Department of Chemistry, University of Oxford, Oxford, United Kingdom.
Department of Biochemistry, University of Oxford, Oxford, United Kingdom.
Sci Rep. 2017 Mar 9;7:44068. doi: 10.1038/srep44068.
ATP synthases (ATPases) are enzymes that produce ATP and control the pH in the cell or cellular compartments. While highly conserved over different species, ATPases are structurally well-characterised but the existence and functional significance of many post-translational modifications (PTMs) is not well understood. We combined a range of mass spectrometric techniques to unravel the location and extent of PTMs in the chloroplast ATP synthase (cATPase) purified from spinach leaves. We identified multiple phosphorylation and acetylation sites and found that both modifications stabilise binding of ε and δ subunits. Comparing cross-linking of naturally modified cATPase with the in vitro deacetylated enzyme revealed a major conformational change in the ε subunit in accord with extended and folded forms of the subunit. Locating modified residues within the catalytic head we found that phosphorylated and acetylated residues are primarily on α/β and β/α interfaces respectively. By aligning along different interfaces the higher abundance acetylated residues are proximal to the regulatory sites while the lower abundance phosphorylation sites are more densely populated at the catalytic sites. We propose that modifications in the catalytic head, together with the conformational change in subunit ε, work in synergy to fine-tune the enzyme during adverse conditions.
ATP 合酶（ATPases）是产生 ATP 并控制细胞或细胞区室中 pH 值的酶。尽管在不同物种中高度保守，但 ATP 合酶在结构上具有很好的特征，但许多翻译后修饰（PTMs）的存在和功能意义尚不清楚。我们结合了一系列质谱技术，从菠菜叶片中纯化的叶绿体 ATP 合酶（cATPase）中揭示 PTM 的位置和程度。我们鉴定了多个磷酸化和乙酰化位点，并发现这两种修饰都稳定了 ε 和 δ 亚基的结合。比较天然修饰的 cATPase 与体外去乙酰化酶的交联，发现 ε 亚基发生了主要的构象变化，与亚基的延伸和折叠形式一致。在催化头部定位修饰的残基时，我们发现磷酸化和乙酰化残基主要位于 α/β 和 β/α 界面上。通过沿着不同的界面排列，丰度较高的乙酰化残基更接近调节位点，而丰度较低的磷酸化位点在催化位点上更密集。我们提出，催化头部的修饰以及 ε 亚基的构象变化，协同作用在不利条件下精细调节酶的功能。
