Structure of the inner kinetochore CCAN complex assembled onto a centromeric nucleosome

Yan, Kaige; Yang, Jing; Zhang, Ziguo; McLaughlin, Stephen H.; Chang, Leifu; Fasci, Domenico; Ehrenhofer‐Murray, Ann E.; Heck, Albert J. R.; Barford, David

doi:10.1038/s41586-019-1609-1

Cited by 116 publications

(279 citation statements)

References 65 publications

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“…Previous studies have reported molecular structures, determined by cryo-EM or crystallography, of Ctf3-C with Mcm16/22-N from S. cerevisiae and of Ctf3-N with Mcm16/22-C from related yeast species Hu et al, 2019;Zhang et al, 2020). In cryo-EM reconstructions of the complete Ctf19c, only the Ctf3-C module is well-resolved Yan et al, 2019). The new Ctf3c model shows how these structural modules relate to each other and provides a high-resolution model of S. cerevisiae Ctf3-N with Mcm16/22-C.…”

Section: Molecular Model Of the Complete Ctf3cmentioning

confidence: 99%

The structural basis for kinetochore stabilization by Cnn1/CENP-T

Hinshaw

2020

Preprint

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Chromosome segregation depends on a regulated connection between spindle microtubules and centromeric DNA. The kinetochore, a massive modular protein assembly, mediates this connection and also serves as a signaling hub that integrates and responds to changing cues during the cell cycle. Kinetochore functions evolve as the cell cycle progresses, culminating in the assurance of a persistent chromosome-microtubule connection during anaphase, when sister chromatids must transit into daughter cells uninterrupted. We previously determined the structure of the Ctf19 complex, a group of kinetochore proteins at the centromeric base of the kinetochore. We now present a high-resolution structure of a Ctf19 complex sub-assembly involved in centromere-microtubule contact: the Ctf3 complex bound to the Cnn1-Wip1 heterodimer. The resulting composite model of the Ctf19 complex and live-cell imaging experiments provide a mechanism for Cnn1-Wip1 recruitment to the kinetochore. The mechanism suggests feedback regulation of Ctf19 complex assembly and unanticipated similarities in kinetochore organization between yeast and vertebrates.

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Section: Molecular Model Of the Complete Ctf3cmentioning

confidence: 99%

The structural basis for kinetochore stabilization by Cnn1/CENP-T

Hinshaw

2020

Preprint

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“…The extensive works have revealed the structures and functions of many CCAN components . The recent cryo‐EM structures of Saccharomyces cerevisiae CCAN complex have elucidated how the subunits are integrated into the inner kinetochore CCAN . However, further analyses are still required to clarify the detailed and dynamic interactions among the CCAN.…”

Section: Introductionmentioning

confidence: 99%

“…We previously reported the N‐terminal half structure of fungal CENP‐I that forms a ternary complex with C‐terminal heterodimer of fungal CENP‐H/K . The recent cryo‐EM structures revealed another half of Ctf3 (CENP‐I homolog), which forms complex with N‐terminal heterodimer of Mcm16/22 (CENP‐H/K homolog) . However, the intramolecular interaction and structure details of CENP‐I are less discussed.…”

Section: Introductionmentioning

confidence: 99%

Structural insights into the intramolecular interactions of centromere protein CENP‐I

Zhao

Cao

et al. 2020

J of Molecular Recognition

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In mitosis, the accurate segregation of sister chromosomes relies on kinetochore, a multiple subunits complex assembled on centromere of each sister chromosome. As a core component of inner kinetochore, CENP-I plays important functions to mediate kinetochore assembly and supports the faithful chromosome segregation. The structures of the N-terminus and C-terminus of CENP-I homologs in complex with CENP-H/K have been reported, respectively. Unfortunately, the intramolecular interactions of CENP-I are poorly understood, and how CENP-I interacts with CENP-M remains unknown. Here, we verified a unique helix α11, which forms the intramolecular interactions with N-terminal HEAT repeats in fungal CENP-I. Deletion of the helix α11 exposed the hydrophobic surface and resulted in the in vitro protein aggregation of N-terminal HEAT repeats of fungal CENP-I. The corresponding helix and its intramolecular interaction are highly conserved in human CENP-I. Deletion of the corresponding helix in human CENP-I dramatically reduced the functional activity to interact with CENP-H and CENP-M. Mutations of the conserved residues on the helix in human CENP-I significantly weakened the binding to CENP-M, but not CENP-H, in HeLa cells. Therefore, our findings for the first time unveiled a conserved helix of CENP-I, which is important for the intramolecular interaction and function, and would be helpful for understanding the structure basis of how CENP-I mediates the kinetochore assembly during cell cycle and mitosis. K E Y W O R D S centromere, computational protein structure, crystal structure, intramolecular interaction, kinetochore, protein-protein interaction

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“…A complementary strategy is to reconstruct kinetochore complexes using recombinant proteins, as demonstrated by the tour-de-force studies that assembled the entire linkage between human CENP-A nucleosomes, inner kinetochore components, and KMN ( Figure 1B) (Weir et al 2016, Pesenti et al 2018. In a similar vein, a recent study using recombinant Saccharomyces cerevisiae proteins demonstrated that 14 CCAN subunits have the ability to self-assemble in the presence of CENP-A nucleosomes (Yan et al 2019). Another study with yeast proteins revealed selfassembly of protein chains consisting of CENP-A nucleosomes, CENP-C and CENP-QU (Okp1/Ame1 complex) scaffolds, and the KMN components Mis12 and Ndc80 complexes (Hamilton et al 2020).…”

Section: Introductionmentioning

confidence: 99%

Reconstitution of human kinetochore in mitotic cell extracts reveals permitted and restricted assembly steps

Tarasovetc

Allu

Cheeseman

et al. 2020

Preprint

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Assembly of a functional kinetochore is critical for accurate chromosome segregation. Hierarchical recruitment of soluble components during kinetochore assembly is a highly regulated mitotic event, but the underlying steps are not well understood. In yeast and Xenopus egg extracts, soluble kinetochore components can spontaneously assemble into microtubule-binding subcomplexes. Although the molecular interactions among specific kinetochore components are evolutionary conserved in eukaryotes, it remains unclear which de novo assembly steps are permitted in extracts of mitotic human cells. By analyzing the recruitment of GFP-fused kinetochore proteins from human mitotic cell extracts to inner kinetochore components immobilized on microbeads, we reconstructed the interaction between CENP-C and CENP-A–containing nucleosomes. However, subsequent phospho-dependent binding of the Mis12 complex was less efficient, whereas binding of the Ndc80 complex was inhibited. Consistently, the microtubule-binding activity of native kinetochore components, as well as those assembled using a combination of native and recombinant human proteins, was weaker than that of recombinant Ndc80 complex alone. Such inhibitory mechanisms that prevent interactions between different kinetochore components are likely to guard against spurious formation of kinetochores in the cytosol of mitotic human cells, and imply existence of specific regulatory mechanisms that permit these interactions at the assembling kinetochore.

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Structure of the inner kinetochore CCAN complex assembled onto a centromeric nucleosome

Cited by 116 publications

References 65 publications

The structural basis for kinetochore stabilization by Cnn1/CENP-T

The structural basis for kinetochore stabilization by Cnn1/CENP-T

Structural insights into the intramolecular interactions of centromere protein CENP‐I

Reconstitution of human kinetochore in mitotic cell extracts reveals permitted and restricted assembly steps

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