Joshi S, Cao G J, Nath C, Shah J
Boston Biomedical Research Institute, Boston, Massachusetts 02114, USA.
Biochemistry. 1997 Sep 9;36(36):10936-43. doi: 10.1021/bi9704109.
Earlier studies on oligomycin sensitivity conferring protein (OSCP) of bovine mitochondrial ATP synthase (F1Fo) indicated that a deletion mutant form (CD-10), lacking the last 10 amino acid residues (K181-L190), was unable to bind to the Fo segment, or reconstitute energy-linked reactions in OSCP-depleted F1Fo complexes [Joshi et al. (1996) Biochemistry 35, 12094-12103]. So far as known, the K181-L190 region of all mammalian species of OSCP harbors four charged residues at positions 181, 184, 187, and 188, while secondary structure predictions suggest that the K178-M186 region has a high propensity to form a helix [Ovchinnikov et al. (1984) FEBS Lett. 166, 19-22; Higuti et al. (1993) Biochim. Biophys. Acta 1172, 311-314; Grinkevich et al. ( 1994) Biol. Membr. 11, 310-323; Engelbrecht et al. (1991) Z. Naturforsch., C: Biochem., Biophys., Biol.,Virol. 46, 759-764]. Present studies were undertaken to clarify the role of individual amino acids in the K181-L190 region in OSCP-stimulated energy coupling. Our data show that simultaneous replacements of all four charged residues by uncharged but polar glutamines, or of K181-R184 by apolar alanines, had no significant influence either on the total alpha-helix content of the mutant forms or on the ability of mutant OSCPs to couple energy-linked reactions. However, a substitution of the K181-M186 region by six proline residues led to complete loss in the coupling activity of the resultant mutant. A detailed analysis of the 6-proline mutant form revealed that the variant was indistinguishable from WT OSCP with respect to expression characteristics, affinity for S-Sepharose, and ability to interact with F1, but was unable to complex with the Fo segment. These studies suggest that the global protein structure was not destabilized. The helix potential prediction analyses showed that the 6-proline OSCP displayed a marked decrease in the helix-forming propensity in the region corresponding to residues 175-190. Quantitative CD analyses to measure helical content demonstrated that both of the mutant forms 6-proline-OSCP and CD-10 had a somewhat lower alpha-helical content compared to WT protein, while synthetic peptides corresponding in sequence to the K178-L190 region displayed a high propensity to form a helix. Taken together, these results suggest that the C-terminal end of OSCP encompasses an alpha-helix which is crucial for high-affinity interactions of the C-terminal end of this subunit with Fo in the F1Fo enzyme.
早期对牛线粒体ATP合酶（F1Fo）的寡霉素敏感性赋予蛋白（OSCP）的研究表明，一种缺失最后10个氨基酸残基（K181 - L190）的缺失突变体形式（CD - 10），无法与Fo片段结合，也不能在缺乏OSCP的F1Fo复合物中重建能量偶联反应[乔希等人（1996年）《生物化学》35卷，12094 - 12103页]。据目前所知，所有哺乳动物物种的OSCP的K181 - L190区域在181、184、187和188位含有四个带电荷的残基，而二级结构预测表明K178 - M186区域具有形成螺旋的高倾向性[奥夫钦尼科夫等人（1984年）《欧洲生物化学学会联合会快报》166卷，19 - 22页；日uti等人（1993年）《生物化学与生物物理学报》1172卷，311 - 314页；格林凯维奇等人（1994年）《生物膜》11卷，310 - 323页；恩格尔布雷希特等人（1991年）《自然科学》C辑：生物化学、生物物理学、生物学、病毒学46卷，759 - 764页]。目前的研究旨在阐明K181 - L190区域中单个氨基酸在OSCP刺激的能量偶联中的作用。我们的数据表明，将所有四个带电荷的残基同时替换为不带电荷但具有极性的谷氨酰胺，或者将K181 - R184替换为非极性的丙氨酸，对突变体形式的总α - 螺旋含量或突变型OSCP偶联能量偶联反应的能力均无显著影响。然而，将K181 - M186区域替换为六个脯氨酸残基导致所得突变体的偶联活性完全丧失。对6 - 脯氨酸突变体形式的详细分析表明，该变体在表达特征、对S - 琼脂糖的亲和力以及与F1相互作用的能力方面与野生型OSCP无差异，但无法与Fo片段形成复合物。这些研究表明整体蛋白质结构并未不稳定。螺旋倾向性预测分析表明，6 - 脯氨酸OSCP在对应于残基175 - 190的区域中形成螺旋的倾向性显著降低。用于测量螺旋含量的定量圆二色性分析表明，6 - 脯氨酸 - OSCP和CD - 10这两种突变体形式与野生型蛋白相比，α - 螺旋含量均略低，而与K178 - L190区域序列对应的合成肽具有形成螺旋的高倾向性。综上所述，这些结果表明OSCP的C末端包含一个α - 螺旋，该螺旋对于该亚基C末端与F1Fo酶中的Fo进行高亲和力相互作用至关重要。
