赖氨酸乙酰转移酶7的冷冻电镜全长结构分析

doi:10.3969/j.issn.1674-8115.2023.09.004

上海交通大学学报（医学版） ›› 2023, Vol. 43 ›› Issue (9): 1099-1106.doi: 10.3969/j.issn.1674-8115.2023.09.004

• 论著 · 基础研究 • 上一篇

赖氨酸乙酰转移酶7的冷冻电镜全长结构分析

郑国培¹(), 曹骎², 沈键锋¹()

^1.上海交通大学医学院附属第九人民医院眼科，上海 200011
^2.上海交通大学生命科学技术学院Bio-X研究院，上海 200030

收稿日期:2023-02-26 接受日期:2023-04-10 出版日期:2023-09-28 发布日期:2023-09-28
通讯作者: 沈键锋 E-mail:zhengguopei@sjtu.edu.cn;jfshen@shsmu.edu.cn
作者简介:郑国培（1996—），男，硕士；电子信箱：zhengguopei@sjtu.edu.cn。
基金资助:
国家自然科学基金面上项目(81972667);上海交通大学医学院“双百人”项目(20191817)

Structural analysis of full-length lysine acetyltransferase 7 by cryo-electron microscopy

ZHENG Guopei¹(), CAO Qin², SHEN Jianfeng¹()

^1.Department of Ophthalmology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
^2.Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai 200030, China

Received:2023-02-26 Accepted:2023-04-10 Online:2023-09-28 Published:2023-09-28
Contact: SHEN Jianfeng E-mail:zhengguopei@sjtu.edu.cn;jfshen@shsmu.edu.cn
Supported by:
General Program of National Natural Science Foundation of China(81972667);“Two-hundred Talents” Program of Shanghai Jiao Tong University School of Medicine(20191817)

摘要/Abstract

摘要：

目的·利用冷冻电镜技术分析人源性赖氨酸乙酰转移酶7（lysine acetyltransferase 7，KAT7）的全长蛋白结构，获得人源KAT7的轮廓信息。方法·使用pGEX-4T1载体和人源KAT7全长基因构建重组蛋白表达质粒pGEX-4T1-GST-KAT7，在原核蛋白表达体系BL21（DE3）中表达KAT7蛋白，使用GST亲和层析获得GST-KAT7重组蛋白；在利用TEV蛋白酶去除GST蛋白标签后，通过HiLoad 16/600 Superdex 75 pg体积排阻色谱进一步分离纯化KAT7蛋白。将获取的蛋白样品利用蛋白质印迹法（Western blotting）对KAT7进行鉴定，使用负染电镜筛选样品并初步观察蛋白形貌；使用冷冻电镜收集数据，利用冷冻电镜数据分析软件CryoSparc挑选蛋白颗粒并分析KAT7全长蛋白的空间结构；通过UCSF Chimera软件将蛋白质数据库（Protein Data Bank，PDB）中KAT7的MYST结构域模型（5GK9）、AlphaFold预测模型与生成的结构模型进行匹配分析。结果·利用亲和层析成功纯化人源性KAT7的全长蛋白，并通过体积排阻色谱获得高纯度的KAT7蛋白；在通过Western blotting鉴定KAT7后，利用负染电镜、冷冻电镜及单颗粒重构技术初步解析了KAT7全长蛋白的空间结构，并通过三维优化处理获得了分辨率约为10 ?的初步三维结构模型；KAT7全长蛋白空间结构呈不规则的半环状，已有的MYST结构域模型（PDB：5GK9）可匹配入KAT7全长模型的C端部分，调整后的AlphaFold预测模型也可匹配KAT7全长结构模型。结论·利用冷冻电镜技术初步分析了人源性KAT7的全长蛋白空间结构模型。

关键词: 赖氨酸乙酰转移酶, 蛋白质翻译后修饰, 乙酰化, 冷冻电镜技术

Abstract:

Objective ·To analyze the full-length protein structure of human-derived lysine acetyltransferase 7 (KAT7) using cryo-electron microscopy (Cryo-EM) and to obtain the profile information of human-derived KAT7. Methods ·The recombinant protein expression plasmid pGEX-4T1-GST-KAT7 was constructed by using the pGEX-4T1 vector and the full-length gene of human-derived KAT7, and the KAT7 protein was expressed in the prokaryotic protein expression system BL21 (DE3). The GST-KAT7 recombinant protein was obtained by using GST affinity chromatography. After removing the GST protein tag with TEV protease, KAT7 was further isolated and purified by HiLoad 16/600 Superdex 75 pg volume exclusion chromatography. The obtained protein samples were identified by Western blotting, and the samples were screened. The protein morphology was observed under negative-stain electron microscopy, and data were collected by using Cryo-EM. The protein particles were selected and the spatial structure of the full-length KAT7 was analyzed with the Cryo-EM analysis software CryoSparc. The MYST structural domain model (5GK9) in the Protein Data Bank (PDB) and AlphaFold prediction model of KAT7 were matched with the generated structural model by UCSF Chimera software. Results ·The full-length protein of human-derived KAT7 was successfully purified by affinity chromatography, and high purity KAT7 was obtained by volume exclusion chromatography. After identifying KAT7 by Western blotting, the spatial structure of KAT7 full-length protein was initially resolved by Cryo-EM and single-particle reconstruction techniques, and a preliminary three-dimensional structure model with a resolution of about 10 ? was obtained by three-dimensional optimization. The spatial structure of KAT7 full-length protein was irregular and semi-loop-shaped, and the existing MYST domain model (PDB: 5GK9) can be matched into the C-terminal part of the KAT7 full-length model. The adjusted AlphaFold prediction model can also match the KAT7 full-length structure model. Conclusion ·A preliminary analysis of the spatial structure model of full-length protein of human-derived KAT7 is performed by using Cryo-EM.

Key words: lysine acetyltransferase, post-translational protein modification, acetylation, cryo-electron microscopy

中图分类号:

Q518.2

郑国培, 曹骎, 沈键锋. 赖氨酸乙酰转移酶7的冷冻电镜全长结构分析[J]. 上海交通大学学报（医学版）, 2023, 43(9): 1099-1106.

ZHENG Guopei, CAO Qin, SHEN Jianfeng. Structural analysis of full-length lysine acetyltransferase 7 by cryo-electron microscopy[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(9): 1099-1106.

图/表 3

图1 KAT7的表达与纯化Note： A. KAT7 recombinant protein expression plasmid construction. GST-KAT7 recombinant protein expression plasmid containing GST protein tag with TEV protease restriction site was constructed by using PGEX-4T1 plasmid. B. Expression and purification of GST-KAT7 protein. The recombinant protein GST-KAT7 was expressed by Escherichia coli. BL21 (DE3), and the protein was purified by using GST affinity chromatography, while expressing the GST tag alone was taken as a negative control. C. Purification of KAT7 protein using volume exclusion chromatography. The KAT7 protein after excision of the GST tag was purified by using volume exclusion chromatography HiLoad 16/600 Superdex 75 pg, and peak samples were collected. D. KAT7 samples were separated by SDS-PAGE and determined by Coomassie brilliant blue staining, and the gels were photographed by using visualizer. E. Western blotting assay was performed on KAT7 samples to determine the purity and specificity.

Fig 1 Expression and purification of KAT7

图2 KAT7的冷冻电镜分析Note： A. The representative negative-staining electron micrograph image of KAT7. B. The representative cryo-electron microscopy micrograph image of KAT7. C. Representative two-dimensional class averages obtained from electron microscopy particles of KAT7. D. Angular distributions of particles for the final reconstruction of KAT7. E. The FSC curves corresponding to the KAT7 three-dimensional structural model. The DC term is 0 Hz and is equivalent to the average of all the samples in the window.

Fig 2 Cryo-electron microscopy analysis of KAT7

图3 KAT7全长结构分析Note： A. Surface view of the electron micrograph density map of KAT7 shown in six orthogonal views. The three-dimensional model size of KAT7 is denoted on the left. B. The crystal structure of MYST motif (5GK9) was docked into corresponding mass of KAT7 shown in three orthogonal views. C. The AlphaFold prediction structure of KAT7 was docked into full-length KAT7 model. D. The N-terminal structural domain (amino acid residues 183?335, yellow) and MYST domain (amino acid residues 336?611, red) of the AlphaFold prediction model were docked to the full-length KAT7 model separately.

Fig 3 Full-length KAT7 structural analysis

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赖氨酸乙酰转移酶7的冷冻电镜全长结构分析

Structural analysis of full-length lysine acetyltransferase 7 by cryo-electron microscopy

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