Cryo‐EM structure of the CENP‐A nucleosome in complex with phosphorylated CENP‐C
2021; Springer Nature; Volume: 40; Issue: 5 Linguagem: Inglês
10.15252/embj.2020105671
ISSN1460-2075
AutoresMariko Ariyoshi, Fumiaki Makino, Reito Watanabe, Reiko Nakagawa, Takayuki Kato, Keiichi Namba, Yasuhiro Arimura, Risa Fujita, Hitoshi Kurumizaka, Eiichi Okumura, Masatoshi Hara, Tatsuo Fukagawa,
Tópico(s)RNA and protein synthesis mechanisms
ResumoArticle19 January 2021free access Transparent process Cryo-EM structure of the CENP-A nucleosome in complex with phosphorylated CENP-C Mariko Ariyoshi Mariko Ariyoshi orcid.org/0000-0002-1361-2642 Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, JapanThese authors contributed equally to this work Search for more papers by this author Fumiaki Makino Fumiaki Makino orcid.org/0000-0001-9512-7087 Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan JEOL Ltd., Akishima, Tokyo, JapanThese authors contributed equally to this work Search for more papers by this author Reito Watanabe Reito Watanabe Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan Search for more papers by this author Reiko Nakagawa Reiko Nakagawa orcid.org/0000-0002-6178-2945 Laboratory for Phyloinformatics, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Hyogo, Japan Search for more papers by this author Takayuki Kato Takayuki Kato orcid.org/0000-0002-8879-6685 Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan Institute of Protein Research, Osaka University, Suita, Osaka, Japan Search for more papers by this author Keiichi Namba Keiichi Namba orcid.org/0000-0003-2911-5875 Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan RIKEN Center for Biosystems Dynamics Research (BDR) and SPring-8 Center, and JEOL YOKOGUSHI Research Alliance Laboratories, Osaka University, Suita, Osaka, Japan Search for more papers by this author Yasuhiro Arimura Yasuhiro Arimura orcid.org/0000-0002-1903-6076 Laboratory of Chromatin Structure and Function, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Risa Fujita Risa Fujita Laboratory of Chromatin Structure and Function, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Hitoshi Kurumizaka Hitoshi Kurumizaka orcid.org/0000-0001-7412-3722 Laboratory of Chromatin Structure and Function, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Ei-ichi Okumura Ei-ichi Okumura Laboratory of Cell and Developmental Biology, Graduate School of Bioscience, Tokyo Institute of Technology, Yokohama, Japan Search for more papers by this author Masatoshi Hara Masatoshi Hara orcid.org/0000-0001-8433-1111 Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan Search for more papers by this author Tatsuo Fukagawa Corresponding Author Tatsuo Fukagawa [email protected] orcid.org/0000-0001-8564-6852 Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan Search for more papers by this author Mariko Ariyoshi Mariko Ariyoshi orcid.org/0000-0002-1361-2642 Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, JapanThese authors contributed equally to this work Search for more papers by this author Fumiaki Makino Fumiaki Makino orcid.org/0000-0001-9512-7087 Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan JEOL Ltd., Akishima, Tokyo, JapanThese authors contributed equally to this work Search for more papers by this author Reito Watanabe Reito Watanabe Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan Search for more papers by this author Reiko Nakagawa Reiko Nakagawa orcid.org/0000-0002-6178-2945 Laboratory for Phyloinformatics, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Hyogo, Japan Search for more papers by this author Takayuki Kato Takayuki Kato orcid.org/0000-0002-8879-6685 Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan Institute of Protein Research, Osaka University, Suita, Osaka, Japan Search for more papers by this author Keiichi Namba Keiichi Namba orcid.org/0000-0003-2911-5875 Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan RIKEN Center for Biosystems Dynamics Research (BDR) and SPring-8 Center, and JEOL YOKOGUSHI Research Alliance Laboratories, Osaka University, Suita, Osaka, Japan Search for more papers by this author Yasuhiro Arimura Yasuhiro Arimura orcid.org/0000-0002-1903-6076 Laboratory of Chromatin Structure and Function, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Risa Fujita Risa Fujita Laboratory of Chromatin Structure and Function, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Hitoshi Kurumizaka Hitoshi Kurumizaka orcid.org/0000-0001-7412-3722 Laboratory of Chromatin Structure and Function, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Ei-ichi Okumura Ei-ichi Okumura Laboratory of Cell and Developmental Biology, Graduate School of Bioscience, Tokyo Institute of Technology, Yokohama, Japan Search for more papers by this author Masatoshi Hara Masatoshi Hara orcid.org/0000-0001-8433-1111 Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan Search for more papers by this author Tatsuo Fukagawa Corresponding Author Tatsuo Fukagawa [email protected] orcid.org/0000-0001-8564-6852 Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan Search for more papers by this author Author Information Mariko Ariyoshi1, Fumiaki Makino1,2, Reito Watanabe1, Reiko Nakagawa3, Takayuki Kato1,4, Keiichi Namba1,5, Yasuhiro Arimura6, Risa Fujita6, Hitoshi Kurumizaka6, Ei-ichi Okumura7, Masatoshi Hara1 and Tatsuo Fukagawa *,1 1Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan 2JEOL Ltd., Akishima, Tokyo, Japan 3Laboratory for Phyloinformatics, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Hyogo, Japan 4Institute of Protein Research, Osaka University, Suita, Osaka, Japan 5RIKEN Center for Biosystems Dynamics Research (BDR) and SPring-8 Center, and JEOL YOKOGUSHI Research Alliance Laboratories, Osaka University, Suita, Osaka, Japan 6Laboratory of Chromatin Structure and Function, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan 7Laboratory of Cell and Developmental Biology, Graduate School of Bioscience, Tokyo Institute of Technology, Yokohama, Japan *Corresponding author. Tel: +81 6 6879 4428; Fax: +81 6 6879 4427; E-mail: [email protected] The EMBO Journal (2021)40:e105671https://doi.org/10.15252/embj.2020105671 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Synopsis Phosphorylation of kinetochore protein CENP-C regulates its binding to the CENP-A nucleosome. Cryo-EM reveals how CENP-C phosphorylation regulates CENP-A binding and provides insights into a dynamic kinetochore assembly mechanism during mitosis. The C-terminal region of CENP-C adopts a stable fold upon CENP-A nucleosome binding. CDK1 phosphorylation of CENP-C stabilizes the complex structure with the CENP-A nucleosome via an intramolecular interaction. CENP-C binds the RG loop of the CENP-A nucleosome, which is recognized by another CENP-A-binding protein, CENP-N. The stable CENP-A- CENP-C complex excludes CENP-N from the CENP-A nucleosome. Introduction The centromere is an essential genomic region for accurate chromosome segregation. In most organisms, the centromere is localized to a particular locus on each chromosome by sequence-independent epigenetic mechanisms. The histone H3 variant centromere protein A (CENP-A) is a key epigenetic marker for centromere specification (Allshire & Karpen, 2008; Black & Cleveland, 2011; Perpelescu & Fukagawa, 2011; Westhorpe & Straight, 2013; Fukagawa & Earnshaw, 2014). CENP-A forms a nucleosome with other canonical histones (H2A/B and H4) that share a fundamental architecture with a canonical histone H3-containing nucleosome (Black & Cleveland, 2011; Tachiwana et al, 2011). However, downstream kinetochore components are specifically associated with CENP-A nucleosomes, but not with H3 nucleosomes, to form a functional kinetochore structure (Foltz et al, 2006; Izuta et al, 2006; Okada et al, 2006; Hori et al, 2008; Amano et al, 2009; Nishino et al, 2012; Weir et al, 2016; Yan et al, 2019). Therefore, to facilitate recognition by kinetochore proteins, the CENP-A nucleosome should be structurally different from the H3 nucleosome. The functional sites of CENP-A have been identified at the N-terminal, RG loop, and C-terminal tail regions in the primary sequence (Black & Cleveland, 2011; Tachiwana et al, 2011). Furthermore, structural information on the CENP-A nucleosome bound to kinetochore proteins is essential for an in-depth understanding of the mechanism by which kinetochore proteins distinguish the CENP-A nucleosome from the canonical histone H3 nucleosome. Among kinetochore proteins, CENP-C (Kato et al, 2013b; Falk et al, 2015; Falk et al, 2016; Guo et al, 2017; Ali-Ahmad et al, 2019; Watanabe et al, 2019) and CENP-N (Carroll et al, 2009; Pentakota et al, 2017; Chittori et al, 2018; Tian et al, 2018; Allu et al, 2019) are direct CENP-A binding proteins. Binding of these kinetochore proteins to the CENP-A nucleosome triggers the assembly of other kinetochore proteins. Therefore, it is critical to address how CENP-C and CENP-N, individually and/or together, specifically bind to the CENP-A nucleosome. Various structural studies have been carried out to understand the CENP-A nucleosome recognition mechanisms of CENP-C and/or CENP-N (Carroll et al, 2009; Kato et al, 2013b; Pentakota et al, 2017; Chittori et al, 2018; Tian et al, 2018; Ali-Ahmad et al, 2019; Allu et al, 2019; Watanabe et al, 2019). These studies have revealed several fundamental structural aspects of the molecular interfaces between the CENP-A nucleosome and CENP-C and CENP-N. First, the central domain and CENP-C motif in human CENP-C, which share a sequence motif essential for binding to the CENP-A nucleosome, recognize the CENP-A C-terminal tail and acidic patch of H2A/B in the CENP-A nucleosome. Second, the CENP-N N-terminal domain binds to the RG loop in the CENP-A target domain (CATD) of CENP-A. Third, CENP-C and CENP-N simultaneously bind to the CENP-A nucleosome because their CENP-A binding sites do not overlap. These findings have provided important information concerning the kinetochore architecture in vertebrate cells. However, several questions remain unanswered and need to be addressed. Since relatively short regions of CENP-C containing either the central domain or the CENP-C motif have been used for these structural studies (Kato et al, 2013b; Ali-Ahmad et al, 2019; Allu et al, 2019), it is still unclear whether and how other regions of CENP-C contribute to its binding to the CENP-A nucleosome. Moreover, we have demonstrated that CDK1-mediated CENP-C phosphorylation outside of the CENP-C motif facilitates CENP-C binding to the CENP-A nucleosome (Watanabe et al, 2019). Thus, a question arises regarding the mechanism by which phosphorylation contributes to CENP-A-CENP-C binding. Furthermore, although previous studies have suggested that CENP-C and CENP-N recognize distinct regions on the CENP-A nucleosome and these proteins might simultaneously bind to the CENP-A nucleosome (Allu et al, 2019), it is unknown whether the longer CENP-C fragment and CENP-N bind to the same surface of the CENP-A nucleosome without any structural collision. To address these questions, we performed a structural analysis of the chicken CENP-A nucleosome in complex with a longer and phosphorylated chicken CENP-C C-terminal fragment (amino acid (aa) 601–864, termed CENPC-CT in this paper), including the CENP-C motif (aa 655–675, termed CENPC motif in this paper) in the absence or presence of the CENP-N N-terminal domain (aa 1–211, termed CENPN-NT in this paper) using cryo-electron microscopy (cryo-EM) single particle image analysis combined with a biochemical approach. The cryo-EM structure of the CENP-A nucleosome bound to the short CENPC motif peptide and CENPN-NT was also determined. In the present study, we demonstrate a unique and well-defined structure of CENPC-CT, featuring a stable CENP-A-CENP-C interaction. Of note, the phosphorylation of T651CENP-C stabilizes the CENP-A-bound structure of CENPC-CT through an intramolecular interaction with R656CENP-C in the CENPC motif. At the newly identified CENP-A-CENP-C interface, the C-terminal folded region extending from the CENPC motif associates with the RG loop of the CENP-A nucleosome, which is also recognized by CENP-N. Collectively, our data imply the binding of CENP-C and CENP-N to the CENP-A nucleosome is mutually exclusive and phosphorylation-dependent. These structural features of the CENP-A nucleosome complexes provide new insights into kinetochore assembly mechanisms. Results The longer C-terminal fragment of CENP-C stably associates with the CENP-A nucleosome CENP-C has multiple domains that include the N-terminal Mis12 complex binding domain, CENP-L/N and CENP-H/I/K/M binding domains, CENPC motif, and C-terminal dimerization domain (Cupin domain) (Fig 1A; Klare et al, 2015; Hara et al, 2018; Watanabe et al, 2019). The entire domain organization of chicken CENP-C is similar to that of human CENP-C, except for the absence of the second CENP-A binding domain (the central domain) in the middle of human CENP-C (Fig 1A; Kato et al, 2013a). However, as the central domain is not widely conserved (Watanabe et al, 2019), we focused on CENPC-CT containing the CENPC motif to analyze their CENP-A binding and purified chicken CENPC-CT (aa 601–864) as a recombinant protein (Fig 1A). We also prepared a shorter peptide (aa 643–683), including the CENPC motif (CM peptide, Fig 1A). We reconstituted chicken CENP-A nucleosomes, whereby the CENP-A N-terminal region was replaced by the H3 N-terminal region to stabilize the nucleosome (Watanabe et al, 2019). First, we assessed the binding ability of CENPC-CT to the CENP-A nucleosome using an electrophoretic mobility shift assay (EMSA). CENPC-CT efficiently bound to the CENP-A nucleosome at a molar ratio of 1:1, but did not bind to the canonical H3 nucleosome, even after adding four times excess of CENPC-CT than the H3 nucleosome (Fig 1B). We also performed similar assays using the CM peptide. While excess CM peptide (more than 18:1 molar ratio) bound to the CENP-A nucleosome, it also bound to the H3 nucleosome (Fig 1B), indicating that specific binding to the CENP-A nucleosome was lost due to excess amounts of the CM peptide. As the CENPC motif binds to the acidic patch of histone H2A/B, excess CM peptide can bind to this region in the H3 nucleosome. However, as CENPC-CT specifically binds to the CENP-A nucleosome at a molar ratio of 1:1, other CENP-C regions in the C-terminal fragment must facilitate the stable and specific binding of CENP-C to the CENP-A nucleosome. Figure 1. CENP-A nucleosome binding of the C-terminal fragment of CENP-C Schematic diagram of the functional regions of chicken and human CENP-C molecules. The canonical CENPC motifs for CENP-A binding (aa 655-675 in chicken CNEP-C, aa 738-758 in human CENP-C) are colored pink. Another CENP-A binding region (central domain) in human CENP-C is colored light pink. The C-terminal fragment (aa 601-864: CENPC-CT) and the CENPC motif-containing peptide (aa 643-683; CM peptide) derived from chicken CNEP-C, which were used for the in vitro CENP-A nucleosome-binding assay, are diagrammed. Electrophoretic mobility shift assay (EMSA) examination of the binding affinity of CENPC-CT and CM peptide to a CENP-A nucleosome. Binding to a canonical H3 nucleosome was also examined using EMSA. Cryo-EM density map of the CENP-A nucleosome in complex with CENPC-CT (CA-CCCT complex) at a 4.5 Å resolution. The side views of the CA-CCCT complex along the two-fold axis are shown. The density corresponding to each molecule in the complex is color-coded, as indicated in the figure. Cryo-EM structure of the CENP-A nucleosome-binding region of CENPC-CT. The left panel shows the cartoon representation of the structure of CENPC-CT bound to the CENP-A nucleosome with the cryo-EM density map. The densities derived from CENPC-CT and CENP-A molecules are color-coded in magenta and pale green, respectively. The functional elements in the CENP-A nucleosome-binding region of CENPC-CT are depicted in the right panel. In addition to the CENPC motif, which recognizes the H2A/H2B acidic patch and the CENP-A C-terminal tail (C-tail), the CM upstream and CM downstream regions are identified in the CENP-A bound structure of CENPC-CT. See also Fig EV2C. Download figure Download PowerPoint To directly examine which regions of CENP-C are involved in stable and specific binding to the CENP-A nucleosome, we purified the CENP-A nucleosome in complex with CENPC-CT (CA-CCCT complex) and performed cryo-EM single particle image analysis of the structure of the complex. Although the CA-CCCT complexes appeared to be formed in vitro (Fig 1B), most of them were aggregated and/or fell apart, when the sample was concentrated and applied to EM grids. Well-distributed particles of the CA-CCCT complex suitable for cryo-EM single particle image analysis were seldom detected in the micrographs. We have previously demonstrated that phosphorylation of CENP-C by CDK1 facilitates CENP-A binding (Watanabe et al, 2019) and hypothesized that phosphorylated CENPC-CT would form a more stable CA-CCCT complex suitable for cryo-EM single particle image analysis. Accordingly, using CENPC-CT phosphorylated by CDK1, we obtained the appropriate number of particles distributed on the EM grid. The three-dimensional (3D) reconstitution of the CA-CCCT complex was determined at a 6.78 Å resolution (Table 1 and Fig EV1A). The EM density of the CA-CCCT complex revealed the phosphorylated CENPC-CT fragment bound to each side of the CENP-A nucleosome (Fig EV2A). Hereafter, we used this phosphorylated CENPC-CT fragment for further cryo-EM single particle image analysis. CENPC-CT contains the dimeric Cupin domain at the extreme C-terminal end (Fig 1A). The stoichiometry of dimeric CENPC-CT bound to the single CENP-A nucleosome was not clear in our cryo-EM analysis. However, based on size exclusion chromatography analyses of CENP-A nucleosome complexes, it is likely that a CENPC-CT dimer associates with each face of the CENP-A nucleosome (Appendix Fig S1), leading to a stoichiometry of 4 CENPC-CT fragments per each CENP-A nucleosome (Appendix Fig S1B-b). Only one protomer out of the CENPC-CT dimer clearly binds CENP-A. Table 1. Statistics for Cryo-EM single particle image analysis and structure refinement. Cryo-EM density map CA-CCCT (4.5Å) (EMDB-30239) (PDB 7BY0) Asymmetric CA-CCCT-CNNT (EMDB-30241) CA-CCpep-CNNT (EMDB-30237) (PDB 7BXT) CA-CCCT (6.78Å) (EMDB-30240) Data collection and processing Sample CA-CCCT-CNNT complex CA-CCpep-CNNT complex CA-CCCTcomplex Magnification 50,000 50,000 50,000 50,000 Voltage (kV) 200 200 200 200 Electron exposure (e–/Å2 ) 100 100 100 100 Defocus range (µm) −0.3 (−7.0) −0.3 (−7.0) −0.3 (−7.0) −0.3 (−7.0) Pixel size (Å) 1.10 1.10 1.47 1.09 Symmetry C2 C1 C2 C2 Initial particle images (n) 3,385,783 3,385,783 4,192,300 3,423,115 Final particle images (n) 38,542 3,959 118,294 39,542 Map resolution (Å) 4.5 7.8 4.2 6.78 FSC threshold 0.143 0.143 0.143 0.143 Refinement CCmap_model 0.87 0.84 Model compositions Nonhydrogen atoms 12,693 15,715 Protein residues 872 1,204 Nucleic acids 285 290 R.m.s. deviations Bond lengths (Å) 0.007 0.010 Angles (°) 0.939 1.169 Ramachandran plot Favored (%) 96.79 93.36 Allowed (%) 3.21 6.64 Outliers (%) 0.00 0.00 Model validation Rotamer outliers (%) 0.15 0.39 Clash score 11.07 9.69 Cβ outliers (%) 0.00 0.00 CaBLAM outliers (%) 2.55 3.93 FSC, Fourier shell correlation; R.m.s. deviations, root-mean-square deviations. Click here to expand this figure. Figure EV1. Cryo-EM single particle image analysis of CA-CCCT, CA-CCCT-CNNT, and CA-CCpep-CNNT complexes Cryo-EM density map of the chicken CA-CCCT complex at a 6.78 Å resolution, colored according to the local resolution estimated by RELION in the left panel. Gold-standard Fourier shell correlation (FSC) curve of the Cryo-EM density map is displayed in the right panel. Reported resolution was based on the FSC = 0.143 criterion. Cryo-EM density map and FSC curve of the chicken CA-CCCT complex at a 4.5 Å resolution obtained using the CENP-A nucleosome + CENPC-CT + CENPN-NT sample (major component) are shown as in (A). Cryo-EM density map and FSC curve of the asymmetric chicken CA-CCCT-CNNT complex at 7.8 Å resolution obtained using CA + CCCT+CNNT sample (minor component) are shown as in (A). Cryo-EM density map and FSC curve of the asymmetric chicken CA-CCpep-CNNT complex at a 4.2 Å resolution are shown as in (A). A representative micrograph of the CENP-A complex with CENPC-CT and CENPN-NT. Representative 2D class averages of the CENP-A nucleosome + CENPC-CT + CENPN-NT sample. Flow chart showing the image processing pipeline for the cryo-EM single particle image analysis of the chicken CA-CCCT complex and asymmetric chicken CA-CCCT-CNNT complex. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Cryo-EM structure of the CA-CCCT complex Cryo-EM density map of the CENPC-CT bound to the CENP-A nucleosome at a 6.78 Å resolution. The EM map for CENPC-CT is shown in a pink surface representation together with the ribbon representation of the CENP-A nucleosome. The molecules in the complex are color-coded as indicated in the figure. The right panel shows a slice view along the two-fold axis. The two CENPC-CT fragments symmetrically bind to the CENP-A nucleosome. Comparison of lower (6.78 Å) and higher (4.5 Å) resolution EM densities of the CA-CCCT complex. The map at 4.5 Å resolution is depicted in a surface representation and superposed on the 6.78 Å resolution map shown as a mesh representation (light gray). The superposed maps corresponding to the CENPC-CT are enlarged in a left panel. Detailed views of the cryo-EM density map of the CA-CCCT complex at a 4.5 Å resolution. The map is shown as a mesh representation with the ribbon model of the final cryo-EM structure. Protease sensitivity of CENPC-CT is altered by the presence of the CENP-A nucleosome. Schematic diagram showing Factor Xa cleavage sites in MBP-fused chicken CENPC-CT with its functional domain organization. The possible minor cleavage sites (Gly-Arg sequence) are indicated as cutting site 2 (between residues 642 and 643) and 3 (between residues 682 and 683), in addition to a major cleavage site between CENPC-CT and MBP (site 1). The amino acid sequence of the folded region in CENPC-CT is indicated below. The lower left panel shows the result of SDS–PAGE analysis of limited proteolysis product of CENPC-CT in the absence or presence of the CENP-A nucleosome. Possible fragments generated by Factor Xa digestion are shown in the lower right panel. Bands corresponding to each fragment are indicated in the gel. In the absence of the CENP-A nucleosome, bands of the limited proteolysis products (b, c, e, f, and g) were observed. These bands were not observed in the presence of CENP-A nucleosome. Download figure Download PowerPoint In addition to CENPC-CT, CENPN-NT also binds to the CENP-A nucleosome (Carroll et al, 2009; Pentakota et al, 2017; Chittori et al, 2018; Allu et al, 2019). Therefore, we reconstituted the CENP-A nucleosome in complex with phosphorylated chicken CENPC-CT and chicken CENPN-NT (aa 1–211) (CA-CCCT-CNNT complex) and analyzed their structure using cryo-EM (Table 1). Cryo-EM single particle image analysis of the CA-CCCT-CNNT complex showed a heterogeneous population of particles. We observed the CA-CCCT complex as the major component (94% of total analyzed particles) even in the presence of CENPN-NT and the asymmetrical CA-CCCT-CNNT complex as a minor component (6% of total analyzed particles) (Fig EV1-EV5). The cryo-EM density maps of the CA-CCCT and asymmetrical CA-CCCT-CNNT complexes were determined at 4.5 and 7.8 Å resolutions, respectively (Figs EV1B and 1C; see details in later sections). Although CENPC-CT did not directly interact with CENP-N, the presence of CENPN-NT improved the particle distribution on the EM grid, leading to a higher-resolution cryo-EM structure of the CA-CCCT complex. An excess amount of CENPC-CT tended to nonspecifically bind to DNA and form aggregates. CENPN-NT may weakly or transiently associate with CENPC-CT or DNA and prevent the formation of such aggregates. Click here to expand this figure. Figure EV3. Conserved CENP-A nucleosome-binding sites in the chicken CENPC-CT fragment Schematic diagram showing functional elements in chicken CENPC-CT. The amino acid sequence of CENPC motif (CM) is enclosed in a pink box. The aligned sequences of rat CENPC motif and human central domain (CD), which were used for previous structural studies, are shown at the bottom. Key residues for CENP-A nucleosome binding, R659, Y667, and W668 in chicken CENP-C, are colored in blue (R659) and magenta (Y667 and W668). Corresponding residues in rat and human CENP-C are also colored. Magnified views of the binding sites for the CENP-A C-terminal region and the H2A/2B acidic patch are presented in the cryo-EM map. Side chains of the key residues are indicated as a stick model. Cryo-EM structure of CENPC-CT bound to the CENP-A nucleosome. The cryo-EM density for CENPC-CT is shown in a mesh representation. The crystal structure of the CENPC motif in complex with the nucleosome (PDB ID: 4X23) is superimposed. The entire backbone structures are well superimposed. Structural comparison between the chicken CA-CCCT complex and the human CD structures (PDB ID: 6MUO and 6SE6) on the CENP-A nucleosome. The structures of CD bound to the CENP-A nucleosome superimposed to that of the CENPC motif in the CA-CCCT complex. CENP-A nucleosome-binding assays with WT or R659A mutant of CENPC-CT. The substitution of R659 residue with alanine (R659A) caused a loss of the CENP-A nucleosome-binding ability. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Association of CENPC-CT with RG loopCENP-A Secondary structure prediction of CENPC-CT. A diagram of chicken CENPC-CT is shown. The secondary structure of the putative CENP-A binding region of CENPC-CT was analyzed by six different programs using a HHpred server https://toolkit.tuebingen.mpg.de/tools/hhpred. Predicted secondary structure elements are indicated by H for helix, E for strand, and D for disordered region. The canonical CENPC motif, with previously determined structure in homologues, is highlighted in pink. A schematic diagram of the cryo-EM structure of CENPC-CT is shown at the bottom. Crosslinking mass spectroscopy (XL-MS) interactions depicted in relation to CENPC-CT and histones, including CENP-A. Color bars represent protein sequences. Black and purple lines show inter- and intra-protein links, respectively. In the right panel, the crosslinked sites between CENP-C and histones are indicated on the CA-CCCT complex structure in which K678CENP-C was linked with K108H2B and K79H4. Detailed XL-MS data are presented in Appendix Fig S2. Alignment of sequences around the CENPC motif region in various species: Gg, chicken; Hs, human; Mm, mouse, and Xl; frog. The CENPC motif and the CM downstream region are depicted by pink and purple boxes, respectively, in the sequence alignment. The residue numbers of human CENP-C are indicated. Schematic diagram of human CENPC-CT wild-type (CENPC-CT WT: aa 687-943) corresponding to chicken CNEPC-CT used for the cryo-EM analysis is depicted. The conserved PSG residues (aa 762-764) in the CM downstream region were substituted with AAA (CENPC-CT 3A762-764) and CENPC-CT in which six residues were deleted (CENPC-CTΔ761-766) are shown. Localization analysis of GFP-fused human CENPC-CT WT and mutants shown in (C) on the mitotic chromosomes in CENP-C knock out human RPE-1 cells. CENP-A was used as a centromere marker. Scale bar indicates 10 μm. Download figure Download PowerPoint
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