1. Structural Biology and Molecular Biophysics

The structure of the Ctf19c/CCAN from budding yeast

  1. Stephen M Hinshaw  Is a corresponding author
  2. Stephen C Harrison  Is a corresponding author
  1. Harvard Medical School, United States
https://doi.org/10.7554/eLife.44239
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  1. Stephen M Hinshaw
  2. Stephen C Harrison
(2019)
The structure of the Ctf19c/CCAN from budding yeast
eLife 8:e44239.
https://doi.org/10.7554/eLife.44239
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Abstract

Eukaryotic kinetochores connect spindle microtubules to chromosomal centromeres. A group of proteins called the Ctf19 complex (Ctf19c) in yeast and the constitutive centromere associated network (CCAN) in other organisms creates the foundation of a kinetochore. The Ctf19c/CCAN influences the timing of kinetochore assembly, sets its location by associating with a specialized nucleosome containing the histone H3 variant Cse4/CENP-A, and determines the organization of the microtubule attachment apparatus. We present here the structure of a reconstituted 13-subunit Ctf19c determined by cryo-electron microscopy at ~4 Å resolution. The structure accounts for known and inferred contacts with the Cse4 nucleosome and for an observed assembly hierarchy. We describe its implications for establishment of kinetochores and for their regulation by kinases throughout the cell cycle.

Data availability

We have deposited the model coordinates and cryo-EM maps in the PDB (6NUW) and EMDB (EMD-0523). Tracking files for imaging experiments are included as a source data file associated with Figure 3.

The following data sets were generated

Article and author information

Author details

  1. Stephen M Hinshaw

    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States
    For correspondence
    hinshaw@crystal.harvard.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4215-5206

  2. Stephen C Harrison

    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States
    For correspondence
    harrison@crystal.harvard.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7215-9393

Funding

Howard Hughes Medical Institute

  • Stephen M Hinshaw
  • Stephen C Harrison

Helen Hay Whitney Foundation

  • Stephen M Hinshaw

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2019, Hinshaw & Harrison

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Stephen M Hinshaw
  2. Stephen C Harrison
(2019)
The structure of the Ctf19c/CCAN from budding yeast
eLife 8:e44239.
https://doi.org/10.7554/eLife.44239

Share this article

https://doi.org/10.7554/eLife.44239

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Further reading

    1. Structural Biology and Molecular Biophysics

    The structure of the yeast Ctf3 complex

    Stephen M Hinshaw, Andrew N Dates, Stephen C Harrison
    Research Advance Updated

    Kinetochores are the chromosomal attachment points for spindle microtubules. They are also signaling hubs that control major cell cycle transitions and coordinate chromosome folding. Most well-studied eukaryotes rely on a conserved set of factors, which are divided among two loosely-defined groups, for these functions. Outer kinetochore proteins contact microtubules or regulate this contact directly. Inner kinetochore proteins designate the kinetochore assembly site by recognizing a specialized nucleosome containing the H3 variant Cse4/CENP-A. We previously determined the structure, resolved by cryo-electron microscopy (cryo-EM), of the yeast Ctf19 complex (Ctf19c, homologous to the vertebrate CCAN), providing a high-resolution view of inner kinetochore architecture (Hinshaw and Harrison, 2019). We now extend these observations by reporting a near-atomic model of the Ctf3 complex, the outermost Ctf19c sub-assembly seen in our original cryo-EM density. The model is sufficiently well-determined by the new data to enable molecular interpretation of Ctf3 recruitment and function.

    1. Structural Biology and Molecular Biophysics

    Water and chloride as allosteric inhibitors in WNK kinase osmosensing

    Liliana R Teixeira, Radha Akella ... Elizabeth J Goldsmith
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    Osmotic stress and chloride regulate the autophosphorylation and activity of the WNK1 and WNK3 kinase domains. The kinase domain of unphosphorylated WNK1 (uWNK1) is an asymmetric dimer possessing water molecules conserved in multiple uWNK1 crystal structures. Conserved waters are present in two networks, referred to here as conserved water networks 1 and 2 (CWN1 and CWN2). Here, we show that PEG400 applied to crystals of dimeric uWNK1 induces de-dimerization. Both the WNK1 the water networks and the chloride-binding site are disrupted by PEG400. CWN1 is surrounded by a cluster of pan-WNK-conserved charged residues. Here, we mutagenized these charges in WNK3, a highly active WNK isoform kinase domain, and WNK1, the isoform best studied crystallographically. Mutation of E314 in the Activation Loop of WNK3 (WNK3/E314Q and WNK3/E314A, and the homologous WNK1/E388A) enhanced the rate of autophosphorylation, and reduced chloride sensitivity. Other WNK3 mutants reduced the rate of autophosphorylation activity coupled with greater chloride sensitivity than wild-type. The water and chloride regulation thus appear linked. The lower activity of some mutants may reflect effects on catalysis. Crystallography showed that activating mutants introduced conformational changes in similar parts of the structure to those induced by PEG400. WNK activating mutations and crystallography support a role for CWN1 in WNK inhibition consistent with water functioning as an allosteric ligand.