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

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10Å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。

Objective·To analyze the full-length protein structure of human-derived lysine acetyltransferase 7(KAT7)using cryoelectron 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 humanderived 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 negativestain 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 10Åwas 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.