NMR structure of the amino-terminal domain from the Tfb1 subunit of TFIIH and characterization of its phosphoinositide and VP16 binding sites
NMR structure of the amino-terminal domain from the Tfb1 subunit of TFIIH and characterization of its phosphoinositide and VP16 binding sites
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DOI：
10.1021/bi050099s
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发表时间：
2005-05-31
期刊：
影响因子：
2.9
通讯作者：
Omichinski, JG
中科院分区：
文献类型：
--
作者：
Di Lello, P;Nguyen, BD;Omichinski, JG
通用转录因子 IIH (TFIIH) 通过其 p62 (Tfb1) 亚基与 TFIIE α 的羧基末端结构域之间的直接相互作用被募集至起始前复合体 (PIC)。 TFIIH 还被证明可以通过与相同的 p62 (Tfb1) 亚基相互作用而与许多转录激活蛋白相互作用。我们已经确定了酵母 TFIIH 的 Tfb1 亚基 (Tfb1 (1-115)) 氨基末端结构域的 NMR 溶液结构。与人类 p62 蛋白的相应结构域一样，Tfb1 (1-115) 包含 PH 结构域折叠，尽管两种功能同源蛋白之间的序列同一性水平较低。此外，我们还进行了体外结合研究，证明 Tfb1 和 p62 的 PH 结构域特异性结合单磷酸化肌醇 [PtdIns(5)P 和 PtdIns(3)P]。 NMR 化学位移图谱表明 Tfb1 (p62) 上的 PtdIns(5)P 结合位点位于 PH 结构域折叠的 β 链 β-β 7 形成的基本口袋中。有趣的是，PtdIns(5)P 结合位点的结构组成与原型 PH 结构域上磷酸肌醇的结合位点组成不同。我们还确定 Tfb1 和 p62 的 PH 结构域足以结合 VP16 的激活结构域。 NMR 化学位移图表明 Tfb1 (p62) PH 结构域内的 VP16 结合位点与 Tfb1 (p62) 上的 PtdIns(5)P 结合位点重叠。这些结果提供了有关 PH 结构域识别磷酸肌醇的新信息，并指出了磷酸肌醇在 VP16 调节中的潜在作用。
General transcription factor IIH (TFIIH) is recruited to the preinitiation complex (PIC) through direct interactions between its p62 (Tfb1) subunit and the carboxyl-terminal domain of TFIIE alpha. TFIIH has also been shown to interact with a number of transcriptional activator proteins through interactions with the same p62 (Tfb1) subunit. We have determined the NMR solution structure of the amino-terminal domain from the Tfb1 subunit of yeast TFIIH (Tfb1 (1-115)). Like the corresponding domain from the human p62 protein, Tfb1 (1-115) contains a PH domain fold despite a low level of sequence identity between the two functionally homologous proteins. In addition, we have performed in vitro binding studies that demonstrate that the PH domains of Tfb1 and p62 specifically bind to monophosphorylated inositides [PtdIns(5)P and PtdIns(3)P]. NMR chemical shift mapping demonstrated that the PtdIns(5)P binding site on Tfb1 (p62) is located in the basic pocket formed by beta-strands beta-beta 7 of the PH domain fold. Interestingly, the structural composition of the PtdIns(5)P binding site is different from the composition of the binding sites for phosphoinositides on prototypic PH domains. We have also determined that the PH domains from Tfb1 and p62 are sufficient for binding to the activation domain of VP16. NMR chemical shift mapping demonstrated that the VP16 binding site within the PH domain of Tfb1 (p62) overlaps with the PtdIns(5)P binding site on Tfb1 (p62). These results provide new information about the recognition of phosphoinositides by PH domains, and point to a potential role for phosphoinositides in VP16 regulation.