Electron microscopic study of Shelterin quinary complex structure in fission yeast

doi:10.3969/j.issn.1674-8115.2022.03.005

Abstract

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

CLC Number:

Q518.2

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.

Figures/Tables 5

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

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	‒	‒

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

Fig 3 Negative-staining electron microscopy analysis of Shelterin complex

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

TrendMD

References 34

1	CECH T R. Beginning to understand the end of the chromosome[J]. Cell, 2004, 116(2): 273-279.
2	DE LANGE T. Shelterin: the protein complex that shapes and safeguards human telomeres[J]. Genes Dev, 2005, 19(18): 2100-2110.
3	DE LANGE T. Shelterin-mediated telomere protection[J]. Annu Rev Genet, 2018, 52: 223-247.
4	CICCONI A, CHANG S. Shelterin and the replisome: at the intersection of telomere repair and replication[J]. Curr Opin Genet Dev, 2020, 60: 77-84.
5	BHARI V K, KUMAR D, KUMAR S, et al. Shelterin complex gene: prognosis and therapeutic vulnerability in cancer[J]. Biochem Biophys Rep, 2021, 26: 100937.
6	LIM C J, CECH T R. Shaping human telomeres: from shelterin and CST complexes to telomeric chromatin organization[J]. Nat Rev Mol Cell Biol, 2021, 22(4): 283-298.
7	PATEL T N, VASAN R, GUPTA D, et al. Shelterin proteins and cancer[J]. Asian Pac J Cancer Prev, 2015, 16(8): 3085-3090.
8	AUGEREAU A, T'KINT DE ROODENBEKE C, SIMONET T, et al. Telomeric damage in early stage of chronic lymphocytic leukemia correlates with shelterin dysregulation[J]. Blood, 2011, 118(5): 1316-1322.
9	SASA G S, RIBES-ZAMORA A, NELSON N D, et al. Three novel truncating TINF2 mutations causing severe dyskeratosis congenita in early childhood[J]. Clin Genet, 2012, 81(5): 470-478.
10	SMITH E M, PENDLEBURY D F, NANDAKUMAR J. Structural biology of telomeres and telomerase[J]. Cell Mol Life Sci, 2020, 77(1): 61-79.
11	CHEN Y. The structural biology of the shelterin complex[J]. Biol Chem, 2019, 400(4): 457-466.
12	CHEN Y, YANG Y T, VAN OVERBEEK M, et al. A shared docking motif in TRF1 and TRF2 used for differential recruitment of telomeric proteins[J]. Science, 2008, 319(5866): 1092-1096.
13	HOFFMAN C S, WOOD V, FANTES P A. An ancient yeast for young geneticists: a primer on the Schizosaccharomyces pombe model system[J]. Genetics, 2015, 201(2): 403-423.
14	ALLSHIRE R C, EKWALL K. Epigenetic regulation of chromatin states in Schizosaccharomyces pombe[J]. Cold Spring Harb Perspect Biol, 2015, 7(7): a018770.
15	KANOH J, ISHIKAWA F. Composition and conservation of the telomeric complex[J]. Cell Mol Life Sci, 2003, 60(11): 2295-2302.
16	LIU J Q, YU C, HU X C, et al. Dissecting fission yeast shelterin interactions via MICro-MS links disruption of shelterin bridge to tumorigenesis[J]. Cell Rep, 2015, 12(12): 2169-2180.
17	ALTSCHULER S E, CROY J E, WUTTKE D S. A small molecule inhibitor of Pot1 binding to telomeric DNA[J]. Biochemistry, 2012, 51(40): 7833-7845.
18	BEREI J, ECKBURG A, MILIAVSKI E, et al. Potential telomere-related pharmacological targets[J]. Curr Top Med Chem, 2020, 20(6): 458-484.
19	XUE J, CHEN H W, WU J, et al. Structure of the fission yeast S. pombe telomeric Tpz1-Poz1-Rap1 complex[J]. Cell Res, 2017, 27(12): 1503-1520.
20	LEI M, PODELL E R, BAUMANN P, et al. DNA self-recognition in the structure of Pot1 bound to telomeric single-stranded DNA[J]. Nature, 2003, 426(6963): 198-203.
21	KIM J K, LIU J Q, HU X C, et al. Structural basis for shelterin bridge assembly[J]. Mol Cell, 2017, 68(4): 698-714.e5.
22	FAIRALL L, CHAPMAN L, MOSS H, et al. Structure of the TRFH dimerization domain of the human telomeric proteins TRF1 and TRF2[J]. Mol Cell, 2001, 8(2): 351-361.
23	LEI M, PODELL E R, CECH T R. Structure of human POT1 bound to telomeric single-stranded DNA provides a model for chromosome end-protection[J]. Nat Struct Mol Biol, 2004, 11(12): 1223-1229.
24	CHEN C, GU P L, WU J, et al. Structural insights into POT1-TPP1 interaction and POT1 C-terminal mutations in human cancer[J]. Nat Commun, 2017, 8: 14929.
25	KIM S H, DAVALOS A R, HEO S J, et al. Telomere dysfunction and cell survival: roles for distinct TIN2-containing complexes[J]. J Cell Biol, 2008, 181(3): 447-460.
26	TANG G, PENG L W, BALDWIN P R, et al. EMAN2: an extensible image processing suite for electron microscopy[J]. J Struct Biol, 2007, 157(1): 38-46.
27	SCHERES S H W. RELION: implementation of a Bayesian approach to cryo-EM structure determination[J]. J Struct Biol, 2012, 180(3): 519-530.
28	PETTERSEN E F, GODDARD T D, HUANG C C, et al. UCSF Chimera: a visualization system for exploratory research and analysis[J]. J Comput Chem, 2004, 25(13): 1605-1612.
29	BURLEY S K, BERMAN H M, KLEYWEGT G J, et al. Protein data bank (PDB): the single global macromolecular structure archive[J]. Methods Mol Biol, 2017, 1607: 627-641.
30	SCOTT H, KIM J K, YU C, et al. Spatial organization and molecular interactions of the Schizosaccharomyces pombe Ccq1-Tpz1-Poz1 shelterin complex[J]. J Mol Biol, 2017, 429(19): 2863-2872.
31	SAVAGE S A, GIRI N, BAERLOCHER G M, et al. TINF2, a component of the shelterin telomere protection complex, is mutated in dyskeratosis congenita[J]. Am J Hum Genet, 2008, 82(2): 501-509.
32	MYLER L R, KINZIG C G, SASI N K, et al. The evolution of metazoan shelterin[J]. Genes Dev, 2021, 35(23/24): 1625-1641.
33	WANG J Y, COHEN A L, LETIAN A, et al. The proper connection between shelterin components is required for telomeric heterochromatin assembly[J]. Genes Dev, 2016, 30(7): 827-839.
34	LIU J Q, HU X C, BAO K H, et al. The cooperative assembly of shelterin bridge provides a kinetic gateway that controls telomere length homeostasis[J]. Nucleic Acids Res, 2021, 49(14): 8110-8119.

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