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

doi:10.3969/j.issn.1674-8115.2022.04.007

Abstract

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

CLC Number:

Q518.2

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.

Figures/Tables 5

TrendMD

References 25

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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

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

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