Cryo-EM structure of the CENP-A nucleosome in complex with phosphorylated CENP-C

Mariko Ariyoshi; Fumiaki Makino; Reito Watanabe; Reiko Nakagawa; Takayuki Kato; Keiichi Namba; Yasuhiro Arimura; Risa Fujita; Hitoshi Kurumizaka; Ei-Ichi Okumura; Masatoshi Hara; Tatsuo Fukagawa

doi:10.15252/embj.2020105671

Cryo-EM structure of the CENP-A nucleosome in complex with phosphorylated CENP-C

EMBO J. 2021 Mar 1;40(5):e105671. doi: 10.15252/embj.2020105671. Epub 2021 Jan 19.

Authors

Mariko Ariyoshi¹, Fumiaki Makino^{1

2}, Reito Watanabe¹, Reiko Nakagawa³, Takayuki Kato^{1

4}, Keiichi Namba^{1

5}, Yasuhiro Arimura⁶, Risa Fujita⁶, Hitoshi Kurumizaka⁶, Ei-Ichi Okumura⁷, Masatoshi Hara¹, Tatsuo Fukagawa¹

Affiliations

¹ Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan.
² JEOL Ltd., Akishima, Tokyo, Japan.
³ Laboratory for Phyloinformatics, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Hyogo, Japan.
⁴ Institute of Protein Research, Osaka University, Suita, Osaka, Japan.
⁵ RIKEN Center for Biosystems Dynamics Research (BDR) and SPring-8 Center, and JEOL YOKOGUSHI Research Alliance Laboratories, Osaka University, Suita, Osaka, Japan.
⁶ Laboratory of Chromatin Structure and Function, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan.
⁷ Laboratory of Cell and Developmental Biology, Graduate School of Bioscience, Tokyo Institute of Technology, Yokohama, Japan.

Abstract

The CENP-A nucleosome is a key structure for kinetochore assembly. Once the CENP-A nucleosome is established in the centromere, additional proteins recognize the CENP-A nucleosome to form a kinetochore. CENP-C and CENP-N are CENP-A binding proteins. We previously demonstrated that vertebrate CENP-C binding to the CENP-A nucleosome is regulated by CDK1-mediated CENP-C phosphorylation. However, it is still unknown how the phosphorylation of CENP-C regulates its binding to CENP-A. It is also not completely understood how and whether CENP-C and CENP-N act together on the CENP-A nucleosome. Here, using cryo-electron microscopy (cryo-EM) in combination with biochemical approaches, we reveal a stable CENP-A nucleosome-binding mode of CENP-C through unique regions. The chicken CENP-C structure bound to the CENP-A nucleosome is stabilized by an intramolecular link through the phosphorylated CENP-C residue. The stable CENP-A-CENP-C complex excludes CENP-N from the CENP-A nucleosome. These findings provide mechanistic insights into the dynamic kinetochore assembly regulated by CDK1-mediated CENP-C phosphorylation.

Keywords: CENP-A; CENP-C; Cryo-EM; kinetochore; phosphorylation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Centromere / metabolism*
Centromere / ultrastructure
Centromere Protein A / metabolism*
Centromere Protein A / ultrastructure
Chickens
Chromosomal Proteins, Non-Histone / metabolism*
Chromosomal Proteins, Non-Histone / ultrastructure
Cryoelectron Microscopy / methods*
Kinetochores / metabolism*
Kinetochores / ultrastructure
Models, Molecular
Nucleosomes / metabolism*
Nucleosomes / ultrastructure
Phosphorylation
Protein Conformation

Substances

Centromere Protein A
Chromosomal Proteins, Non-Histone
Nucleosomes
centromere protein C

Associated data

PDB/7BY0
PDB/7BXT