Structural and functional characterization of a complex between the acidic transactivation domain of EBNA2 and the Tfb1/p62 subunit of TFIIH.
Structural and functional characterization of a complex between the acidic transactivation domain of EBNA2 and the Tfb1/p62 subunit of TFIIH.
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DOI：
10.1371/journal.ppat.1004042
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发表时间：
2014-03
期刊：
影响因子：
6.7
通讯作者：
Omichinski JG
中科院分区：
文献类型：
--
作者：
Chabot PR;Raiola L;Lussier-Price M;Morse T;Arseneault G;Archambault J;Omichinski JG
感染EB病毒（EBV）可导致许多人类疾病，包括霍奇金淋巴瘤和伯基特淋巴瘤。这些EBV相关疾病的发展与九种病毒潜伏蛋白的存在有关，包括核抗原2（EBNA 2）。EBNA 2蛋白通过其激活宿主和病毒基因转录的能力在EBV感染中起关键作用。作为该功能的一部分，EBNA 2通过与其C-末端反式激活结构域（C-terminal transactivation domain，C-terminal）的相互作用与几种宿主转录调节蛋白结合，包括通用转录因子IIH（TFIIH）的Tfb 1/p62（酵母/人）亚基和组蛋白乙酰转移酶CBP（CREB结合蛋白）/p300。在这篇手稿中，我们研究了EBNA 2（残基431-487）与TFIIH和CBP/p300的Tfb 1/p62亚基的酸性双链的相互作用，使用核磁共振（NMR）光谱，等温滴定量热仪（ITC）和酵母中的反式激活研究。NMR研究表明，EBNA 2结合到Tfb 1的普列克底物蛋白同源（PH）结构域（Tfb 1 PH）和残基448-471（EBNA 2448 -471）是必要的和充分的这种相互作用。Tfb 1 PH-EBNA 2448 -471复合物的NMR结构表征表明，EBNA 2的内在无序双螺旋与Tfb 1 PH形成9个残基的α-螺旋复合物。在该螺旋内，三个疏水氨基酸（Trp 458、Ile 461和Phe 462）与Tfb 1 PH进行一系列重要的相互作用，并且它们的重要性在使用EBNA 2突变体的ITC和反式激活研究中得到验证。此外，NMR研究表明，EBNA 2的相同区域也需要结合CBP/p300的KIX结构域。这项研究提供了一个原子水平上的相互作用，涉及EBNA 2与靶宿主蛋白质的结合。此外，Tfb 1 PH-EBNA 2448 -471复合物与结合至Tfb 1 PH的p53和VP 16的结构的比较突出了固有无序的酸性TADs在识别共同靶宿主蛋白中的多功能性。EB病毒（EBV）感染与许多人类疾病有关，核抗原EBNA 2是在EBV相关疾病中起关键作用的九种病毒潜伏蛋白之一。EBNA 2部分地通过其C-末端酸性反式激活结构域（acidic transactivation domain，ETA 2）和许多宿主转录调节蛋白（包括通用转录因子IIH（general transcription factor IIH，TFIIH）和组蛋白乙酰转移酶CBP/p300）之间的相互作用来激活病毒和宿主基因的表达。在这份手稿中，我们证明了EBNA 2结合到pleckstrin同源（PH）结构域从Tfb 1/p62亚基的TFIIH和确定EBNA 2和Tfb 1/p62之间的复合物的三维结构。结构表明，EBNA 2的三个疏水残基在复合物界面处进行关键的相互作用，并且这些相同的残基在与CBP/p300的结合中也起重要作用。EBNA 2-Tfb 1复合物的结构与含有与相同Tfb 1/p62靶标结合的来自其他蛋白质（p53和VP 16）的酸性TADs的复合物的结构的比较突出了这些内在无序结构域的固有多功能性以及关键疏水残基定位的微小变化如何使它们使用不同的功能界面与共同靶标结合。
Infection with the Epstein-Barr virus (EBV) can lead to a number of human diseases including Hodgkin's and Burkitt's lymphomas. The development of these EBV-linked diseases is associated with the presence of nine viral latent proteins, including the nuclear antigen 2 (EBNA2). The EBNA2 protein plays a crucial role in EBV infection through its ability to activate transcription of both host and viral genes. As part of this function, EBNA2 associates with several host transcriptional regulatory proteins, including the Tfb1/p62 (yeast/human) subunit of the general transcription factor IIH (TFIIH) and the histone acetyltransferase CBP(CREB-binding protein)/p300, through interactions with its C-terminal transactivation domain (TAD). In this manuscript, we examine the interaction of the acidic TAD of EBNA2 (residues 431–487) with the Tfb1/p62 subunit of TFIIH and CBP/p300 using nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimeter (ITC) and transactivation studies in yeast. NMR studies show that the TAD of EBNA2 binds to the pleckstrin homology (PH) domain of Tfb1 (Tfb1PH) and that residues 448–471 (EBNA2448–471) are necessary and sufficient for this interaction. NMR structural characterization of a Tfb1PH-EBNA2448–471 complex demonstrates that the intrinsically disordered TAD of EBNA2 forms a 9-residue α-helix in complex with Tfb1PH. Within this helix, three hydrophobic amino acids (Trp458, Ile461 and Phe462) make a series of important interactions with Tfb1PH and their importance is validated in ITC and transactivation studies using mutants of EBNA2. In addition, NMR studies indicate that the same region of EBNA2 is also required for binding to the KIX domain of CBP/p300. This study provides an atomic level description of interactions involving the TAD of EBNA2 with target host proteins. In addition, comparison of the Tfb1PH-EBNA2448–471 complex with structures of the TAD of p53 and VP16 bound to Tfb1PH highlights the versatility of intrinsically disordered acidic TADs in recognizing common target host proteins. Infection with the Epstein-Barr virus (EBV) is linked to a number of human diseases and the nuclear antigen EBNA2 is one of nine viral latent proteins that plays a key role in EBV-linked diseases. EBNA2 activates expression of both viral and host gene in part through interaction between its C-terminal acidic transactivation domain (TAD) and a number of host transcriptional regulatory proteins including the general transcription factor IIH (TFIIH) and the histone acetyltransferase CBP/p300. In this manuscript, we demonstrate that the TAD of EBNA2 binds to the pleckstrin homology (PH) domain from the Tfb1/p62 subunit of TFIIH and determine a three-dimensional structure of a complex between EBNA2 and Tfb1/p62. The structure shows that three hydrophobic residues from the TAD of EBNA2 make key interactions at the complex interface and these same residues also play an important role in the binding to CBP/p300. Comparison of the structure of the EBNA2-Tfb1 complex with complexes containing acidic TADs from other proteins (p53 and VP16) bound to the same Tfb1/p62 target highlights the inherent versatility of these intrinsically disordered domains and how minor variations in positioning of key hydrophobic residues allows them to bind to common targets using different functional interfaces.
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影响因子：
2.9
作者：
Di Lello, P;Nguyen, BD;Omichinski, JG
通讯作者：
Omichinski, JG
影响因子：
3.7
作者：
FAHRAEUS, R;JANSSON, A;RYMO, L
通讯作者：
RYMO, L
影响因子：
64.8
作者：
HAMMERSCHMIDT, W;SUGDEN, B
通讯作者：
SUGDEN, B
影响因子：
2.9
作者：
Houtman, JCD;Higashimoto, Y;Samelson, LE
通讯作者：
Samelson, LE
DOI：
10.1107/s0907444998003254
发表时间：
1998-09-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
Brunger, AT;Adams, PD;Warren, GL
通讯作者：
Warren, GL