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

doi:10.3969/j.issn.1674-8115.2022.04.007

摘要/Abstract

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 GATAD2A (GATA zinc finger domain containing 2A) 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 Expi293F 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 (7AO9, 5FXY) 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 GATAD2A 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 (3D) 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 3D structural model of the human NuRD complex was finally obtained by further 3D refinement at an overall resolution of 17 ? (1 ?=0.1 nm). The existing atomic structural model (PDB: 7AO9, 5FXY) 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

中图分类号:

Q518.2

段昱娟, 黄晶. 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[J]. 上海交通大学学报（医学版）, 2022, 42(4): 455-463.

DUAN Yujuan, HUANG Jing. Negative-stain electron microscopic study of the nucleosome remodeling and deacetylase complex[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(4): 455-463.

图/表 5

图1 NuRD复合物的表达与纯化Note： A. Composition and domain organization of human NuRD complex. The NuRD complex comprised at least six subunits, and there were two or three paralogs for each subunit. The plasmids of MBD3 were constructed with Flag tag at the C-terminus and GATAD2A with His tag at the N-terminus for subsequent protein expression and purification. B. SDS-PAGE analysis of the purified NuRD complex derived sequentially from the Ni-NTA and anti-DYKDDDDK G1 affinity resin. The identities of the major polypeptides and protein size markers were indicated. C. Gel filtration chromatography profile of the NuRD complex on Superose 6 Increase 5/150 GL column. D. SDS-PAGE analysis of fractions across the major peak shown in C. PHD—plant homeodomain finger; BAH—bromo adjacent homology; ELM2—Egl-27 and MTA1 homology 2; SANT—switching defective protein 3 (Swi3),adaptor 2 (Ada2), nuclear receptor co-repressor (N-CoR), transcription factor (TF); ZnF—Zinc finger; R1/R2—RBBP4 recruitment domains; WD40—conserved tryptophan (W) and aspartic acid (D) residues and a repeat length of approximately 40 amino acids; CC—coiled coil.

Fig 1 Expression and purification of the NuRD complex

图2 纯化的NuRD复合物的Western blotting鉴定Note：A. The Coomassie-blue staining of the SDS-PAGE gel clearly showed that the final elution contained all major protein components of the NuRD complex. B?G. Through Western blotting analysis, each component of the NuRD complex was detected by using MTA1 rabbit polyclonal antibody (B), HDAC1 rabbit polyclonal antibody (C), RBBP4 rabbit monoclonal antibody (D), anti-Flag mouse polyclonal antibody (E), anti-His mouse polyclonal antibody (F) and CHD4 rabbit polyclonal antibody (G).

Fig 2 Western blotting identification of the purified NuRD complex

表1 纯化的NuRD复合物样品蛋白组分LC-MS/MS分析结果

Tab 1 Analysis of protein components in the purified NuRD complex by LC-MS/MS

Module	Description		Coverage	Peptide	PSM	Unique peptide	AA
Module	Full name	Protein	Coverage	Peptide	PSM	Unique peptide	AA
Nucleosome remodeling module	Transcriptional repressor p66-α	GATAD2A	71	44	412	41	633
	Methyl-CpG-binding domain protein 3	MBD3	75	27	240	25	291
	Chromodomain-helicase-DNA-binding protein 4	CHD4	58	91	189	74	1 912
	Chromodomain-helicase-DNA-binding protein 3	CHD3	21	27	42	13	2 000
	Chromodomain-helicase-DNA-binding protein 5	CHD5	14	20	41	2	1 954
	Transcriptional repressor p66-β	GATAD2B	6	4	23	1	593
	Methyl-CpG-binding domain protein 2	MBD2	18	5	9	3	411
Histone deacetylase module	Metastasis-associated protein MTA2	MTA2	73	41	142	37	668
	Histone-binding protein RBBP7	RBBP7	78	22	121	16	425
	Metastasis-associated protein MTA1	MTA1	72	47	119	40	715
	Histone deacetylase 2	HDAC2	62	21	107	13	488
	Histone deacetylase 1	HDAC1	56	21	93	13	482
	Histone-binding protein RBBP4	RBBP4	69	17	72	11	425
	Metastasis-associated protein MTA3	MTA3	57	28	64	20	594

图3 NuRD 复合物的负染电镜分析Note： A. A representative negative-staining electron micrograph image of the NuRD complex. B. Representative two-dimensional class averages obtained from electron microscope particles of the NuRD complex. C. Angular distributions of particles for the final reconstruction of the NuRD complex. D. The gold-standard FSC curve calculated between two halves of the NuRD complex dataset.

Fig 3 Negative-staining electron microscopy analysis of the NuRD complex

图4 NuRD复合物三维模型结构和多亚基的定位Note： A. Surface view of the electron micrograph density map of the NuRD complex shown in six orthogonal views. The three-dimensional model size of the NuRD complex is denoted on the left. B. The crystal structures of RBBP4 (5FXY) and the electron microscopy structure of MTA1-HDAC1-RBBP4-MBD2 (7AO9) are docked into corresponding mass of NuRD complex shown in three orthogonal views. MTA1 is colored in cyan, HDAC1 in green, RBBP4 in orange, and MBD2 in yellow.

Fig 4 Three-dimensional reconstruction of NuRD complex and localization of multiple domains

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