The redefined DNA-binding domain of human xeroderma pigmentosum complementation group A: production, crystallization and structure solution
The redefined DNA-binding domain of human xeroderma pigmentosum complementation group A: production, crystallization and structure solution
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重新定义的人色素性干皮病互补组 A 的 DNA 结合结构域：生产、结晶和结构解决方案
DOI：
10.1107/s2053230x18016990
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发表时间：
2019
影响因子：
0.9
通讯作者：
Qian Chengmin
中科院分区：
文献类型：
--
作者：
Lian Fu-Ming;Yang Xiangwei;Yang Wancai;Jiang Yong-Liang;Qian Chengmin
人着色性干皮病互补组A（XPA）是一种支架蛋白，其在DNA损伤验证和募集下游核酸内切酶以促进核苷酸切除修复中的DNA损伤修复中起重要作用。XPA 98 -219（残基98-219）已被鉴定为DNA结合结构域，并且在过去二十年中已被广泛研究。然而，最近的研究已经将DNA结合结构域重新定义为XPA 98 -239（残基98-239）;它发挥比XPA 98 -219显著更高的DNA结合亲和力，并且具有与全长蛋白质非常相似的结合亲和力。本文介绍了人XPA 98 -239的生产、结晶和结构解析。使用由pH 7.0的1.8M柠檬酸三铵组成的沉淀剂获得晶体。 原生X射线衍射数据和锌单波长异常衍射（SAD）数据分别收集到1.93和2.06 μ m的分辨率。 晶体属P3空间群，晶胞参数a = 67.1，B = 67.1，c = 35.6 °，γ = 120.0°。 晶体含量分析表明，在不对称单元中存在一个分子，对应于2.65马修斯系数为2.65 × 3 Da−1，溶剂含量为53.6%。 用AutoSol软件对初始相进行求解，并利用锌SAD自动建立结构模型。初始结构模型覆盖了不对称单元中142个残基中的119个，Rwork为22.15%，Rfree为25.82%。与先前获得的XPA的截短溶液NMR结构（残基98-210）相比，在该初始模型中包含19个残基的C-末端延伸（残基211-229，对应于用于增强DNA结合的重新定义的结构域中的20个额外C-末端残基中的10个）。XPA的原子坐标正在完善中。
Human xeroderma pigmentosum complementation group A (XPA) is a scaffold protein that plays significant roles in DNA-damage verification and in recruiting downstream endonucleases to facilitate the repair of DNA lesions in nucleotide-excision repair. XPA98–219 (residues 98–219) has been identified as a DNA-binding domain and has been extensively studied in the last two decades. However, the most recent studies have redefined the DNA-binding domain as XPA98–239 (residues 98–239); it exerts a remarkably higher DNA-binding affinity than XPA98–219 and has a binding affinity that is quite similar to that of the full-length protein. Here, the production, crystallization and structure solution of human XPA98–239 are described. Crystals were obtained using a precipitant composed of 1.8 M ammonium citrate tribasic pH 7.0. Native X-ray diffraction data and zinc single-wavelength anomalous diffraction (SAD) data were collected to 1.93 and 2.06 Å resolution, respectively. The crystals belonged to space group P3, with unit-cell parameters a = 67.1, b = 67.1, c = 35.6 Å, γ = 120.0°. Crystal-content analysis showed the presence of one molecule in the asymmetric unit, corresponding to a Matthews coefficient of 2.65 Å3 Da−1 and a solvent content of 53.6%. The initial phases were solved and the structure model was automatically built by zinc SAD using the AutoSol program. The initial structure model covered 119 of 142 residues in the asymmetric unit, with an Rwork of 22.15% and an Rfree of 25.82%. Compared with a previously obtained truncated solution NMR structure of XPA (residues 98–210), a 19-residue C-terminal extension (residues 211–229, corresponding to 10 of the 20 extra C-terminal residues in the redefined domain for enhanced DNA binding) was contained in this initial model. Refinement of the atomic coordinates of XPA is ongoing.