Department of Mathematical and Life Sciences, Hiroshima University, Hiroshima, Japan.
Biophys J. 2013 May 21;104(10):2222-34. doi: 10.1016/j.bpj.2013.04.007.
The intrinsically disordered region (IDR) of a protein is an important topic in molecular biology. The functional significance of IDRs typically involves gene-regulation processes and is closely related to posttranslational modifications such as phosphorylation. We previously reported that the Drosophila facilitates chromatin transcription (FACT) protein involved in chromatin remodeling contains an acidic ID fragment (AID) whose phosphorylation modulates FACT binding to nucleosomes. Here, we performed dynamic atomic force microscopy and NMR analyses to clarify how the densely phosphorylated AID masks the DNA binding interface of the high-mobility-group domain (HMG). Dynamic atomic force microscopy of the nearly intact FACT revealed that a small globule temporally appears but quickly vanishes within each mobile tail-like image, corresponding to the HMG-containing IDR. The lifespan of the globule increases upon phosphorylation. NMR analysis indicated that phosphorylation induces no ordered structure but increases the number of binding sites in AID to HMG with an adjacent basic segment, thereby retaining the robust electrostatic intramolecular interaction within FACT even in the presence of DNA. These data lead to the conclusion that the inhibitory effect of nucleosome binding is ascribed to the increase in the probability of encounter between HMG and the phosphorylated IDR.
蛋白质的无规则区域（IDR）是分子生物学中的一个重要课题。IDR 的功能意义通常涉及基因调控过程，并且与磷酸化等翻译后修饰密切相关。我们之前曾报道过，参与染色质重塑的果蝇促进染色质转录（FACT）蛋白含有酸性 ID 片段（AID），其磷酸化调节 FACT 与核小体的结合。在这里，我们进行了动态原子力显微镜和 NMR 分析，以阐明高度迁移率族域（HMG）中密集磷酸化的 AID 如何掩盖 DNA 结合界面。对几乎完整的 FACT 的动态原子力显微镜观察表明，在每个移动的长尾状图像内，一个小球体暂时出现但很快消失，对应于含有 IDR 的 HMG。磷酸化后，小球体的寿命增加。NMR 分析表明，磷酸化不会诱导有序结构，但会增加 AID 与相邻碱性片段的 HMG 的结合位点数量，从而在存在 DNA 的情况下保留 FACT 内强大的静电分子内相互作用。这些数据得出的结论是，核小体结合的抑制作用归因于 HMG 和磷酸化 IDR 之间相遇概率的增加。
