裂殖酵母Shelterin五元复合物的电镜结构研究

doi:10.3969/j.issn.1674-8115.2022.03.005

摘要/Abstract

摘要：

目的·通过电子显微镜（电镜）和负染技术分析裂殖酵母端粒结合蛋白质Shelterin五元复合物（Rap1-Poz1-Tpz1-Ccq1-Pot1）的三维结构。方法·利用大肠埃希菌表达系统在体外重组和表达带有组氨酸-小分子泛素相关修饰物蛋白（histidine-small ubiquitin related modifier，His-SUMO）和谷胱甘肽巯基转移酶（glutathione S-transferase，GST）纯化标签的目的蛋白质，通过两步亲和层析（镍离子金属螯合亲和层析、谷胱甘肽琼脂糖凝胶亲和层析）以及一步凝胶过滤层析（Superose^TM6 10/300 GL）分离纯化目的蛋白质复合物。采用0.75%甲酸双氧铀对蛋白质样品进行染色，用120 kV透射电镜观察，放大倍数设置为92 000倍，收集颗粒分散性较好的负染照片。用EMAN2软件和Relion3.0软件，通过单颗粒重构技术研究裂殖酵母Shelterin复合物的三维结构，最后利用UCSF Chimera软件对裂殖酵母Shelterin复合物的重构模型进行分析。结果·经过两步亲和层析以及凝胶过滤层析获得纯度高、组分齐、均一性良好的重组裂殖酵母Shelterin五元复合物的蛋白质样品；利用120 kV透射电镜获得负染电镜照片83张，从中挑选18 659个蛋白质复合物颗粒进行三维模型构建，初步解析了该复合物的三维结构；将重构模型与已报道的蛋白质组分的高分辨率结构进行分子对接，确定各组分在复合物中的定位情况，揭示该复合物以二聚体的形式存在。结论·构建了裂殖酵母Shelterin五元复合物（Rap1-Poz1-Tpz1-Ccq1-Pot1）的低分辨率的三维模型。

关键词: 端粒, 端粒末端保护, Shelterin复合物, 电子显微镜, 负染

Abstract:

Objective·To study the structure of the Shelterin quinary complex (Rap1-Poz1-Tpz1-Ccq1-Pot1) in fission yeast Schizosaccharomyces pombe (S. pombe)by negative-staining and electron microscopy.

Methods·The Shelterin quinary complex was reconstituted via expression and purification of recombinant proteins in Escherichia coli with histidine-small ubiquitin-related modifier (His-SUMO) and glutathione S-transferase (GST) tags. The target protein complex was isolated via sequential Ni-NTA and glutathione sepharose affinity purification followed by gel filtration chromatography (Superose^TM6 10/300 GL). The protein samples were stained by 0.75% uranium formate, and then observed by 120 kV transmission electron microscope (TEM) with a magnification of 92 000. The micrographs with good particle dispersity were obtained and the 3D structure of Shelterin complex was reconstructed by single particle image analysis by using EMAN2 and Relion3.0. Finally, UCSF Chimera was used to analyze the reconstruction model of Shelterin complex.

Results·The recombinant S. pombe Shelterin quinary complex with high purity, component integrity and good homogeneity was obtained by using a tandem affinity purification scheme with Ni-NTA and GST-based chromatography. Eighty-three micrographs were collected through 120 kV TEM, in which 18 659 particles were picked for 3D model reconstruction. After that, the rough 3D structure of the complex was obtained. Molecular docking of the available crystal structures of Shelterin subcomplex into the reconstructed model revealed that the complex existed in a dimeric form and determined the positioning of each component in the complex.

Conclusion·The low-resolution 3D model of S. pombe Shelterin quinary complex (Rap1-Poz1-Tpz1-Ccq1-Pot1) is obtained.

Key words: telomere, telomere end-protection, Shelterin complex, electron microscopy, negative-staining

中图分类号:

Q518.2

鲁彦伽, 孙虹, 吴振芳, 雷鸣. 裂殖酵母Shelterin五元复合物的电镜结构研究[J]. 上海交通大学学报（医学版）, 2022, 42(3): 290-297.

LU Yanjia, SUN Hong, WU Zhenfang, LEI Ming. Electron microscopic study of Shelterin quinary complex structure in fission yeast[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(3): 290-297.

图/表 5

图1 裂殖酵母Shelterin复合物各组分重组质粒的构建Note： A. Modularized organization of Shelterin in fission yeast. B. Domain organization of the Shelterin quinary complex. Shaded areas indicate the identified bipartite interaction regions. C. Schematic diagram of construction of reconstitution of Shelterin complex in E. coli. OB—OB-fold domain; TRFH—telomeric repeat factors homology domain; BRCT—BRCA1 C-terminal domain; RCT—Rap1 C-terminal domain; CTD—C-terminal domain; Myb—Myb domain.

Fig 1 Construction of recombinant plasmids of each component of S. pombe Shelterin complex

表 1 缓冲液配方

Tab 1 Buffer recipes

Buffer name	NaCl/ （mmol·L^-1）	Tris-HCl/（mmol·L^-1）	Glycerol/ %	Imidazole/（mmol·L^-1）
Lysis buffer	500	25	10	‒
Wash buffer Ⅰ	150	25	‒	20
Wash buffer Ⅱ	150	25	‒	‒
Elution buffer Ⅰ	150	25	‒	250
FPLC buffer	150	25	‒	‒

图2 裂殖酵母Shelterin复合物的体外表达与纯化Note： A. Two-step affinity purification of Shelterin complex. L—lysate; S—supernatant; FT1—Ni-NTA flow through; W1—Ni-NTA buffer wash; E1—Ni-NTA elution; U—6 h ULP1 digestion; FT2—glutathione sepharose flow through; E2—supernatant after GST-3C digestion. B. Gel filtration chromatography profile of the Shelterin complex on SuperoseTM6 10/300 GL column. C. Fractions corresponding to the peaks in the gel-filtration profile examined by Coomassie blue staining of SDS-PAGE. The corresponding elution volumes (mL) are marked above each lane. Be—sample before SuperoseTM6 10/300 GL gel filtration chromatography.

Fig 2 Expression and purification of S. pombe Shelterin complex in vitro

图3 Shelterin复合物的负染电镜分析结果Note： A. A representative negative-staining (NS) image of the S. pombe Shelterin quinary complex (Rap1-Poz1-Tpz1-Ccq1-Pot1). Scale bar=100 nm. B. Representative reference-free 2D class averages of the particles of Shelterin complex extracted from NS images. C. 3D reconstruction of Shelterin complex shown in six orthogonal views. D. Angular distribution of the selected particles used for the reconstruction of Shelterin complex. The colours represent the number of particles used for 3D reconstruction at the projection angle. The closer the colour is to red, the more particles there are; the closer the colour is to blue, the less particles there are.

Fig 3 Negative-staining electron microscopy analysis of Shelterin complex

图4 Shelterin复合物三维重构模型和各亚基的分子对接情况Note： A. Gold standard Fourier shell correlation (FSC) curve of the final reconstructed electron micrograph map of Shelterin quinary complex. B. Volume size of the Shelterin complex. C. Docking of the atomic structures of Pot1OB3-Tpz1PIM, Rap1PBM-Poz1-Tpz1PBM, Tpz1CBM-Ccq1TAD, and Pot1OB1-OB2 complexes into the NS-electron microscopic structure of the Shelterin complex in four orthogonal views.

Fig 4 3D reconstruction of the Shelterin complex and docking of solved structures into the overall envelope

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