DNA-binding domain of myelin-gene regulatory factor: purification, crystallization and X-ray analysis
DNA-binding domain of myelin-gene regulatory factor: purification, crystallization and X-ray analysis
复制标题
髓磷脂基因调节因子的 DNA 结合域：纯化、结晶和 X 射线分析
DOI：
10.1107/s2053230x17007828
复制
发表时间：
2017
影响因子：
0.9
通讯作者：
Shi Ning
中科院分区：
文献类型：
--
作者：
Wu WenYu;Zhen Xiangkai;Shi Ning
在脊椎动物中枢神经系统中，包裹轴突的髓鞘对于动作电位的快速传导至关重要。髓磷脂基因调节因子（MRF）是最近发现的一种髓鞘形成所需的转录因子。MRF的丧失导致脱髓鞘疾病和运动学习缺陷。MRF是一种膜结合转录因子，其经历从内质网膜的自切割。MRF的N-末端含有作为同源三聚体起作用的DNA结合结构域（DBD）。在这项研究中，MRF DBD的克隆，纯化和结晶，以了解调控髓鞘基因转录的分子机制。随后将硒代甲硫氨酸引入晶体中以获得用于MRF DBD结构的相。天然的和硒代蛋氨酸标记的晶体分别显示出2.50和2.51 nm的衍射分辨率。 晶体属P321空间群，晶胞参数a = 104.0，B = 104.0，c = 46.7 °，α = 90，β = 90，γ = 120°。 计算的马修斯系数为3.04 3 Da-1，溶剂含量为59.5%，表明在不对称单元中存在一个MRF DBD分子。
The myelin sheath, which envelops axons in the vertebrate central nervous system, is crucial for the rapid conduction of action potentials. Myelin-gene regulatory factor (MRF) is a recently identified transcription factor that is required for myelin-sheath formation. Loss of MRF leads to demyelinating diseases and motor learning deficiency. MRF is a membrane-bound transcription factor that undergoes autocleavage from the endoplasmic reticulum membrane. The N-terminus of MRF contains a DNA-binding domain (DBD) that functions as a homotrimer. In this study, the MRF DBD was cloned, purified and crystallized in order to understand the molecular mechanism that regulates the transcription of myelin genes. Selenomethionine was subsequently introduced into the crystals to obtain the phases for the MRF DBD structure. The native and selenomethionine-labelled crystals exhibited diffraction to 2.50 and 2.51 Å resolution, respectively. The crystals belonged to space group P321 and the selenomethionine-labelled crystals had unit-cell parameters a = 104.0, b = 104.0, c = 46.7 Å, α = 90, β = 90, γ = 120°. The calculated Matthews coefficient was 3.04 Å3 Da−1 and the solvent content was 59.5%, indicating the presence of one MRF DBD molecule in the asymmetric unit.