cerevisiae V(1)V(O) ATPase.
亚基G是真核生物质子泵V(1)V(O) ATP酶的一个必需的柄部亚基。此前，已以更高分辨率解析了13kDa亚基G的N端区域G(1)(-)(59)的结构。在此，通过溶液核磁共振（NMR）来确定酿酒酵母V(1)V(O) ATP酶亚基G的重组C端区域（G(61)(-)(101)）的结构。该蛋白质在64至100位残基之间形成一个延伸的α螺旋，其中G(61)(-)(101)的前五个和最后一个残基是灵活的。G(61)(-)(101)的表面电荷分布显示，由于结构一侧的正负电荷分布以及另一侧的疏水表面，其C端具有两亲性。疏水表面模式主要由丙氨酸残基形成。在整个亚基G中，丙氨酸残基72、74和81被单个半胱氨酸取代。72位和81位的半胱氨酸形成了二硫键。相比之下，突变体Ala74Cys无法形成交联。结合最近确定的G(1)(-)(59)的NMR溶液结构，本文给出的G(61)(-)(101)溶液结构使我们能够提出酿酒酵母V(1)V(O) ATP酶整个亚基G的首个结构模型。
