Disulfide bridge formation influences ligand recognition by the ATAD2 bromodomain
Disulfide bridge formation influences ligand recognition by the ATAD2 bromodomain
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DOI：
10.1002/prot.25636
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发表时间：
2019-02-01
影响因子：
2.9
通讯作者：
Glass, Karen C.
中科院分区：
文献类型：
--
作者：
Gay, Jamie C.;Eckenroth, Brian E.;Glass, Karen C.
ATP酶家族，含AAA结构域的蛋白2（ATAD 2）具有C末端布罗莫结构域，其作为染色质阅读器结构域起作用，识别核小体内组蛋白尾部上的乙酰化赖氨酸。ATAD 2在许多癌症中过表达，其表达与患者预后不良相关，使其成为有吸引力的治疗靶点和潜在的生物标志物。我们解析了ATAD 2布罗莫结构域的晶体结构，发现它在乙酰赖氨酸结合口袋（Cys 1057-Cys 1079）的碱基附近含有二硫桥。定点突变显示，去除游离C-末端半胱氨酸（C1101）残基大大提高了ATAD 2布罗莫结构域在体外的溶解度。等温滴定量热法实验与Ellman测定结合证明，分子内二硫桥的形成负面影响配体结合亲和力，并改变ATAD 2布罗莫结构域与组蛋白H4 K5 ac肽以及小分子布罗莫结构域配体相互作用的热力学参数。分子动力学模拟表明，ATAD 2布罗莫结构域中二硫桥的形成不会改变乙酰赖氨酸结合口袋的折叠状态或柔性的结构。然而，在检查配体结合亲和力时，或在设计与该乙酰赖氨酸读取器模块相互作用的新溴结构域抑制剂化合物时，应考虑到这种独特的结构特征。
The ATPase family, AAA domain-containing protein 2 (ATAD2) has a C-terminal bromodomain, which functions as a chromatin reader domain recognizing acetylated lysine on the histone tails within the nucleosome. ATAD2 is overexpressed in many cancers and its expression is correlated with poor patient outcomes, making it an attractive therapeutic target and potential biomarker. We solved the crystal structure of the ATAD2 bromodomain and found that it contains a disulfide bridge near the base of the acetyllysine binding pocket (Cys1057-Cys1079). Site-directed mutagenesis revealed that removal of a free C-terminal cysteine (C1101) residue greatly improved the solubility of the ATAD2 bromodomain in vitro. Isothermal titration calorimetry experiments in combination with the Ellman's assay demonstrated that formation of an intramolecular disulfide bridge negatively impacts the ligand binding affinities and alters the thermodynamic parameters of the ATAD2 bromodomain interaction with a histone H4K5ac peptide as well as a small molecule bromodomain ligand. Molecular dynamics simulations indicate that the formation of the disulfide bridge in the ATAD2 bromodomain does not alter the structure of the folded state or flexibility of the acetyllysine binding pocket. However, consideration of this unique structural feature should be taken into account when examining ligand-binding affinity, or in the design of new bromodomain inhibitor compounds that interact with this acetyllysine reader module.