上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

核小体重塑及组蛋白去乙酰化酶复合物的负染电镜结构分析

段昱娟, 黄晶   

  1. 上海交通大学医学院附属第九人民医院上海精准医学研究院
  • 收稿日期:2022-01-13 出版日期:2022-04-26 发布日期:2022-04-26
  • 通讯作者: 黄 晶，电子信箱：huangjing@shsmu.edu.cn。
  • 作者简介:段昱娟 （1997—），女，硕士生；电子信箱：yujuan_duan@sjtu.edu.cn。
  • 基金资助:
    国家自然科学基金 （32022036）。

Negative-stain electron microscopic study of the nucleosome remodeling and deacetylase comp

DUAN Yujuan, HUANG Jing   

  1. Shanghai Institute of Precision Medicine, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai200125, China
  • Received:2022-01-13 Online:2022-04-26 Published:2022-04-26
  • Contact: HUANG Jing, E-mail: huangjing@shsmu.edu.cn
  • Supported by:
    National Natural Science Foundation of China (32022036).

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摘要： 目的·利用负染电镜技术分析人源核小体重塑及组蛋白去乙酰化酶复合物（nucleosome remodeling and deacetylase complex,NuRD复合物）结构，获得人源NuRD复合物的轮廓信息。方法·将C端带有3×Flag标签的MBD3 （methyl-CpG binding domain protein 3）和N端带有10×His标签的GATAD2A （GATA zinc finger domain containing 2A）克隆至pMLink表达载体中，采用聚乙烯亚胺瞬时转染过表达的方式在人源Expi293F悬浮细胞里表达NuRD复合物中的蛋白质组分；依次通过Ni-NTA亲和层析、Flag （DYKDDDDK）标签亲和层析和Superose 6 Increase 5/150凝胶过滤层析分离纯化NuRD复合物；利用蛋白质印迹法（Western blotting）和液相色谱-串联质谱法（liquid chromatography-tandem mass spectrometry,LC-MS/MS）对复合物进行组分鉴定；利用负染电镜技术结合单颗粒重构技术研究NuRD复合物的空间结构；通过UCSF Chimera软件将蛋白质数据库（Protein Data Bank,PDB）中已有亚复合物的原子结构模型（7AO9,5FXY）与生成的结构模型进行自动匹配及比对，预测多个蛋白组分在负染结构模型中的定位。结果·利用两步亲和层析，成功富集了带有纯化标签的MBD3、GATAD2A蛋白及其他内源蛋白组分，通过进一步的凝胶过滤层析分离得到了均一性良好的复合物；通过Western blotting和LC-MS/MS鉴定，确认纯化得到的复合物为组分完整的人源NuRD复合物。利用负染电镜技术及单颗粒重构技术初步解析了NuRD复合物的空间结构，其整体轮廓特征明显，呈现为不对称的长条形；通过进一步的三维优化处理，最终获得了人源NuRD复合物分辨率约为17?（1?=0.1 nm）的初步三维结构模型；已有的亚复合物原子结构模型（PDB:7AO9,5FXY）与NuRD复合物的初步三维结构模型自动匹配后，初步确定了MTA1/2/3 （metastasis-associated protein 1/2/3）、 HDAC1/2 （histone deacetylase 1/2）、 RBBP4/7 （retinoblastoma-binding protein 4/7）及MBD2/3 （methyl-CpG-binding domain protein 2/3）蛋白亚基在NuRD复合物负染结构模型中的定位。结论·利用单颗粒重构技术搭建了人源NuRD复合物的低分辨率负染结构模型。

关键词: 核小体重塑及组蛋白去乙酰化酶复合物, 表观遗传调控, 去乙酰化, 负染电镜技术

Abstract:

Objective·To study the structure of human nucleosome remodeling and deacetylase complex（NuRD complex） with negative-stain electron microscopy to obtain the profile information of human NuRD complex. Methods·Full length MBD3（methyl-CpG binding domain protein 3） and GATAD2 A（GATA zinc finger domain containing 2 A） constructs were cloned into the pMLink vectors with an amino terminal 10×His tag and a carboxyl terminal 3×Flag affinity tag, respectively. Proteins were expressed in human Expi293 F cells grown in suspension cultures by using polyethylenimine as the transfection reagent, and were isolated via affinity purifications with Ni-NTA and anti-DYKDDDDK（Flag） G1 affinity resin sequentially. The complex was further purified by Superose 6 Increase 5/150 gel filtration chromatography to improve the homogeneity, identified by Western blotting and liquid chromatography-tandem mass spectrometry（LC-MS/MS）, and was then studied by negative-stain electron microscopy and single particle analysis. The existing atomic structural model of the subcomplex（7 AO9, 5 FXY） in Protein Data Bank（PDB） was automatically docked into the generated structural model by the UCSF Chimera software to predict the localization of multiple protein components in the structural model. Results·The Flag-tagged MBD3 and His-tagged GATAD2 A proteins could effectively pull down the other endogenous components of the NuRD complex through two-step affinity purifications. The high-purity complex was obtained by gel filtration chromatography and confirmed as the NuRD complex by LC-MS/MS and Western blotting identification. Preliminary study on the three-dimensional（3 D） structure of the NuRD complex was carried out with negative-stain electron microscopy and single particle analysis, which revealed that the NuRD complex was in the obvious shape of a long asymmetrical rod. The 3 D structural model of the human NuRD complex was finally obtained by further 3 D refinement at an overall resolution of 17 ?（1 ? =0.1 nm）. The existing atomic structural model（PDB: 7 AO9, 5 FXY） was automatically docked into the negative staining structural model, and the localizations of MTA1/2/3（metastasis-associated protein 1/2/3）, HDAC1/2（histone deacetylase 1/2）, RBBP4/7（retinoblastoma-binding protein 4/7） and MBD2/3（methyl-CpG-binding domain protein 2/3） subunits in the NuRD complex were preliminarily confirmed. Conclusion·A low-resolution negative-staining structural model of human NuRD complex is obtained by single particle analysis.

Key words: nucleosome remodeling and deacetylase（NuRD） complex, epigenetic regulation, deacetylation, negative-stain electron microscopy

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