Structural and activity characterization of human PHPT1 after oxidative modification.
Structural and activity characterization of human PHPT1 after oxidative modification.
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DOI：
10.1038/srep23658
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发表时间：
2016-04-01
影响因子：
4.6
通讯作者：
Stevens SM Jr
中科院分区：
文献类型：
--
作者：
Martin DR;Dutta P;Mahajan S;Varma S;Stevens SM Jr
磷酸组氨酸磷酸酶1 （PHPT1）是哺乳动物中唯一已知的磷酸组氨酸磷酸酶，可调节几种蛋白质的磷酸组氨酸水平，包括参与信号传导、脂质代谢和钾离子运输的蛋白质。虽然人类PHPT1 （hPHPT1）的高分辨率结构是可用的，并且已经报道了对底物结合和催化活性重要的残基，但对调节hPHPT1活性的翻译后修饰知之甚少。在这里，我们描述了暴露于活性氧过氧化氢（H2O2）时hPHPT1氧化的结构和功能影响。具体而言，采用液相色谱-串联质谱法定量hPHPT1氧化还原敏感残基的位点特异性氧化。本研究结果表明，H2O2暴露诱导hPHPT1在底物结合区域的残基Met95上选择性氧化。然而，明确的溶剂分子动力学模拟预测，Met95氧化对hPHPT1催化位点载子态的结构和动力学只有很小的影响，这表明如果Met95氧化改变了hPHPT1的活性，那么它将通过改变中间态的稳定性来实现。采用一种新的基于质谱的分析方法，我们确定h2o2诱导的氧化不会对hPHPT1功能产生负面影响；这一结果与通常认为蛋白质氧化是典型的功能丧失修饰的概念相反。
Phosphohistidine phosphatase 1 (PHPT1), the only known phosphohistidine phosphatase in mammals, regulates phosphohistidine levels of several proteins including those involved in signaling, lipid metabolism, and potassium ion transport. While the high-resolution structure of human PHPT1 (hPHPT1) is available and residues important for substrate binding and catalytic activity have been reported, little is known about post-translational modifications that modulate hPHPT1 activity. Here we characterize the structural and functional impact of hPHPT1 oxidation upon exposure to a reactive oxygen species, hydrogen peroxide (H2O2). Specifically, liquid chromatography-tandem mass spectrometry was used to quantify site-specific oxidation of redox-sensitive residues of hPHPT1. Results from this study revealed that H2O2 exposure induces selective oxidation of hPHPT1 at Met95, a residue within the substrate binding region. Explicit solvent molecular dynamics simulations, however, predict only a minor effect of Met95 oxidation in the structure and dynamics of the apo-state of the hPHPT1 catalytic site, suggesting that if Met95 oxidation alters hPHPT1 activity, then it will do so by altering the stability of an intermediate state. Employing a novel mass spectrometry-based assay, we determined that H2O2–induced oxidation does not impact hPHPT1 function negatively; a result contrary to the common conception that protein oxidation is typically a loss-of-function modification.