Medical Research Council Laboratory of Molecular Biology, Cambridge, England.
Proc Natl Acad Sci U S A. 2012 Sep 25;109(39):15752-6. doi: 10.1073/pnas.1214176109. Epub 2012 Sep 12.
The tumor suppressor p53 is a homotetramer of 4 × 393 residues. Its core domain and tetramerization domain are linked and flanked by intrinsically disordered sequences, which hinder its full structural characterization. There is an outstanding problem of the state of the tetramerization domain. Structural studies on the isolated tetramerization domain show it is in a folded tetrameric conformation, but there are conflicting models from electron microscopy of the full-length protein, one of which proposes that the domain is not tetramerically folded and the tetrameric protein is stabilized by interactions between the N and C termini. Here, we present methyl-transverse relaxation optimized NMR spectroscopy (methyl-TROSY) investigations on the full-length and separate domains of the protein with its methionine residues enriched with (13)C to probe its quaternary structure. We obtained high-quality spectra of both the full-length tetrameric p53 and its DNA complex, observing the environment at 11 specific methyl sites. The tetramerization domain was as tetramerically folded in the full-length constructs as in the isolated domain. The N and C termini were intrinsically disordered in both the full-length protein and its complex with a 20-residue specific DNA sequence. Additionally, we detected in the interface of the core (DNA-binding) and N-terminal parts of the protein a slow conformational exchange process that was modulated by specific recognition of DNA, indicating allosteric processes.
肿瘤抑制因子 p53 是由 4 个 393 个残基组成的四聚体。其核心结构域和四聚化结构域相连，并被固有无序序列所包围，这阻碍了其充分的结构特征描述。其中存在一个关于四聚化结构域状态的突出问题。对分离的四聚化结构域的结构研究表明，它处于折叠的四聚体构象，但电子显微镜的全长蛋白质模型存在冲突，其中一个模型提出该结构域没有四聚折叠，四聚体蛋白质通过 N 端和 C 端之间的相互作用稳定。在这里，我们通过甲基横向弛豫优化的 NMR 光谱学（methyl-TROSY）对该全长和分离结构域进行了研究，其中蛋白的甲硫氨酸残基用 (13)C 标记以探测其四级结构。我们获得了全长四聚体 p53 及其 DNA 复合物的高质量光谱，观察了 11 个特定甲基位点的环境。四聚化结构域在全长构建体中与在分离的结构域中一样是四聚折叠的。全长蛋白质及其与 20 个残基特定 DNA 序列的复合物中，N 端和 C 端都是固有无序的。此外，我们在蛋白质的核心（DNA 结合）和 N 端部分的界面检测到一个缓慢的构象交换过程，该过程受到 DNA 的特异性识别的调节，表明存在变构过程。
