Mapping Interactions of the Intrinsically Disordered C-Terminal Regions of Tetrameric p53 by Segmental Isotope Labeling and NMR.
Mapping Interactions of the Intrinsically Disordered C-Terminal Regions of Tetrameric p53 by Segmental Isotope Labeling and NMR.
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DOI：
10.1021/acs.biochem.2c00528
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发表时间：
2022-12-06
期刊：
影响因子：
2.9
通讯作者：
Wright, Peter E.
中科院分区：
文献类型：
--
作者：
Krois, Alexander S.;Park, Sangho;Martinez-Yamout, Maria A.;Dyson, H. Jane;Wright, Peter E.
肿瘤抑制蛋白p53的c端区包含三个结构域，即核定位信号（NLS）、四聚化结构域（TET）和c端调控结构域（CTD），它们对p53的功能至关重要。全长p53中这些结构域的结构和相互作用的表征一直受到p53四聚体的总体大小和灵活性的限制。利用反式内嵌蛋白剪接，我们生成了全长p53构建体，其中c端区域用15N同位素标记用于NMR分析，使我们能够在全长蛋白的背景下获得c端结构域的原子水平信息。NLS和CTD残基的共振线宽较窄，表明这些区域主要是溶剂暴露和动态无序的，而折叠四聚域的共振线宽超出检测范围。CTD的两个区域，横跨369-374和381-388残基，赖氨酸含量高，与p53识别元件侧面区域的DNA进行动态和序列无关的相互作用。当侧翼区域的长度在识别元件的两侧延伸到大约20个碱基对时，dna结合状态的种群增加。利用含有TAZ2和乙酰转移酶结构域的转录共激活子CBP的结构，对K372、K373和K382进行乙酰化，抑制CTD与DNA的相互作用。这项工作提供了对全长四聚体内内在无序的p53 c端区域的行为和CTD介导DNA结合和特异性的分子基础的高分辨率见解。
The C-terminal region of the tumor suppressor protein p53 contains three domains, nuclear localization signal (NLS), tetramerization domain (TET), and C-terminal regulatory domain (CTD), that are essential for p53 function. Characterization of the structure and interactions of these domains within full-length p53 has been limited by the overall size and flexibility of the p53 tetramer. Using trans-intein splicing, we have generated full-length p53 constructs in which the C-terminal region is isotopically labeled with 15N for NMR analysis, allowing us to obtain atomic-level information on the C-terminal domains in the context of the full-length protein. Resonances of NLS and CTD residues have narrow linewidths, showing that these regions are largely solvent exposed and dynamically disordered, whereas resonances from the folded tetramerization domain are broadened beyond detection. Two regions of the CTD, spanning residues 369-374 and 381-388 and with high lysine content, make dynamic and sequence-independent interactions with DNA in regions that flank the p53 recognition element. The population of DNA-bound states increases as the length of the flanking regions is extended up to approximately 20 base pairs on either side of the recognition element. Acetylation of K372, K373, and K382, using a construct of the transcriptional coactivator CBP containing the TAZ2 and acetyltransferase domains, inhibits interaction of the CTD with DNA. This work provides high resolution insights into the behavior of the intrinsically disordered C-terminal regions of p53 within the full-length tetramer and the molecular basis by which the CTD mediates DNA binding and specificity.
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影响因子：
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作者：
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影响因子：
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DOI：
10.1073/pnas.1903077116
发表时间：
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影响因子：
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通讯作者：
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