Kaige Yan1, Jing Yang1, Ziguo Zhang1, Stephen H McLaughlin1, Leifu Chang1,2, Domenico Fasci3, Ann E Ehrenhofer-Murray4, Albert J R Heck3, David Barford5. 1. MRC Laboratory of Molecular Biology, Cambridge, UK. 2. Department of Biological Sciences, Purdue University, West Lafayette, IN, USA. 3. Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Utrecht, The Netherlands. 4. Humboldt-Universität zu Berlin, Institut für Biologie, Berlin, Germany. 5. MRC Laboratory of Molecular Biology, Cambridge, UK. dbarford@mrc-lmb.cam.ac.uk.
Abstract
In eukaryotes, accurate chromosome segregation in mitosis and meiosis maintains genome stability and prevents aneuploidy. Kinetochores are large protein complexes that, by assembling onto specialized Cenp-A nucleosomes1,2, function to connect centromeric chromatin to microtubules of the mitotic spindle3,4. Whereas the centromeres of vertebrate chromosomes comprise millions of DNA base pairs and attach to multiple microtubules, the simple point centromeres of budding yeast are connected to individual microtubules5,6. All 16 budding yeast chromosomes assemble complete kinetochores using a single Cenp-A nucleosome (Cenp-ANuc), each of which is perfectly centred on its cognate centromere7-9. The inner and outer kinetochore modules are responsible for interacting with centromeric chromatin and microtubules, respectively. Here we describe the cryo-electron microscopy structure of the Saccharomyces cerevisiae inner kinetochore module, the constitutive centromere associated network (CCAN) complex, assembled onto a Cenp-A nucleosome (CCAN-Cenp-ANuc). The structure explains the interdependency of the constituent subcomplexes of CCAN and shows how the Y-shaped opening of CCAN accommodates Cenp-ANuc to enable specific CCAN subunits to contact the nucleosomal DNA and histone subunits. Interactions with the unwrapped DNA duplex at the two termini of Cenp-ANuc are mediated predominantly by a DNA-binding groove in the Cenp-L-Cenp-N subcomplex. Disruption of these interactions impairs assembly of CCAN onto Cenp-ANuc. Our data indicate a mechanism of Cenp-A nucleosome recognition by CCAN and how CCAN acts as a platform for assembly of the outer kinetochore to link centromeres to the mitotic spindle for chromosome segregation.
In eukaryotes, accurate chromosome segregation in mitosis and meiosis maintains genome stability and prevents aneuploidy. Kinetochores are large protein complexes that, by assembling onto specialized Cenp-A nucleosomes1,2, function to connect centromeric chromatin to microtubules of the mitotic spindle3,4. Whereas the centromeres of vertebrate chromosomes comprise millions of DNA base pairs and attach to multiple microtubules, the simple point centromeres of budding yeast are connected to individual microtubules5,6. All 16 budding yeast chromosomes assemble complete kinetochores using a single Cenp-A nucleosome (Cenp-ANuc), each of which is perfectly centred on its cognate centromere7-9. The inner and outer kinetochore modules are responsible for interacting with centromeric chromatin and microtubules, respectively. Here we describe the cryo-electron microscopy structure of the Saccharomyces cerevisiae inner kinetochore module, the constitutive centromere associated network (CCAN) complex, assembled onto a Cenp-A nucleosome (CCAN-Cenp-ANuc). The structure explains the interdependency of the constituent subcomplexes of CCAN and shows how the Y-shaped opening of CCAN accommodates Cenp-ANuc to enable specific CCAN subunits to contact the nucleosomal DNA and histone subunits. Interactions with the unwrapped DNA duplex at the two termini of Cenp-ANuc are mediated predominantly by a DNA-binding groove in the Cenp-L-Cenp-N subcomplex. Disruption of these interactions impairs assembly of CCAN onto Cenp-ANuc. Our data indicate a mechanism of Cenp-A nucleosome recognition by CCAN and how CCAN acts as a platform for assembly of the outer kinetochore to link centromeres to the mitotic spindle for chromosome segregation.
The 14 subunit-CCAN complex assembled onto specialized Cenp-A nucleosomes (Cenp-A
substituted for histone H3) reconstituted using either an S. cerevisiae
centromere sequence or the Widom 601 sequence, with both complexes eluting at similar
volumes on size exclusion chromatography (Extended Data
Fig. 1a-e). In contrast, CCAN did not assemble onto a canonical H3
nucleosome, indicating the specificity of the CCAN – Cenp-ANuc
interaction (Extended Data Fig. 1b, f).
Cryo-electron micrographs of CCAN–Cenp-ANuc (using the more stable
601-Cenp-A nucleosome) revealed a heterogeneous population of particles that by 3D
classification were identified as monomeric free CCAN, a monomer of CCAN in complex with
Cenp-ANuc, and dimeric CCAN (Extended Data
Figs 2 and 3). A 3D reconstruction of
free monomeric CCAN was determined to 3.5 Å resolution (Fig. 1, Extended Data Figs 2
and 3 and Extended
Data Table 1). Clearly defined EM density for the majority of amino acid side
chains (Extended Data Fig. 4 and Extended Tables 1 and 2) allowed building and refinement of the complete atomic model of
CCAN, guided by existing models of individual CCAN subunits. The
CCAN–Cenp-ANuc complex at 4.15 Å was built by docking apo
CCAN and a nucleosome into the CCAN–Cenp-ANuc EM reconstruction (Fig. 2 and Extended
Data Table 1). A cryo-EM reconstruction of uncross linked
CCAN–Cenp-ANuc, at lower resolution (Extended Data Fig. 5a), matched that of the cross-linked structure, whereas
the free CCAN dimer, determined at 8.6 Å (Extended
Data Fig. 5b), resembles the 4.25 Å structure of S.
cerevisiae CCAN [10].
Compared with the latter study, the Nkp1 and Nkp2 subunit assignments differ.
Extended Data Figure 1
Reconstituted S. cerevisiae
CCAN–Cenp-ANuc complexes.
a, Size exclusion chromatogram profiles (Agilent Bio
SEC-5 column) for (i) CCAN, (ii) CCAN–Cenp-A nucleosome (with 601)
complex, (iii) Cenp-A nucleosome (with 601), (iv) H3 nucleosome (with 601)
and (v) H3N-Cenp-ANuc (with 601). b,
Comparative size exclusion chromatogram profiles (Agilent Bio SEC-5 column)
for CCAN–Cenp-ANuc with the Cenp-A nucleosome wrapped with
either the (i) 147 bp Widom 601 positioning sequence
(CCAN–Cenp-ANuc (601) – as in (a))
or (ii) a 153 bp S. cerevisiae centromeric
Cen3 sequence (CCAN–Cenp-ANuc
(Cen3)). Both complexes eluate at the same volume. CCAN
and the H3 nucleosome do not form a complex (iii). c, Coomassie
blue-stained SDS PAGE gel of the 14 subunit CCAN complex. d,
Coomassie blue-stained SDS PAGE gel of Cenp-ANuc (601). Lane E32:
Ethidium bromide stained gel of fraction 32. e,
CCAN–Cenp-ANuc (601) complex. Lane E13: Ethidium
bromide stained gel of fraction 13. SEC chromatograms in (a).
f, SDS PAGE gel of CCAN and H3 nucleosome (601) SEC run
shown in (b). g-j, Coomassie blue-stained SDS PAGE
gels of various Cenp-H, I and K segments co-expressed with Cenp-TW and
purified with a double Strep tag on the tagged Cenp-I subunit, indicated by
*. j, Shows that the HFDs of Cenp-TW (Cenp-THFDW)
interacts with the Cenp-HIKHead. These results confirm the
assignments of the Cenp-H, K and I subunits in our cryo-EM maps.
k, Schematic of the organization of
CCAN–Cenp-ANuc subunits and sub-complexes and
connections to the outer kinetochore Mis12 and Ndc80 complexes. Lines
indicate sub-complex connections. The two pathways connecting
Cenp-ANuc to the Ndc80 complex and microtubules are indicated
as P1 and P2 (thick lines to Ndc80). Subunits of the essential P1 pathway
are labelled black and indicated with blue shading, whereas subunits of the
non-essential P2 pathway are labelled white and indicated with yellow
shading. The P2 pathway becomes essential when the P1 pathway is defective
through defects in Dsn1 phosphorylation [9]. The experiments shown in a-j were
performed independently in triplicate with similar results. For gel source
data see Supplementary
Fig. 1.
Extended Data Figure 2
Cryo-EM data of the S. cerevisiae
CCAN–Cenp-ANuc complex.
a, A typical cryo-electron micrograph of
CCAN–Cenp-ANuc, representative of 9,002 micrographs.
b, Galleries of 2D classes of CCAN, representative of 100
2D classes. c, Galleries of 2D classes of
CCAN–Cenp-ANuc, representative of 150 2D classes.
Outlined in red are 2-D class averages for the C2-symmetric dimeric
CCAN-Cenp-ANuc complex viewed in the plane of the C2-symmetry
axis. Only a few views were observed, precluding a 3D reconstruction.
Cryo-EM grids partially destabilize CCAN – Cenp-ANuc
interactions, resulting in a very low abundance of dimeric
CCAN–Cenp-ANuc particles (~0.03% of total). The
two-fold symmetry axes of the dimeric CCAN-Cenp-ANuc complex are
shown as dashed arrows. Experiments for data in b and
c were performed independently twelve times with similar
results. d, Fourier shell correlation (FSC) curves shown for
the cryo-EM reconstructions of CCAN–Cenp-ANuc complexes:
apo CCAN, mask1 (Cenp-OPQU+, Cenp-LN), mask2 (Cenp-HIK, Cenp-LN,
sub-Cenp-OP), CCAN–Cenp-ANuc. Mask1 and mask2 used for
multi-body refinement are defined in (h) and (i)
in and Methods. e, Angular distribution plot of
CCAN–Cenp-ANuc particles. f, Local
resolution map of CCAN. g, Local resolution map of
CCAN–Cenp-ANuc. h, Local resolution map
of mask1 (Cenp-OPQU+, Cenp-LN). i, Local resolution map of
mask2 (Cenp-HIK, Cenp-LN, sub-Cenp-OP).
Extended Data Figure 3
Workflow of 3D classification of the CCAN–Cenp-ANuc
cryo-EM data set.
a, After initial 2D classification ~1.4 million
particles were sorted by 3D classification into apo CCAN (52%) and the
CCAN–Cenp-ANuc complex (48%). For apo CCAN, 4% existed
as dimers (black box) and 19% showed an ordered head-group
(Cenp-HIKHead) for the Cenp-HIK–TW sub-complex (blue
box). A mask was applied to the CCAN–Cenp-ANuc EM map to
exclude the structurally variable Cenp-HIKHead domain for
reconstruction of the 4.15 Å structure. b, Details of
the four masks used for multi-body refinement. c, A small 3D
class of CCAN–Cenp-ANuc revealing density attached to
Cenp-HIKHead contacting the DNA gyre of Cenp-ANuc
was assigned as Cenp-THFDW.
Figure 1
Structure of the S. cerevisiae CCAN complex.
a, Cryo-EM density map and b, Cartoon representation of
CCAN. 11 subunits are assigned. ‘N’ and ‘C’ indicate
the N- and C-termini of Cenp-QU, Nkp1 and Nkp2. c, Details of the
Cenp-HIK–Cenp-LN modules. Residues of Cenp-I are visible from 320
onwards. d, Cryo-EM density for the complete Cenp-HIK module
showing Cenp-HIKHead from the CCAN dimer EM 3D class (Extended Data Figs 3a and 5b).
Extended Data Table 1
Cryo-EM data collection, refinement and validation statistics.
CCAN(EMDB -4580)(PDB
6QLE)
CCAN–Cenp-ANuc(EMDB-4579)(PDB
6QLD)
Mask1(EMDB-4581)(PDB
6QLF)
Mask2(EMDB-4971)
Data collection and
processing
Magnification
75,000
75,000
75,000
75,000
Voltage (kV)
300
300
300
300
Electron exposure
(e–/Å2)
32
32
32
32
Defocus range (μm)
2.0-2.8
2.0-2.8
2.0-2.8
2.0-2.8
Pixel size (Å)
1.09
1.09
1.09
1.09
Symmetry imposed
C1
C1
C1
C1
Initial particle images (no.)
1,796,016
1,796,016
1,796,016
1,796,016
Final particle images (no.)
618,459
193,882
618,459
618,459
Map resolution (Å)
3.55
4.15
3.45
3.83
FSC
threshold
0.143
0.143
0.143
0.143
Map resolution range (Å)
3.0-5.5
3.5-7.0
3.0-5.5
3.0-5.5
Refinement
lnitial model used (PDB code)
5MU3, 6EQT, 4JE3, 5W94
3AN2, 4X23, 5MU3, 6EQT, 4JE3, 5W94
5MU3, 6EQT, 4JE3, 5W94
5MU3, 6EQT, 4JE3, 5W94
Model resolution (Å)
3.5
4.0
3.3
-
0.143 FSC
threshold
Model resolution range (Å)
50 - 3.0
50 - 3.6
50 - 3.0
-
Map sharpening B factor
(Å2)
-139
-108
-135
-172
Model composition
Non-hydrogen atoms
18,058
29,183
13,541
-
Protein
residues
2,401
3,172
1,790
-
Ligands
0
248
0
-
B factors
(Å2)
Protein
78.6
82.2
67.2
-
Ligand
-
245.8
-
-
R.m.s. deviations
Bond
lengths (Å)
0.004
0.004
0.005
-
Bond
angles (°)
0.798
0.793
0.828
-
Validation
MolProbity
score
1.39
1.57
1.45
-
Clashscore
2.78
4.80
2.99
-
Poor
rotamers (%)
0.11
0.08
0.19
-
Ramachandran plot
Favored
(%)
95.30
94.78
94.76
-
Allowed
(%)
4.60
5.02
5.04
-
Disallowed
(%)
0.10
0.20
0.20
-
Extended Data Figure 4
Cryo-EM density maps of apo CCAN.
a, Portion of cryo-EM map for the coiled coils of
Cenp-H and Cenp-K. A selection of highly conserved intersubunit residues
defined in (b, c) are labelled. These residues are well defined
in EM density, consistent with the structure. b, c, Multiple
sequence alignment of the coiled-coil regions of b, Cenp-H and
c, Cenp-K. d-f, Portions of cryo-EM maps for:
d, Cenp-LN. e, Cenp-I. f,
Nkp1-Nkp2. The chain assignments and polarity of Cenp-H, Cenp-I and Cenp-K
of our structure agree with the cryo-EM structure of yeast Ctf3 (PDB 6OUA)
[61].
Extended Data Table 2
Table of CCAN subunits.
Details of structured regions of CCAN subunits built into the
cryo-EM density maps are indicated, including regions built as polyAla. The
calculated molecular masses for CCAN and Cenp-ANuc complexes are
(i) CCAN: 543.3 kDa, (ii) CCAN dimer: 1.09 MDa, (iii) Cenp-ANuc:
223 kDa, (iv) (CCAN)1−Cenp-ANuc: 0.766 MDa and
(v) (CCAN)2−Cenp-ANuc 1.31 MDa.
Structure of the S. cerevisiae
CCAN–Cenp-ANuc complex.
a, Cryo-EM density map of CCAN–Cenp-ANuc.
Cenp-AN: residues 111-129. b, c, Two views of a
cartoon representation of CCAN–Cenp-ANuc. Cenp-ANuc
wraps ~105 bp of DNA, leaving 20 bp of DNA unwrapped at both ends
(coloured yellow for the ordered terminal segment). Supplementary Video 1.
d-f, Three views of the cryo-EM density of a 3D sub-class of
the overall CCAN–Cenp-ANuc 3D class, before application of the
mask used to refine the cryo-EM map shown in (a) (Extended Data Fig. 3a), highlighting contacts
to Cenp-ANuc. d, The Cenp-HIKHead module
contacts Cenp-A. e, Cenp-THFDW contacts the DNA gyre of
Cenp-ANuc. f, N-terminal region of Cenp-QU contacts
Cenp-A and H4.
Extended Data Figure 5
Cryo-EM densities of CCAN and CCAN–Cenp-ANuc
complexes.
a, Cryo-EM reconstruction of
CCAN–Cenp-ANuc from uncross-linked sample at 8.6
Å resolution. b, Cryo-EM map of dimeric CCAN (also Extended Data Fig. 3a - black box).
Subunits are colour-coded as in Fig. 1.
The 3.5 Å monomeric free CCAN coordinates were rigid-body docked into
the cryo-EM map. c, Cartoon representation of the S.
cerevisiae MIND complex [15] (right) showing a striking similarity to the coiled
coils of Cenp-QU-Nkp1-Nkp2 of CENP-OPQU+ (left). d, View of the
4.7 Å resolution cryo-EM map of free Cenp-HIK with fitted coordinates
from CCAN. e, In the context of CCAN, Cenp-HIKHead
rotates to accommodate Cenp-ANuc. The two conformations of
Cenp-HIK from the apo CCAN and CCAN–Cenp-ANuc complexes
were superimposed onto their rigid portion of Cenp-HIK (C-terminal region of
Cenp-I – shown for apo CCAN) to indicate the conformational
variability of Cenp-HIKHead between the two states. Subunits of
Cenp-HIKHead of CCAN–Cenp-ANuc are coloured
lighter. f, Cryo-EM density of Cenp-ANuc showing the
Cenp-C motif of Cenp-C.
The arrangement of the three sub-complexes of CCAN; Cenp-LN, Cenp-OPQU+ and
Cenp-HIK–TW (Extended Data Table 2),
generates a ‘Y’-shaped structure (Fig. 1a,
b). The Cenp-N subunit, located at the centre of the ‘Y’, is
the coordinating element of CCAN, consistent with it forming a critical node at the
centromere–kinetochore interface [11]. Cenp-OPQU+, which has an elongated shape and generates the stem
and one arm of the ‘Y’, interacts mainly with Cenp-N. Cenp-L also forms an
extensive interface with Cenp-N, and contributes the major point of contact with
Cenp-HIK–TW. Together, Cenp-L and Cenp-HIK–TW generate the opposite arm of
the ‘Y’ (Fig. 1a, b). The six-subunit
Cenp-OPQU+ module shares four subunits in common with vertebrate Cenp-OPQUR, and its
structure in CCAN resembles the negative stain reconstruction of human OPQUR [12]. The long N-terminal regions of Cenp-O
and Cenp-P, disordered in the K. lactis crystal structure [13], become more structured through
interactions with Cenp-HIK and Cenp-N (Fig. 1b, c).
Four subunits of Cenp-OPQU+ (Cenp-Q, Cenp-U, Nkp1 and Nkp2) form extended
α-helices that associate in a parallel, inter-weaved fashion to create an
irregular coiled-coil α-helical bundle. This shares a striking similarity to the
outer kinetochore complex Mis12 [14,15] (Extended Data Fig. 5c). Nkp1 and Nkp2 create an outer layer of
α-helices in Cenp-OPQU+, which are likely substituted by Cenp-R in vertebrates
[12].The Cenp-HIK module (Fig. 1c), which
resembles the free Cenp-HIK complex (Extended Data Fig.
5d), is dominated by the C-terminal HEAT repeats of Cenp-I (Extended Data Fig. 4e). The coiled-coil
α-helices of Cenp-H and Cenp-K run anti-parallel to Cenp-I (Fig. 1c and Extended Data Fig.
4a-c). The base of Cenp-HIK is a four α-helical bundle comprising the
N-termini of Cenp-H and Cenp-K. The flexible head domain, visible in free Cenp-HIK
(Cenp-HIKHead), and a small population of CCAN particles (Extended Data Figs 3c and 5b, d), matches the shape of the crystal structure of the N-terminal
Cenp-I HEAT repeats that are associated with the C-termini of both Cenp-H and Cenp-K
[16] (Fig. 1d). The Cenp-TW sub-complex, comprising the histone-fold
domain (HFD) subunits Cenp-T and Cenp-W, was not clearly resolved in cryo-EM maps of
CCAN and CCAN–Cenp-ANuc. Cenp-TW associates with Cenp-HIK in solution,
in agreement with previous studies [11,17], and Cenp-THFDW interacts
equally well with a complex comprising Cenp-HIKHead (Extended Data Fig. 1g-j), indicating that the HFDs of Cenp-TW
directly interact with Cenp-HIKHead.The relative organization of CCAN subunits in our cryo-EM reconstruction is in
agreement with that defined from the de novo assembly of the S.
cerevisiae kinetochore [9]
(Extended Data Fig. 1k), and consistent with a
negative stain EM reconstruction of the human HIKM-LN-OPQUR complex [12]. To assess the validity of our
structure, we performed cross-linking mass spectrometry (XL-MS) analysis of the
complexes. Numerous intra and inter-subunit cross-links were identified (Extended Data Fig. 6a, b and Supplementary Tables 1 and 2).
Mapping these cross-links onto CCAN and CCAN–Cenp-ANuc, for which both
lysines of the cross-linked pair are defined, showed that 95% of the detected crosslinks
are within the expected linker distance constraints (Extended Data Fig. 6c-f).
Extended Data Figure 6
Cross-linking mass spectrometry analysis of the CCAN and
CCAN–Cenp-ANuc complexes.
a, b, Circular plots displaying all the identified
cross-links for CCAN (a) and CCAN–Cenp-ANuc
(b). Inter- and intra-subunit cross-links are indicated in
red and blue, respectively c, d, Histogram plots showing the
Cα-Cα distance distribution of the cross-links that could be
mapped onto the CCAN (c), and CCAN–Cenp-ANuc
structures (d). 95% of the mapped cross-links satisfy the
cross-linker imposed distance restraint of 30 Å indicated with a
dashed red line. e, f, Cross-links mapped onto the CCAN
(e) and CCAN–Cenp-ANuc complex
(f). Inter and intra-subunit cross-links are indicated in
red and blue, respectively. Cross-links exceeding the cross-linker imposed
distance restraint of 30 Å are indicated in yellow. g,
Residues on CCAN shown by XL-MS that cross-link with Cenp-C are indicated on
the CCAN structure. Red spheres: cross-links in the
CCAN–Cenp-ANuc complex. Yellow spheres: additional
cross-links unique to apo CCAN. The experiments shown in a and
b were performed independently in triplicate with similar
results.
Kinetochores assemble onto Cenp-ANuc
[9,18], the hallmark of centromeric chromatin, with the CCAN subunits
Cenp-C and Cenp-N directing this assembly [19,20]. In the
CCAN–Cenp-ANuc complex, Cenp-ANuc is an octameric
nucleosome, with DNA wrapped as a left-handed super-helix (Fig. 2 and Supplementary
Video 1), shown previously for free Cenp-ANuc
[8,21-23]. Also
consistent with these reports is that compared with canonical H3 nucleosomes, in the
CCAN–Cenp-ANuc complex, the DNA gyre of Cenp-ANuc is
more loosely wrapped. In CCAN–Cenp-ANuc, only 105 bp of DNA encircle
the Cenp-A-octamer, contrasting with 147 bp for canonical nucleosomes [24] (Figs
2 and 3a-c). A total of 20 bp of DNA are
unwrapped equally at each DNA terminus of Cenp-ANuc. One of the unwrapped DNA
termini, well defined in cryo-EM density, interacts with CCAN, whereas the other is
disordered (Fig. 2a). We observe clearly defined
α-helical density for the N-terminal segment of one Cenp-A subunit
(Cenp-AN), inserted between the unwrapped DNA duplex and DNA gyre (Figs 2a and 3d).
Figure 3
Cenp-LN interacts with the unwrapped DNA duplex of
Cenp-ANuc.
a, b. Two orthogonal views showing the unwrapped DNA duplex of
Cenp-ANuc engaged by the DNA-binding groove of the Cenp-LN
sub-complex. c, Surface of Cenp-LN showing positive electrostatic
potential of the DNA-binding groove. The canonical S.
cerevisiae H3 nucleosome (orange, PDB: 1ID3 [24]) wraps 147 bp of DNA compared
with the 105 bp wrapped by the S. cerevisiae Cenp-A nucleosome
(yellow). d, Zoomed-view showing insertion of the N-terminus of
Cenp-A (Cenp-AN) between the unwrapped DNA duplex and DNA gyre of
Cenp-ANuc. Arg67 of the Cenp-N Pyrin domain inserts into the DNA
major groove.
In the CCAN–Cenp-ANuc complex (Fig. 2 and Supplementary
Video 1), Cenp-ANuc inserts end-on into the
‘Y’-shaped opening of CCAN with each arm of CCAN embracing opposite sides
of the nucleosome. This positions the Cenp-LN module to form extensive contacts with the
unwrapped DNA duplex at one of the termini of the Cenp-ANuc DNA gyre (Fig. 2). Cenp-LN adopts a ‘U’-shaped
structure creating an evolutionarily conserved positively charged groove that engages
the unwrapped DNA (Fig. 3c and Extended Data Fig. 7a-c). The DNA duplex runs along the Cenp-LN
groove, exiting opposite to the nucleosome (Figs 2
and 3a-c). Cenp-HIKHead also functions
in Cenp-ANuc recognition because in the CCAN–Cenp-ANuc
complex, EM density corresponding to Cenp-HIKHead–Cenp-TW contacts the
DNA gyre of Cenp-ANuc, with Cenp-I in close proximity to Cenp-A (Fig. 2d, Extended Data
Fig. 3c and Supplementary
Video 1). Compared with apo-CCAN, Cenp-HIKHead–Cenp-TW
rotates ~90° to accommodate Cenp-ANuc (Extended Data Fig. 5e). Previous studies suggested that the
vertebrate Cenp-TWSX heterotetramer forms a nucleosome-like particle to interact with
DNA [25]. However, this is not
compatible with Cenp-TW of budding yeast exactly co-localizing with centromeric
Cenp-ANuc, in a Cenp-I-dependent manner [17]. The HFDs of Cenp-TW were assigned to EM density
associated with Cenp-HIKHead contacting the DNA gyre of Cenp-ANuc,
visible in a minor 3D class of CCAN–Cenp-ANuc (Fig. 2e and Extended Data Fig.
3c). On the opposite side of CCAN to Cenp-HIK, the N-terminal regions of
Cenp-Q and Cenp-U contact the DNA gyre of Cenp-ANuc, and the N-termini of
Cenp-A and H4 (Fig. 2c, f). This is consistent with
the Cenp-QU dimer binding DNA [26] and
recognising the posttranslational status of the N-terminus of Cenp-A [27], and further validated by our XL-MS
data revealing Cenp-Q crosslinks to H2A and H2B (Extended
Data Fig. 6b).
Extended Data Figure 7
The S. cerevisiae Cenp-ANuc nucleosome is
unwrapped.
a-c, The positively-charged electrostatic potential of
the DNA-binding groove of Cenp-LN sub-complex is conserved in S.
cerevisiae, S. pombe and H.
sapiens. S. pombe and H.
sapiens are modelled structures. d, Cenp-N
interacts with ScCenp-ANuc in the context of
CCAN differently from the interaction of free human Cenp-N with
Cenp-ANuc. The Cenp-N subunit of the human
Cenp-N–Cenp-A nucleosome structure (PDB: 6C0W [29]) was superimposed onto
Cenp-N of the S. cerevisiae
CCAN–Cenp-ANuc structure. In this mode of
Cenp-N–Cenp-ANuc interactions, Cenp-ANuc
would clash with Cenp-OPQU+ and Cenp-N of CCAN. e, Structure
ScH3Nuc (PDB: 1ID3 [24]) and f,
Cenp-ANuc (this work). g, sequence alignment of
the N-terminal regions of ScH3 and Cenp-A (Cse4) histones.
For the chimeric H3N-Cenp-ANuc, residues 1-50 of
ScH3 were substituted for residues 1-140 of
ScCenp-A. A similar approach was used for vertebrate
Cenp-ANuc
[23].
Cenp-N engages Cenp-ANuc in the budding yeast
CCAN–Cenp-ANuc complex differently from how the isolated
vertebrate Cenp-N subunit interacts with Cenp-ANuc through the L1 loop of
Cenp-A and the adjacent DNA gyre [28,29]. Because of steric clashes, the
interaction of Cenp-N with Cenp-ANuc revealed in these studies is not
compatible with the position of Cenp-N in the context of the CCAN complex (Extended Data Fig. 7d). Binding of
Cenp-ANuc at this interface of CCAN, as proposed [10], would require substantial
conformational changes of CCAN. The discrepancy between our structure and that of the
vertebrate system, may either reflect genuine species differences in
CCAN–Cenp-ANuc architectures, or result from the vertebrate
Cenp-N–Cenp-ANuc structure representing an intermediate in the
CCAN–Cenp-ANuc assembly pathway, in accordance with
CCAN–Cenp-ANuc remodelling during the cell cycle [11].Cenp-C also determines kinetochores–Cenp-ANuc interactions
[20] and we found that Cenp-C is
required for stable assembly onto Cenp-A-Cen3 nucleosomes (data not
shown), although not Cenp-A-601 nucleosomes (Fig.
4b). Cenp-C interacts with Cenp-A through its Cenp-C motif (Extended Data Fig. 5f), similar to vertebrates
[30]. However, the regions of
Cenp-C associated with CCAN were not visible in the cryo-EM map. XL-MS data indicate
that Cenp-C participates in multiple interactions with CCAN (Extended Data Figs 6a, b, g and Supplementary Tables 1 and
2).
Figure 4
The Cenp-N DNA binding groove is required for stable CCAN –
Cenp-ANuc interactions.
a, Surface of the Cenp-LN module showing the Cenp-N DNA binding
groove engaging the unwrapped DNA, indicating the 13 mutated Arg and Lys
residues of Cenp-N. Inset: overview of CCAN–Cenp-ANuc showing
the Cenp-A L1 loop. b, Size exclusion chromatograms of various
CCANΔCenp-C–Cenp-ANuc complexes. Wild
type CCANΔCenp-C forms a complex with Cenp-ANuc,
but mutating the Cenp-N DNA binding groove weakens CCAN –
Cenp-ANuc interactions (Extended
Data Fig. 8c, d). The binding of both CCANΔCenp-C
and CCANΔCenp-C-Cenp-NMut to
H3N-Cenp-ANuc is severely disrupted, with little
complex formed (Extended Data Fig. 8g, h).
The positions of complexes are indicated by arrows. (CCANΔC =
CCANΔCenp-C). This experiment was performed independently
in triplicate with similar results. c, The DNA-binding groove
functions in vivo. Wild type Cenp-N
(CHL4) rescues the growth defect of the
chl4Δ cse4-R73A mutant strain at 37 °C,
whereas the Cenp-NMut (chl4) does not.
WT: wild type strain. This experiment was performed independently ten times with
similar results. d, Western blot demonstrates that
Cenp-NWT and Cenp-NMut are expressed at equivalent
levels in the chl4Δ cse4-R73A mutant strain.
e, Loading control. Coomassie-blue stained gel shows dynein and
acetyl-CoA carboxylase. Experiments in d and e were
performed independently in triplicate times with similar results.
f, Two views showing a representation of dimeric
CCAN–Cenp-ANuc complex with the second CCAN protomer
generated by the dyad symmetry of Cenp-ANuc. Sites of contact to the
outer kinetochore (KT) (through Cenp-U and Cenp-T) are indicated. For gel source
data see Supplementary Fig.
1.
To test the validity of the CCAN–Cenp-ANuc structure, we
mutated 13 Arg and Lys residues in Cenp-N that line the Cenp-LN–DNA binding
groove (Fig. 4a) and tested the ability of the
mutant CCAN to assemble onto Cenp-ANuc. To avoid complications of Cenp-C
interacting with Cenp-ANuc, we used CCAN without Cenp-C
(CCANΔCenp-C). The Cenp-N mutant did not impair
CCANΔCenp-C assembly, and similar to CCAN,
CCANΔCenp-C binds to Cenp-A-601 nucleosomes, but not H3
nucleosomes (Fig. 4b and Extended Data Figs 8a-c and 9a,
b). The Cenp-N mutant disrupted CCANΔCenp-C –
Cenp-ANuc interactions (Fig. 4b and
Extended Data Fig. 8d). In contrast, mutating
the L1 loop of Cenp-A did not disrupt the binding of CCANΔCenp-C to
Cenp-ANuc (Extended Data Figs 8e
and 9a).
Extended Data Figure 8
SDS PAGE gels of
CCANΔCenp-C–Cenp-ANuc
complexes.
a-h, Coomassie-blue stained SDS PAGE gels of various
CCANΔCenp-C–Cenp-ANuc complexes.
Corresponding SEC chromatogram is shown in Fig. 4b and Extended Data Fig.
9a. a, b, Mutating the Cenp-N DNA binding groove did
not impair CCANΔCenp-C assembly. c, Wild type
CCANΔCenp-C forms a complex with Cenp-ANuc.
d, Mutating the Cenp-N DNA binding groove disrupts
CCANΔCenp-C – Cenp-ANuc
interactions. e, Mutating the L1 loop of Cenp-A did not
destabilize CCANΔCenp-C – Cenp-ANuc
interactions. f, Deletion of the N-terminus of Cenp-A (1-129)
(ΔNCenp-ANuc) did not impair
CCANΔCenp-C – Cenp-ANuc
interactions. h, Both CCANΔCenp-C and
CCANΔCenp-C-Cenp-NMut bound poorly to
H3N-Cenp-ANuc. The experiments shown were
performed independently in triplicate with similar results. For gel source
data see Supplementary
Fig. 1.
Extended Data Figure 9
Testing of CCANΔCenp-C binding to
Cenp-ANuc.
a, Comparative size exclusion chromatogram profiles
(Agilent Bio SEC-5 column) for wild type CCANΔCenp-C and
the Cenp-NMut of CCANΔCenp-C to
Cenp-ANuc and its modifications
(Cenp-ANuc-L1Nuc,
ΔNCenp-ANuc,
H3N-Cenp-ANuc) and H3Nuc. Mutating the
L1 loop (Cenp-A-L1Nuc) of Cenp-A or deletion of the N-terminal
129 residues (ΔNCenp-ANuc) did not destabilize
CCANΔCenp-C – Cenp-ANuc
interactions. In contrast, CCAN with the Cenp-NMut bound less
well and both CCAN and CCAN-Cenp-NMut bound hardly at all to
H3N-Cenp-ANuc. (CCANΔC =
CCANΔCenp-C). Associated SDS PAGE gels in Extended Data Fig. 8 and Extended Data Fig. 9b). b,
Coomassie-blue stained SDS PAGE gel showed that
CCANΔCenp-C did not associated with H3Nuc.
c, Micrococcal nuclease digestion of Cenp-ANuc,
H3Nuc and H3N-Cenp-ANuc. 601 DNA is
shown as a control. The H3Nuc and
H3N-Cenp-ANuc protect a similar and longer length
of DNA compared with Cenp-ANuc. d, Model of CBF3
[60] bound to
CCAN–Cenp-ANuc indicating that CBF3 would not
associate with a fully assembled kinetochore, consistent with proteomic data
[62]. The
experiments shown in a-c were performed independently in
triplicate with similar results. For gel source data see Supplementary Fig.
1.
We then assessed the role of the unwrapped DNA termini of Cenp-ANuc
in mediating CCAN – Cenp-ANuc interactions. Because the
αN-helix of the H3 histone stabilizes the wrapped DNA termini of canonical H3
nucleosomes [22,24], to create a more closed, highly wrapped
Cenp-ANuc, we substituted the N-terminal 50 residues of H3 for the
N-terminal 140 residues of Cenp-A, creating a chimeric H3N-Cenp-A (Extended Data Fig. 7e-g). The resultant
H3N-Cenp-ANuc wrapped a similar length of DNA as
H3Nuc (~147 bp) (Extended Data Fig.
9c). The affinity of CCANΔCenp-C for
H3N-Cenp-ANuc was severely disrupted, such that
CCANΔCenp-C was substantially dissociated from
H3N-Cenp-ANuc (Fig. 4b
and Extended Data Fig. 8g). Binding of
H3N-Cenp-ANuc to CCANΔCenp-C was completely
disrupted with the Cenp-N mutant (Fig. 4b and Extended Data Figs 8h). The reduced affinity of CCAN
for H3N-Cenp-ANuc is not due to the lack of the Cenp-A N-terminus
because CCAN bound to Cenp-ANuc and ΔNCenp-ANuc
equally well (Fig. 4b and Extended Data Fig. 8c, f). These biochemical studies confirm the
CCAN – Cenp-ANuc cryo-EM structure showing that CCAN interacts with
the unwrapped DNA termini of Cenp-ANuc, and that a major role of the Cenp-LN
DNA-binding groove is to engage the unwrapped DNA gyre of Cenp-ANuc (Fig. 3c).Disruption of the budding yeast Cenp-N gene (CHL4) causes
chromosome loss and instability, without affecting viability [31]. However, combining a chl4 deletion
with either mutation of Cenp-A (CSE4), or deletion of other kinetochore
subunits, results in synthetic growth defects and lethality [9,27]. Cenp-N is an
essential gene in S. pombe and humans. To investigate the in
vivo consequences of disrupting the DNA-binding groove of Cenp-LN, we
tested whether the synthetic growth defect of the chl4Δ
cse4-R37A mutant at 37 °C [27] is rescued by Cenp-NMut. Whereas wild type Cenp-N
rescued the growth defect of the chl4Δ cse4-R37A mutant, the
Cenp-NMut did not (Fig. 4c-e). This
result demonstrates a functional role for the Cenp-LN DNA-binding groove, and together
with our biochemical data (Fig. 4b and Extended Data Fig. 8), supports the CCAN –
Cenp-ANuc architecture we report here. In budding yeast,
Cenp-ANuc is linked to the outer kinetochore Ndc80 complex and associated
microtubules through a pathway comprising the essential proteins Cenp-C, Cenp-QU and the
Mis12 complex, and by a second pathway involving Cenp-TW and Cenp-N [9] (Extended
Data Fig. 1k). The location of Cenp-N at the centre of CCAN is consistent
with these two pathways. The unwrapped DNA termini of Cenp-ANuc contribute to
stabilizing the CCAN – Cenp-ANuc complex through the Cenp-LN DNA
binding groove, augmented by contacts of both Cenp-A and the Cenp-ANuc DNA
gyre with Cenp-C (Extended Data Fig. 5f), Cenp-LN
(Fig. 3c), Cenp-TW, Cenp-HIKHead and
Cenp-QU [27] (Fig. 2d-f).In the cryo-EM reconstruction, Cenp-ANuc is associated with a single
CCAN, whereas the expected stoichiometry is two CCANs to Cenp-ANuc
[32]. SEC-MALS and AUC confirmed the
reconstituted CCAN–Cenp-ANuc is consistent with two CCANs per
Cenp-ANuc (Extended Data Fig.
10a-g). In a generated model of dimeric CCAN–Cenp-ANuc, two
CCAN complexes associate through their tips of the ‘Y’, creating a slot
that perfectly accommodates Cenp-ANuc that is inserted vertically (Fig. 4f). The two CCAN complexes cradle
Cenp-ANuc with its unwrapped DNA duplexes stretched out, over-lying
CCAN’s DNA-binding surface, consistent with XL-MS cross-links between Cenp-Q and
Cenp-TW (Extended Data Fig. 6b). Extensive 2D
classification of the cryo-EM data identified 2D classes of dimeric
CCAN–Cenp-ANuc particles with two-fold symmetry axes (Extended Data Fig. 2c). These particles correspond
closely to the calculated reprojections of the proposed dimeric
CCAN–Cenp-ANuc complex (Extended
Data Fig. 10h). Cryo-EM grids destabilize CCAN – Cenp-ANuc,
resulting in a very low abundance of dimeric CCAN–Cenp-ANuc
particles.
Extended Data Figure 10
S. cerevisiae CCAN–Cenp-ANuc comprises
two CCAN complexes in solution.
The predicted mass of (CCAN)2–Cenp-ANuc
is 1.31 MDa, (CCAN)1–Cenp-ANuc is 0.77 MDa and
that for a CCAN dimer 1.09 MDa (Extended Data Table 2).
Representative SEC-MALS data for a, cross-linked S.
cerevisiae CCAN–Cenp-ANuc complex, run
independently in triplicate with similar results, average molecular mass is
1.23 MDa [(CCAN)2–Cenp-ANuc]. b,
uncross-linked S. cerevisiae
CCAN–Cenp-ANuc complex, run independently
in triplicate with similar results, with average masses of 1.38 MDa
[(CCAN)2–Cenp-ANuc] and 526 kDa
[(CCAN)1]. c, S. cerevisiae
CCAN alone, run independently in duplicate with similar results, with
average masses of 839 kDa for the leading edge (green) and 650 kDa for the
trailing edge (magenta) suggesting a non-resolved monomer-dimer equilibrium.
Velocity analytical ultracentrifugation of d, cross-linked and
e, uncross-linked S. cerevisiae
CCAN–Cenp-ANuc complexes with residuals to the fits
below of a c(s) distribution model: f, for the cross-linked
complex, the major species sediments at 15.8 S (Sw,20 = 26.1 S)
with a minor species at 12.1 S (Sw,20 = 20.0 S) that corresponds
to calculated masses of 1.34 MDa
[(CCAN)2–Cenp-ANuc] and 896 kDa [possibly
(CCAN)1–Cenp-ANuc] respectively with a
fitted value of 1.761 for the frictional ratio; g, for
uncross-linked samples, the major species is resolved into two species that
sediment at 14.3 S (Sw,20 = 22.6 S) and 15.7 S (Sw,20
= 24.9 S) with a minor species at 12.3 S (Sw,20 = 19.4 S) which
gave masses of 1.32 MDa [(CCAN)2–Cenp-ANuc] and
1.15 MDa [(CCAN)2] for the major species and 716 kDa
[(CCAN)1–Cenp-ANuc] for the minor species.
The experiments shown in d-g were performed independently in
triplicate with similar results. h, Examples of two 2D class
averages showing the dimeric CCAN–Cenp-ANuc particles
viewed in the plane of the C2 symmetry axis (red outline) (data from Extended Data Fig. 2c) and the 2D
reprojections of a modelled dimeric CCAN–Cenp-ANuc based
on the CCAN–Cenp-ANuc cryo-EM reconstruction (yellow
outline) (Extended Data Fig. 10i).
There is a close correspondence in shape and dimensions between the
calculated reprojections and the observed 2D classes. The two-fold symmetry
axes of the dimeric CCAN-Cenp-ANuc complex are shown as dashed
arrows. i, j, Two alternative models for how CCAN assembled
onto a Cenp-A nucleosome would interact with the outer
kinetochore–microtubule interface (Supplementary Video
2). i, In scenario (1), CCAN interacts with the outer
kinetochore from the same side as the DNA-binding surface. Microtubules
attached to the outer kinetochore would hoist CCAN from below the over-lying
nucleosome and out-stretched DNA. j, In scenario (2), the
microtubule-outer kinetochore interface contacts CCAN from the opposite side
to the CCAN-DNA binding surface. Outer-KT (outer-kinetochore): KMN network
and microtubule attachment complexes: Dam1/DASH (budding yeast) and Ska
proteins of vertebrates. The combined dimension of dimeric
CCAN–Cenp-ANuc (32 nm) matches that of the hub at the
centre of the yeast kinetochore [63].
In S. cerevisiae, the CBF3 complex engages the CDEIII element
of the ~125 bp centromere to direct Cenp-A/Cse4 nucleosome deposition. Modelling
indicates that only when bound to a single CCAN promoter can Cenp-ANuc
simultaneously accommodate CBF3 (Extended Data Fig.
9d), suggesting that CBF3 would not associate with a fully assembled
kinetochore.The dimeric CCAN–Cenp-ANuc complex suggests two possibilities
for how a kinetochore-attached microtubule would segregate centromeric chromatin (Extended Data Fig. 10i, j and Supplementary Video 2). In one
scenario, CCAN attaches to the microtubule through the outer kinetochore, using the same
face as its DNA-binding surface (Extended Data Fig.
10i). This would sandwich the DNA between CCAN and the outer kinetochore, a
possibility compatible with the long flexible linkers that attach CCAN to the outer
kinetochore. As the microtubule pulls on the kinetochore, CCAN would hoist the
over-lying DNA. Alternatively, microtubules could attach to CCAN from the opposite face
to its DNA-binding surface, so the chromosome is pulled from behind the inner
kinetochore (Extended Data Fig. 10j). Because
vertebrate Cenp-ANuc also wraps between 100-120 bp (of α-satellite
DNA) [22], with nucleosome unwrapping
enhanced by Cenp-C [33], and the human
CCAN architecture [12] is similar to
yeast, it is likely that the mechanism of recognition of the specialized Cenp-A
nucleosome, we describe here for the budding yeast inner kinetochore, is evolutionarily
conserved.
Methods
Cloning, expression, purification and reconstitution of recombinant
CCAN–Cenp-ANuc nucleosome complex
Cloning
The genes for CTF19, OKP1, MCM21, AME1, NKP1,
NKP2, CTF3, MCM16, MCM22, CNN1, WIP1, MIF2, CHL4 and
IML3 (MCM19) (Extended Data Table 2 for vertebrate Cenp conversion)
were amplified by PCR from Saccharomyces cerevisiae genomic
DNA and cloned into a pU1 plasmid using a modified Multibac expression
system [34]. The intron in
MCM21 was deleted by USER methodology. A double StrepII
tag together with a TEV cleavage site was attached to the C-termini of Ame1,
Ctf3, Chl4, Mif2 and Cnn1 proteins. For expression of the Cenp-OPUQ+ complex
(COMA+: Ctf19, Okp1, Mcm21, Ame1, Nkp1 and Nkp2) gene expression cassettes
in pU1 were subsequently cloned into a pF2 vector [34]. The gene expression cassettes for
CTF3, MCM16, MCM22, CNN1 and WIP1 were cloned into pF2
to generate the Cenp-HIK–TW complex.
Cenp-HIK-TW complexes
To test which regions of Cenp-H, Cenp-I and Cenp-K interact with
each other and with Cenp-TW, the following fragments of Cenp-H, Cenp-I and
Cenp-K were constructed: Cenp-I (residues 1-308) (Cenp-IN),
Cenp-H (residues 137-182) (Cenp-HC), Cenp-H (residues 130-239)
(Cenp-KC) and combinations of Cenp-H, Cenp-I and Cenp-K,
together with Cenp-TW were for assembled into the pU1 plasmid for Multibac
expression [34] for
co-expression using the insect cell/baculovirus system. A double StrepII tag
was added to C-terminus of Cenp-I.To test the role of the positively-charged DNA-binding groove of
Cenp-N for Cenp-A nucleosome interactions, a total of 13 Arg and Lys
mutations were introduced into CHL4 (Cenp-NMut)
by total gene synthesis (GeneArt/Thermo Fischer):
chl4
Cenp-NMut was combined with Cenp-L to generate a
Cenp-NMut-Cenp-L co-expression baculovirus.The baculoviruses for expression of Cenp-OPQU+, Cenp-HIK–TW,
Cenp-C and Cenp-LN were prepared for expression using the insect
cell-baculovirus system [34].The cDNA encoding for Saccharomyces cerevisiae CSE4 (S.
cerevisiae CENP-A), H2A, H2B and H4 histone genes were
synthesized (GeneArts/Thermo Fisher) with optimized codons for expression in
Escherichia coli and were subsequently cloned into
pET28A with a TEV protease cleavable N-terminal His6 tag. For the
recombinant Cse4 octamer (ScCenp-A octamer), four
expression cassettes for CSE4, H2A, H2B and H4 histone
genes were subsequently cloned into a single pET28 plasmid by USER
methodology for E. coli expression. For
ScH3 octamer purification CSE4 was
replaced by the H3 gene. The Cenp-A L1 loop mutant
(Cenp-AL1:
cse4) and
cse4 (ΔNCenp-A)
were expressed for producing Cenp-AL1 and
Cenp-AΔN octamers and nucleosomes, respectively. The
chimeric H3N-Cenp-A histone comprises a fusion of residues 1-50
of ScH3 with residues 141-229 of CSE4. The
H3N-Cenp-A histone (molecular mass 15.74 kDa) was used to
generate H3N-Cenp-ANuc-601 by the same procedure as
for Cenp-ANuc.
Expression and purification
Complexes of Cenp-OPQU+, Cenp-HIK, Cenp-HIK–TW, Cenp-LN and
Cenp-C were expressed individually in High-5 insect cells (Trichoplusia ni:
expression system). The High-5 insect cell line was not tested for
mycoplasma contamination and was not authenticated. The cells were harvested
48 h after infection. The lysate was loaded onto a Strep-Tactin ®
Column (Qiagen) and the complexes were eluted with 2.5 mM desthiobiotin
(Sigma) in a buffer of 50 mM Tris.HCl (pH 8.0), 200 mM NaCl, 1 mM DTT. The
StrepII-tag was cleaved using TEV protease overnight at 4˚C. The
proteins and complexes were further purified on Resource Q anion exchange
and size exclusion chromatography in a buffer of 20 mM Hepes (pH 8.0), 200
mM NaCl, 2 mM DTT. Free Cenp-HIK was cross-linked using 0.05% glutaraldehyde
for 8 min on ice and quenched with 50 mM Tris.HCl (pH 8.0), then further
purified using Superose 6 size exclusion chromatography. The proteins and
complexes were collected, concentrated, frozen in liquid nitrogen and stored
at -80˚C. The stable 14-subunit CCAN complex was reconstituted by
combining individually purified CCAN sub-complexes; Cenp-LN, Cenp-OPQU
together with the budding yeast-specific Nkp1 and Nkp2 subunits
(Cenp-OPQU+), Cenp-HIK–TW and Cenp-C.For Cenp-HIK-TW assembly assays, a combination of full length and
either their N or C terminal fragments of Cenp-I, Cenp-H and Cenp-K were
co-expressed together with Cenp-T and Cenp-W or with Cenp-THFD
(residues 268-361) and Cenp-W. Affinity purified complexes were analysed
using SDS-PAGE analysis.The ScCenp-A octamer was prepared by co-expression
of CSE4, H2A, H2B and H4 in
B834
E. coli cells. The harvested cell pellet was lysed in a
buffer of 50 mM Tris.HCl (pH 8.0), 2 M NaCl. The ScCenp-A
octamer was isolated by Ni-NTA affinity chromatography, eluted with
imidazole in 2 M NaCl buffer. The octamer was further purified by S200 size
exclusion chromatography, concentrated to 3 mg/mL in a buffer of 10 mM
Tris.HCl (pH 7.5), 2 M NaCl, 1 mM EDTA and 2 mM DTT and frozen in liquid
nitrogen and stored at -80˚C.For DNA fragment preparation, NEB Stable E. coli
cells containing a plasmid with a multiple copy (20x) of the 147 base pair
Widom 601 sequence flanked by EcoRV sites in a pUC18 backbone (gift from
Fabrizio Martino, MRC-LMB) were cultured in LB broth with ampicillin. The
plasmid was isolated by using the Plasmid Giga Kit (Qiagen). The Widom 601
fragment was purified with a 1 mL resource Q anion exchange chromatography
column (GE Healthcare Life Sciences) after over-night digestion with
EcoRV-HF (NEB). The purified DNA was precipitated, dissolved,
buffer-exchanged and stored in a buffer of 2 M NaCl, 10 mM Tris.HCl (pH
7.5), 1 mM EDTA, 2 mM DTT at -20˚C. CEN3 DNA
fragment was prepared by the primer-extension method. Two oligos used were:
CEN3F ATAAGTCACA TGATGATATT TGATTTTATT ATATTTTTAA AAAAAGTAAA AAATAAAAAG
TAGTTTATTT TTAAAAAATA AAATTTAAAA and CEN3R TTCAATGAAA TATATATTTC TTACTATTTC
TTTTTTAACT TTCGGAAATC AAATACACTA ATATTTTAAA TTTTATTTTT TAAAAATAAA CTA
(Sigma-Aldrich). The fragment was produced in a one step extension at 68
˚C for 1 min. The final product of the 153 base pair
CEN3 (ATAAGTCACA TGATGATATT TGATTTTATT ATATTTTTAA
AAAAAGTAAA AAATAAAAAG TAGTTTATTT TTAAAAAATA AAATTTAAAA TATTAGTGTA TTTGATTTCC
GAAAGTTAAA AAAGAAATAG TAAGAAATAT ATATTTCATT GAA) fragment was purified using
a 1 mL resource Q anion exchange chromatography and stored in a buffer of 2
M NaCl, 10 mM Tris.HCl (pH 7.5), 1 mM EDTA, 2 mM DTT at -20˚C.
ScCenp-A nucleosome and derivatives preparation
ScCenp-A, Cenp-A-L1Mut,
ΔNCenp-A, H3N-Cenp-A and H3 histone
octamers were wrapped by gradient dialysis from 2 M NaCl to 100 mM NaCl
buffer with 10 mM Tris.HCl (pH 7.5), 1 mM EDTA and 2 mM DTT.
ScCenp-A octamer was mixed with either 601 DNA or
CEN3 DNA, at 7.8 μM concentration. The mixture
in the dialysis tube was inserted into a 500 mL beaker containing 500 mL
buffer of 2 M NaCl, 10 mM Tris.HCl (pH 7.5), 1 mM EDTA, 2 mM DTT. The NaCl
concentration in the dialysis buffer was gradually decreased to 100 mM using
an Akta pump at 1.5 mL min-1 for 16 hours at 4˚C. The
mixture was further dialysed against the buffer of 100 mM NaCl, 10 mM
Tris.HCl (pH 7.5), 1 mM EDTA, 2 mM DTT for 4 hours at 4°C. The
ScCenp-A nucleosome and derivatives were stored at
4°C.
Reconstitution of CCAN−Cenp-A nucleosome complex
The CCAN–Cenp-A nucleosome complex was reconstituted by
mixing purified Cenp-C and Cenp-LN with Cenp-A nucleosome followed by
Cenp-HIK–TW and Cenp-OPQU+. The stoichiometry of CCAN sub-complexes
to Cenp-ANuc was adjusted so that CCAN sub-complexes were in
excess, as judged by their separation from CCAN–Cenp-ANuc
by size exclusion chromatography. The mixed sample was dialysed over-night
in a buffer of 10 mM Hepes (pH 8.0), 80 mM NaCl, 1 mM EDTA and 0.5 mM TCEP
at 4˚C. CCAN–Cenp-ANuc was purified by Superose 6
size exclusion chromatography. For cryo-EM analysis,
CCAN−Cenp-ANuc was cross-linked with 5 mM BS3 (Thermo
Fisher Scientific) for one hour on ice and quenched with 50 mM Tris and then
subjected to further size exclusion chromatography with an Agilent Bio SEC-5
column (Agilent Technologies) before preparing cryo-EM grids. Mild
cross-linking of CCAN−Cenp-ANuc reduced dissociation of
CCAN from Cenp-ANuc during preparation of cryo-EM grids. To
assess whether cross-linked created artefacts, we also collected a cryo-EM
data set using uncross-linked CCAN–Cenp-ANuc.
SEC analysis of CCAN−Cenp-ANuc complexes
To analyse the formation and stability of
CCAN−Cenp-ANuc complexes and mutants in CCAN and
Cenp-A, all CCAN−Cenp-ANuc complexes were assembled as
above (with or without Cenp-C) and then applied to an Agilent Bio SEC-5 size
exclusion chromatography column. The eluted fractions were analysed on SDS
PAGE gels and stained with Coomassie Blue and ethidium bromide to detect
proteins and DNA. For assembly of the CCAN−Cenp-ANuc
complexes, the concentration of Cenp-ANuc was 1.6 μM, and
that for the individual CCAN sub-complexes (1.6 μM).
Multi-angle light scattering
SEC-MALS was performed using a Wyatt MALS system. CCAN alone,
uncross-linked and BS3 cross-linked CCAN−Cenp-ANuc
complexes were injected onto an Agilent Bio SEC-5 column gel filtration
column pre-equilibrated in 10 mM Hepes (pH 7.5), 80 mM NaCl, 1 mM EDTA and
0.5 mM TCEP. The light scattering and protein concentration at each point
across the peaks in the chromatograph were used to determine the absolute
molecular mass from the intercept of the Debye plot using Zimm’s
model as implemented in the ASTRA v5.3.4.20 software (Wyatt Technologies).
To determine inter-detector delay volumes, band-broadening constants and
detector intensity normalization constants for the instrument, we used
aldolase as a standard prior-to sample measurement. Data were plotted with
the program PRISM v8.2.0 (GraphPad Software Inc.).
Analytical ultracentrifugation
Uncross-linked and BS3 cross-linked CCAN–Cenp-ANuc
complex at approximately 1 mg/mL in 10 mM Hepes (pH 7.5), 80 mM NaCl, 1 mM
EDTA and 0.5 mM TCEP were subjected to velocity sedimentation at 40,000 rpm
at 4 ˚C in an An50Ti rotor using an Optima XL-I analytical
ultracentrifuge (Beckmann). The data were analysed in SEDFIT 16.1 [35] using a c(s) distribution
model. The partial-specific volumes (v-bar) were calculated using Sednterp
(v20130813 beta) (Dr Thomas Laue, University of New Hampshire). The density
and viscosity of the buffer were determined with a DMA 4500M density meter
(Anton Parr) and an AMVn viscometer (Anton Paar). Data were plotted with the
program GUSSI [36].
Micrococcal nuclease digestion assay
Nucleosomes were digested for 40 min with 1 unit of MNase (NEB) per
microgram of DNA at room temperature (22 °C). Reactions were
terminated with the addition of excess EGTA. The digested nucleosome
mixtures were loaded onto an agarose gel and stained to visualize the
DNA.
Yeast strains and growth analysis
The S. cerevisiae strain with a
chl4 deletion and cse4-R37A mutation
(chl4Δ cse4-R37A), AEY4992
(MATα ade2-101 lys2 his3-11,15 trp1-1
leu2-3,112 ura3-1 can1-100 chl4∆::kanMX cse4-R37A) and
wild type S. cerevisiae strain (W303)
(MATα ade2-101 his3-11,15 trp1-1 leu2-3,112
ura3-1) were described and authenticated in [27,37]. Yeast strains do not have mycoplasma and were not
tested for mycoplasma contamination. Cenp-NWT and
Cenp-NMut strains were created by transforming AEY4992
[27,37] with a 2µ origin plasmid pYes2
incorporating either CHL4 or
chl4
(chl4)
with CHL4’s native promoter, a C-terminal double
StrepII-tag on Chl4, and the URA3 selection marker. The
transformed cells were selected on synthetic media lacking uracil, and the
presence of the plasmid-encoded CHL4 was verified by PCR
using a primer pair over-spanning the CHL4 and
URA3 genes. Cells were grown in drop-out uracil (SC-U)
medium at 30°C and spotted in tenfold dilution steps on YPED plates.
The plates were incubated at either 30°C or 37°C for three
days.
Immunoprecipitation and Western blotting for detecting Cenp-N expression
in the chl4Δ cse4-R37A yeast
Six litres of synthetic SC-U culture were inoculated with the
chl4Δ cse4-R37A yeast strain transformed with
the pYes2 plasmid expressing either wild type or mutant Cenp-N with a
C-terminal double StrepII-tag (and empty vector control) and harvested at
OD600 of ~0.8. Pelleted cells were lysed in buffer (50
mM Tris, pH 8.0, 300 mM NaCl, 1 mM EDTA, 1 mM DTT) and the cleared lysate
was loaded onto a 1 mL Streptactin column. Fractions were eluted with 5 mM
desthiobiotin and analysed by SDS PAGE. Western blotting was performed with
an anti-Strep antibody (MCA2489P, Bio-Rad) that detected the C-terminal
double StrepII-tag on Cenp-N. Total protein was analysed by Coomassie blue
staining for loading controls (normalized loading).
Electron microscopy data collection
3.0 μl of the CCAN−Cenp-ANuc complex at a
concentration of ~1 mg/mL was applied to glow-discharged copper 300
mesh Quantifoil R1.2/1.3 holey carbon grids (Quantifoil Micro Tools GmbH)
(no carbon support). The grids were flash frozen by being plunged into
liquid ethane using an FEI Vitrobot Mark IV (waiting time, 20 s, blotting
time, 2 s). EM image stacks were collected with Falcon III cameras in
counting mode on four different FEI Titan Krios electron microscopes at a
nominal magnification of 75 K (yielding pixel sizes of 1.065Å, 1.070
Å, 1.085 Å, 1.090 Å, respectively). The images were
recorded at a dose rate of 0.6 electrons per pixel per second and the total
exposure time was 60 s (75 frames) with the FEI automated low-dose
data-collection program EPU. Defocus varied from -2.0 to -2.8µm with
an interval of 0.2 µm.For the isolated Cenp-HIK sample, freshly purified Cenp-HIK complex
was first visualized by negative-staining EM to check the sample quality.
Aliquots of 3 µl samples at ~0.2 mg/mL were applied onto
glow-discharged Quantifoil R1.2/1.3 300-mesh holey carbon grids. The grids
were incubated for 30 s at 4 °C and 100% humidity and then blotted
for 8 s and plunged into liquid ethane using an FEI Vitrobot III. Grids made
in this way showed strong preferred orientation. To overcome this problem,
we treated the Cenp-HIK complex with 0.025% glutaraldehyde for 10 min on ice
before size exclusion chromatography purification. More views were observed
after this treatment, allowing us to reconstruct the 3D structure.For the isolated Cenp-HIK sub-complex, images were collected using
EPU with a Falcon III detector in counting mode. 910 micrographs were
collected using a dose rate of 0.5 electrons per pixel per second and a
total exposure time of 60 s. Each micrograph was recorded into a movie stack
of 75 frames. Calibrated physical pixel size is 1.38 Å/pixel.
Image processing
Movie frames were first aligned using MotionCor2 [38]. CTF parameters were
estimated with Gctf [39].
The initial template-free particle picking was performed with Gautomatch
(developed by Kai Zhang, http://www.mrc-lmb.cam.ac.uk/kzhang/Gautomatch/). Subsequent
image processing was carried out using RELION 2.1 and RELION 3.0 [40,41]. A subset of 556 micrographs (of 1582) was used for
Gautomatch template-free particle picking, and the resulting 119,143
coordinates were imported into RELION 2.1 for particle extraction and
reference-free 2D classification. Selected averages from the 2D
classification were used for an initial model reconstruction with
SIMPLE-PRIME [42]. These 2D
class averages were used for template-based particle auto-picking in
Gautomatch for the entire dataset. The extracted particles were subject to
two rounds of reference-free 2D classifications resulting in a dataset of
1,385,496 particles from the combined total of 9,002 micrographs. A tandem
cascade of 3D classifications against the model built with SIMPLE-PRIME
[42] was performed,
and initial iterations were performed without angular search restriction for
each round of classification. After removing the bad particles, 424,577
particles were assigned to CCAN, whereas 193,882 were assigned to the
CCAN−Cenp-ANuc, which were used for the subsequent
Baysian polishing, multi-body refinement, and the final map refinement and
atomic coordinate refinement. Beam-tilt parameters of the particles were
estimated based on the individual dataset, and they were applied during the
Baysian polishing of each dataset in RELION 3.0. 3D refinements and
multi-body refinements were performed with the polished particle stacks
after merging all the datasets. The dataset including all the particles
generated the highest resolution reconstruction with an overall CCAN mask.
The final resolutions for CCAN and CCAN–Cenp-ANuc are 3.55
Å and 4.15 Å, respectively, based on the gold-standard
FSC=0.143 criterion [43]
(Extended Data Fig. 2d).To identify dimeric CCAN-Cenp-ANuc particles, five 2D
classes, whose 2D averages of CCAN-Cenp-ANuc (Extended Data Fig. 2c) showed smeared
density in close proximity to Cenp-ANuc, were selected for
further analyses. The selected particles (10, 553 particles) were subject to
a tandem cassette of 2D classifications, resulting in 556 particles, which
showed clear C2-symmetry 2D averages. These particles were re-extracted from
the micrographs with a box size of 400 pixels to accommodate the bigger
symmetric particles. The re-extracted particles were then subject to further
2D classification, and classified into 20 classes, generating the
representative symmetric 2D averages shown in the red box of Extended Data Fig. 2c. The reprojections
of the modelled dimeric-CCAN-Cenp-ANuc map (filtered to 20
Å resolution) were generated with relion_project. The projections are
shown as Extended Data Fig. 10h. The
small number of particles and highly preferred orientation on the EM grid
(in the plane of the 2-fold symmetry axis) precluded a 3D
reconstruction.
Multi-body refinement
To improve map resolution we performed multi-body refinement (MBR)
in RELION 3.0 [41]. Two
masks were generated. Mask1 comprised Cenp-LN-OPQU+, excluding Cenp-HIK.
Mask2 comprised Cenp-HIK and portions of Cenp-N, L, O and P, (Extended Data Figs 2h, i and 3b). The resultant maps were determined
at 3.45 Å and 3.83 Å resolution, respectively. To further
improve regions at the periphery of Cenp-OPQU+, partial signal subtracted
particles (Cenp-HIK subtracted) were used for a second round of multi-body
refinement. Mask3 included part of Cenp-N and N-terminal regions of Cenp-Q,
Cenp-U, Nkp1 and Nkp2 with small regions of Cenp-O and Cenp-P. Mask4
comprised Cenp-OP, Cenp-LN and C-terminal regions of Cenp-QU, Nkp1 and Nkp2.
Multi-body refinement based on mask3 and mask4 resulted in 3.92 Å and
3.49 Å maps, respectively. The resultant maps derived using
multi-body refinement based on the four masks showed significantly improved
definition of EM densities and were used for model building (Extended Data Figs 2d, h, i and 3b). Careful choice of the boundaries of
mask2 was critical to optimizing the EM density quality for Cenp-HIK.
Including specific regions of Cenp-N, L, O and P within mask2 was critical
to generating maps that allowed side chain definition of the coiled-coil
regions of Cenp-H and Cenp-K (Extended Data
Fig. 4a). This defined the correct assignment and polarity of
these chains. MBR also improved definition of side chains in the base of
Cenp-HIK. The subsequent multi-body refinement using mask3 and mask4
improved side chain definition for the peripheral regions of Cenp-OPQU+.
Portions of the EM density map are shown in Extended Data Fig. 4. A 3D class (4% of total apo-CCAN)
corresponding to dimeric apo-CCAN was determined at 10 Å resolution
(Extended Data Fig. 3a).For the uncross-linked dataset, the same procedures were applied.
123,215 particles from 1,586 micrographs were used for the final
reconstruction of a map at 7.8 Å resolution for the
CCAN–Cenp-ANuc complex (Extended Data Fig. 5a).For the isolated Cenp-HIK complex, the same procedure was applied.
374,158 particles were used for the final reconstruction of a map at 4.3
Å resolution for Cenp-HIK complex.Before visualization, a negative B factor determined with RELION 2.1
was applied to the density map for sharpening. The modulation transfer
function (MTF) of the detector was corrected in the post-processing step
with RELION 3.0 [40]. The
local resolution was estimated with RELION 3.0 [40].
Model building and structure refinement
Apo-CCAN
EM density maps were visualized in COOT[44] and Chimera [45]. The crystal structure of K.
lactis Cenp-OPQ (PDB:5MU3) [46] (equivalent to S. cerevisiae
Cenp-O residues 159 to 362, S. cerevisiae Cenp-P residues
148 to 361 and S. cerevisiae Cenp-Q residues 320 to 342)
and structures of S. cerevisiae Cenp-N (residues 374 to
450), Cenp-L (PDB:4JE3) [47]
and human Cenp-N N-terminal domain (NTD) (PDB: 6EQT) [29] (equivalent to residues 12
to 260 of S. cerevisiae Cenp-N) were fitted into the
cryo-EM density maps of apo-CCAN, with refitting and mutating to the
S. cerevisiae sequence for Cenp-NNTD,
Cenp-O, Cenp-P and Cenp-Q. Based on the excellent quality of the EM
densities, atomic models of Nkp1, Nkp2, Cenp-U, Cenp-Q, Cenp-H (residues 7
to 136), Cenp-I (residues 321 to 728) and Cenp-K (residues 4 to 128) and the
inter-domain region of Cenp-N (residues 261 to 373) were built de novo. Only
short stretches of Cenp-Q (residues 161 to 216) and Cenp-U (residues 131 to
155) were built as polyAla (Extended Data
Table 2). The secondary-structural and disordered regions of the
protein sequences were analysed with PHYRE2 [48] and PSIPred [49]. A model for the Cenp-HIK head domain was
based on the crystal structure of regions of the Cenp-HIK assembly from
C. thermophilum and T. terrestris (PDB
5Z08) [16] corresponding to
S. cerevisiae Cenp-H (residues Asp143 to Ile181),
Cenp-I (residues Leu5 to Ala241) and Cenp-K (residues Ala136 to Thr236) and
derived using PHYRE2 [48].
The 3.5 Å monomeric free CCAN coordinates were rigid-body docked into
the cryo-EM map The Cenp-HIK head domain was fitted to EM density of the
dimeric apo-CCAN. A linker region that connects Cenp-NNTD with
Cenp-NCTD, not present in crystal structures, was built
de novo.
CCAN−Cenp-ANuc
The CCAN complex model was then fit into the
CCAN–Cenp-ANuc cryo EM map. The nucleosome was
modelled on the S. cerevisiae H3 nucleosome (PDB: 1ID3)
[50] with S.
cerevisiae Cenp-A modelled on H. sapiens
Cenp-A (PDB: 3AN2) [22] and
mutated to the S. cerevisiae Cenp-A sequence, and the 601
Widom DNA sequence (PDB: 3LZ0) [51]. The Cenp-C model (PDB: 4X23) [30] in the centromeric
nucleosome was rigid body-docked into the EM density.The apo-CCAN and CCAN–Cenp-ANuc models (excluding
the Cenp-HIK head domains) were optimized by several rounds of real-space
refinement using PHENIX (phenix.real_space_refine) [52]. Standard stereochemical
and secondary structural constraints were applied during the real-space
refinement. The final models were evaluated with COOT[44], PHENIX[52] and MolProbity (http://molprobity.biochem.duke.edu/) [53]. Figures were prepared
using ChimeraX [54],
Chimera[45], and
PyMOL (Molecular Graphics System, 2.0.3, Schrodinger, LLC). Details of the
fitted and refined coordinates in Extended
Data Table 2. Multiple sequence alignments were performed and
displayed using JALVIEW [55].
Cross-linking mass spectrometry analysis
To assess the validity of our structure, we performed cross-linking
mass spectrometry (XL-MS) analysis of the complexes [56]. Three independent
crosslinking reactions were performed for each sample. The CCAN or
CCAN–Cenp-ANuc complexes in 20 mM HEPES pH 7.5, 80 mM
NaCl and at a concentration of 3 mg/mL were crosslinked with 1 mM DSSO for
15 min at room temperature. Each reaction was quenched with Tris.HCl (pH
8.0) to 50 mM and supplemented with urea to 8 M. The samples were reduced by
addition of DTT at a final concentration of 10 mM for 1 hour at room
temperature, and alkylated for 0.5 hour at room temperature in the dark by
addition of iodoacetamide to 50 mM. Protein digestion was performed with
Lys-C at an enzyme-to-protein ratio of 1:75 (w/w) at 30 °C for 3
hours, then the samples were diluted in 50 mM ammonium bicarbonate and
further digested with trypsin at an enzyme-to-protein ratio of 1:75 (w/w) at
37 °C for 16 hours. The digested samples were acidified with formic
acid to 1%, desalted using home-made C18 stage tips, dried and stored at
−80 °C for further use.Each sample was analysed by LC-MS/MS using an Agilent 1290 Infinity
System (Agilent Technologies) in combination with an Orbitrap Fusion Lumos
(Thermo Scientific). Reverse phase chromatography was carried out using a
100-μm inner diameter 2-cm trap column (packed in-house with
ReproSil-Pur C18-AQ, 3 μm) coupled to a 75-μm inner diameter
50 cm analytical column (packed in-house with Poroshell 120 EC-C18, 2.7
μm) (Agilent Technologies). Mobile-phase solvent A consisted of 0.1%
formic acid in water, and mobile-phase solvent B consisted of 0.1% formic
acid in 80% acetonitrile. A 180 min gradient was used, and start and end
percentage buffer B adjusted to maximize the samples separation.MS acquisition was performed using the MS2_MS3 strategy: the MS1
scan was recorded in Orbitrap at a resolution of 60000, the selected
precursors were fragmented in MS2 with CID and the crosslinker signature
peaks recorded at a resolution of 30000. The fragments displaying the mass
difference specific for DSSO were further fragmented in a MS3 scan in the
ion trap (IT) [57]. Each
sample was analysed with Proteome Discoverer 2.3 (version 2.3.0.522) with
the XlinkX nodes integrated [57] and searching against databases generated after
bottom-up analysis of the samples. The crosslink output (Supplementary Tables 1 and
2) was subsequently visualized using the xVis [58] web tool and the
crosslinks mapped onto the cryo-EM structures of CCAN and CCAN–Cenp-A
using PyMOL (Molecular Graphics System, 2.0.3, Schrodinger, LLC) (Extended Data Fig. 6e-g). The
cross-linking mass spectrometry raw files, the associated output and
databases are deposited through the ProteomeXchange Consortium [59].
Modelling the
CCAN–Cenp-ANuc–CBF3–Cen3
complex
To model CCAN and CBF3 simultaneously bound to the Cenp-A
nucleosome, we docked the free unwrapped DNA duplex of the
CCAN–Cenp-ANuc complex onto the
CBF3–Cen3 coordinates (PDB: 6GYS) [60], matching the minor and
major grooves of both complexes. To avoid overlap of CBF3 and CCAN, the dyad
symmetry axis of the Cenp-A nucleosome is positioned seven nucleotides
upstream of the midpoint of CDEII of the Cen3 sequence.
Modelling H. sapiens and S. pombe
Cenp-LN complexes
To generate the H. sapiens Cenp-LN complex we used
residues 1-207 from PDB 6EQT [29], and modelled residues 208-338 and Cenp-N by one-to-one
threading in PHYRE2 [48]
using S. cerevisiae Cenp-LN as a template. S.
pombe Cenp-LN was modelled with PHYRE2 [48] using S.
cerevisiae Cenp-LN as a template. The electrostatic potential
of S. cerevisiae, S. pombe and H.
sapiens Cenp-LN complexes were calculated and displayed in
PyMOL (Molecular Graphics System, 2.0.3, Schrodinger, LLC).
Reconstituted S. cerevisiae
CCAN–Cenp-ANuc complexes.
a, Size exclusion chromatogram profiles (Agilent Bio
SEC-5 column) for (i) CCAN, (ii) CCAN–Cenp-A nucleosome (with 601)
complex, (iii) Cenp-A nucleosome (with 601), (iv) H3 nucleosome (with 601)
and (v) H3N-Cenp-ANuc (with 601). b,
Comparative size exclusion chromatogram profiles (Agilent Bio SEC-5 column)
for CCAN–Cenp-ANuc with the Cenp-A nucleosome wrapped with
either the (i) 147 bp Widom 601 positioning sequence
(CCAN–Cenp-ANuc (601) – as in (a))
or (ii) a 153 bp S. cerevisiae centromeric
Cen3 sequence (CCAN–Cenp-ANuc
(Cen3)). Both complexes eluate at the same volume. CCAN
and the H3 nucleosome do not form a complex (iii). c, Coomassie
blue-stained SDS PAGE gel of the 14 subunit CCAN complex. d,
Coomassie blue-stained SDS PAGE gel of Cenp-ANuc (601). Lane E32:
Ethidium bromide stained gel of fraction 32. e,
CCAN–Cenp-ANuc (601) complex. Lane E13: Ethidium
bromide stained gel of fraction 13. SEC chromatograms in (a).
f, SDS PAGE gel of CCAN and H3 nucleosome (601) SEC run
shown in (b). g-j, Coomassie blue-stained SDS PAGE
gels of various Cenp-H, I and K segments co-expressed with Cenp-TW and
purified with a double Strep tag on the tagged Cenp-I subunit, indicated by
*. j, Shows that the HFDs of Cenp-TW (Cenp-THFDW)
interacts with the Cenp-HIKHead. These results confirm the
assignments of the Cenp-H, K and I subunits in our cryo-EM maps.
k, Schematic of the organization of
CCAN–Cenp-ANuc subunits and sub-complexes and
connections to the outer kinetochore Mis12 and Ndc80 complexes. Lines
indicate sub-complex connections. The two pathways connecting
Cenp-ANuc to the Ndc80 complex and microtubules are indicated
as P1 and P2 (thick lines to Ndc80). Subunits of the essential P1 pathway
are labelled black and indicated with blue shading, whereas subunits of the
non-essential P2 pathway are labelled white and indicated with yellow
shading. The P2 pathway becomes essential when the P1 pathway is defective
through defects in Dsn1 phosphorylation [9]. The experiments shown in a-j were
performed independently in triplicate with similar results. For gel source
data see Supplementary
Fig. 1.
Cryo-EM data of the S. cerevisiae
CCAN–Cenp-ANuc complex.
a, A typical cryo-electron micrograph of
CCAN–Cenp-ANuc, representative of 9,002 micrographs.
b, Galleries of 2D classes of CCAN, representative of 100
2D classes. c, Galleries of 2D classes of
CCAN–Cenp-ANuc, representative of 150 2D classes.
Outlined in red are 2-D class averages for the C2-symmetric dimeric
CCAN-Cenp-ANuc complex viewed in the plane of the C2-symmetry
axis. Only a few views were observed, precluding a 3D reconstruction.
Cryo-EM grids partially destabilize CCAN – Cenp-ANuc
interactions, resulting in a very low abundance of dimeric
CCAN–Cenp-ANuc particles (~0.03% of total). The
two-fold symmetry axes of the dimeric CCAN-Cenp-ANuc complex are
shown as dashed arrows. Experiments for data in b and
c were performed independently twelve times with similar
results. d, Fourier shell correlation (FSC) curves shown for
the cryo-EM reconstructions of CCAN–Cenp-ANuc complexes:
apo CCAN, mask1 (Cenp-OPQU+, Cenp-LN), mask2 (Cenp-HIK, Cenp-LN,
sub-Cenp-OP), CCAN–Cenp-ANuc. Mask1 and mask2 used for
multi-body refinement are defined in (h) and (i)
in and Methods. e, Angular distribution plot of
CCAN–Cenp-ANuc particles. f, Local
resolution map of CCAN. g, Local resolution map of
CCAN–Cenp-ANuc. h, Local resolution map
of mask1 (Cenp-OPQU+, Cenp-LN). i, Local resolution map of
mask2 (Cenp-HIK, Cenp-LN, sub-Cenp-OP).
Workflow of 3D classification of the CCAN–Cenp-ANuc
cryo-EM data set.
a, After initial 2D classification ~1.4 million
particles were sorted by 3D classification into apo CCAN (52%) and the
CCAN–Cenp-ANuc complex (48%). For apo CCAN, 4% existed
as dimers (black box) and 19% showed an ordered head-group
(Cenp-HIKHead) for the Cenp-HIK–TW sub-complex (blue
box). A mask was applied to the CCAN–Cenp-ANuc EM map to
exclude the structurally variable Cenp-HIKHead domain for
reconstruction of the 4.15 Å structure. b, Details of
the four masks used for multi-body refinement. c, A small 3D
class of CCAN–Cenp-ANuc revealing density attached to
Cenp-HIKHead contacting the DNA gyre of Cenp-ANuc
was assigned as Cenp-THFDW.
Cryo-EM density maps of apo CCAN.
a, Portion of cryo-EM map for the coiled coils of
Cenp-H and Cenp-K. A selection of highly conserved intersubunit residues
defined in (b, c) are labelled. These residues are well defined
in EM density, consistent with the structure. b, c, Multiple
sequence alignment of the coiled-coil regions of b, Cenp-H and
c, Cenp-K. d-f, Portions of cryo-EM maps for:
d, Cenp-LN. e, Cenp-I. f,
Nkp1-Nkp2. The chain assignments and polarity of Cenp-H, Cenp-I and Cenp-K
of our structure agree with the cryo-EM structure of yeast Ctf3 (PDB 6OUA)
[61].
Cryo-EM densities of CCAN and CCAN–Cenp-ANuc
complexes.
a, Cryo-EM reconstruction of
CCAN–Cenp-ANuc from uncross-linked sample at 8.6
Å resolution. b, Cryo-EM map of dimeric CCAN (also Extended Data Fig. 3a - black box).
Subunits are colour-coded as in Fig. 1.
The 3.5 Å monomeric free CCAN coordinates were rigid-body docked into
the cryo-EM map. c, Cartoon representation of the S.
cerevisiae MIND complex [15] (right) showing a striking similarity to the coiled
coils of Cenp-QU-Nkp1-Nkp2 of CENP-OPQU+ (left). d, View of the
4.7 Å resolution cryo-EM map of free Cenp-HIK with fitted coordinates
from CCAN. e, In the context of CCAN, Cenp-HIKHead
rotates to accommodate Cenp-ANuc. The two conformations of
Cenp-HIK from the apo CCAN and CCAN–Cenp-ANuc complexes
were superimposed onto their rigid portion of Cenp-HIK (C-terminal region of
Cenp-I – shown for apo CCAN) to indicate the conformational
variability of Cenp-HIKHead between the two states. Subunits of
Cenp-HIKHead of CCAN–Cenp-ANuc are coloured
lighter. f, Cryo-EM density of Cenp-ANuc showing the
Cenp-C motif of Cenp-C.
Cross-linking mass spectrometry analysis of the CCAN and
CCAN–Cenp-ANuc complexes.
a, b, Circular plots displaying all the identified
cross-links for CCAN (a) and CCAN–Cenp-ANuc
(b). Inter- and intra-subunit cross-links are indicated in
red and blue, respectively c, d, Histogram plots showing the
Cα-Cα distance distribution of the cross-links that could be
mapped onto the CCAN (c), and CCAN–Cenp-ANuc
structures (d). 95% of the mapped cross-links satisfy the
cross-linker imposed distance restraint of 30 Å indicated with a
dashed red line. e, f, Cross-links mapped onto the CCAN
(e) and CCAN–Cenp-ANuc complex
(f). Inter and intra-subunit cross-links are indicated in
red and blue, respectively. Cross-links exceeding the cross-linker imposed
distance restraint of 30 Å are indicated in yellow. g,
Residues on CCAN shown by XL-MS that cross-link with Cenp-C are indicated on
the CCAN structure. Red spheres: cross-links in the
CCAN–Cenp-ANuc complex. Yellow spheres: additional
cross-links unique to apo CCAN. The experiments shown in a and
b were performed independently in triplicate with similar
results.
The S. cerevisiae Cenp-ANuc nucleosome is
unwrapped.
a-c, The positively-charged electrostatic potential of
the DNA-binding groove of Cenp-LN sub-complex is conserved in S.
cerevisiae, S. pombe and H.
sapiens. S. pombe and H.
sapiens are modelled structures. d, Cenp-N
interacts with ScCenp-ANuc in the context of
CCAN differently from the interaction of free human Cenp-N with
Cenp-ANuc. The Cenp-N subunit of the human
Cenp-N–Cenp-A nucleosome structure (PDB: 6C0W [29]) was superimposed onto
Cenp-N of the S. cerevisiae
CCAN–Cenp-ANuc structure. In this mode of
Cenp-N–Cenp-ANuc interactions, Cenp-ANuc
would clash with Cenp-OPQU+ and Cenp-N of CCAN. e, Structure
ScH3Nuc (PDB: 1ID3 [24]) and f,
Cenp-ANuc (this work). g, sequence alignment of
the N-terminal regions of ScH3 and Cenp-A (Cse4) histones.
For the chimeric H3N-Cenp-ANuc, residues 1-50 of
ScH3 were substituted for residues 1-140 of
ScCenp-A. A similar approach was used for vertebrate
Cenp-ANuc
[23].
SDS PAGE gels of
CCANΔCenp-C–Cenp-ANuc
complexes.
a-h, Coomassie-blue stained SDS PAGE gels of various
CCANΔCenp-C–Cenp-ANuc complexes.
Corresponding SEC chromatogram is shown in Fig. 4b and Extended Data Fig.
9a. a, b, Mutating the Cenp-N DNA binding groove did
not impair CCANΔCenp-C assembly. c, Wild type
CCANΔCenp-C forms a complex with Cenp-ANuc.
d, Mutating the Cenp-N DNA binding groove disrupts
CCANΔCenp-C – Cenp-ANuc
interactions. e, Mutating the L1 loop of Cenp-A did not
destabilize CCANΔCenp-C – Cenp-ANuc
interactions. f, Deletion of the N-terminus of Cenp-A (1-129)
(ΔNCenp-ANuc) did not impair
CCANΔCenp-C – Cenp-ANuc
interactions. h, Both CCANΔCenp-C and
CCANΔCenp-C-Cenp-NMut bound poorly to
H3N-Cenp-ANuc. The experiments shown were
performed independently in triplicate with similar results. For gel source
data see Supplementary
Fig. 1.
Testing of CCANΔCenp-C binding to
Cenp-ANuc.
a, Comparative size exclusion chromatogram profiles
(Agilent Bio SEC-5 column) for wild type CCANΔCenp-C and
the Cenp-NMut of CCANΔCenp-C to
Cenp-ANuc and its modifications
(Cenp-ANuc-L1Nuc,
ΔNCenp-ANuc,
H3N-Cenp-ANuc) and H3Nuc. Mutating the
L1 loop (Cenp-A-L1Nuc) of Cenp-A or deletion of the N-terminal
129 residues (ΔNCenp-ANuc) did not destabilize
CCANΔCenp-C – Cenp-ANuc
interactions. In contrast, CCAN with the Cenp-NMut bound less
well and both CCAN and CCAN-Cenp-NMut bound hardly at all to
H3N-Cenp-ANuc. (CCANΔC =
CCANΔCenp-C). Associated SDS PAGE gels in Extended Data Fig. 8 and Extended Data Fig. 9b). b,
Coomassie-blue stained SDS PAGE gel showed that
CCANΔCenp-C did not associated with H3Nuc.
c, Micrococcal nuclease digestion of Cenp-ANuc,
H3Nuc and H3N-Cenp-ANuc. 601 DNA is
shown as a control. The H3Nuc and
H3N-Cenp-ANuc protect a similar and longer length
of DNA compared with Cenp-ANuc. d, Model of CBF3
[60] bound to
CCAN–Cenp-ANuc indicating that CBF3 would not
associate with a fully assembled kinetochore, consistent with proteomic data
[62]. The
experiments shown in a-c were performed independently in
triplicate with similar results. For gel source data see Supplementary Fig.
1.
S. cerevisiae CCAN–Cenp-ANuc comprises
two CCAN complexes in solution.
The predicted mass of (CCAN)2–Cenp-ANuc
is 1.31 MDa, (CCAN)1–Cenp-ANuc is 0.77 MDa and
that for a CCAN dimer 1.09 MDa (Extended Data Table 2).
Representative SEC-MALS data for a, cross-linked S.
cerevisiae CCAN–Cenp-ANuc complex, run
independently in triplicate with similar results, average molecular mass is
1.23 MDa [(CCAN)2–Cenp-ANuc]. b,
uncross-linked S. cerevisiae
CCAN–Cenp-ANuc complex, run independently
in triplicate with similar results, with average masses of 1.38 MDa
[(CCAN)2–Cenp-ANuc] and 526 kDa
[(CCAN)1]. c, S. cerevisiae
CCAN alone, run independently in duplicate with similar results, with
average masses of 839 kDa for the leading edge (green) and 650 kDa for the
trailing edge (magenta) suggesting a non-resolved monomer-dimer equilibrium.
Velocity analytical ultracentrifugation of d, cross-linked and
e, uncross-linked S. cerevisiae
CCAN–Cenp-ANuc complexes with residuals to the fits
below of a c(s) distribution model: f, for the cross-linked
complex, the major species sediments at 15.8 S (Sw,20 = 26.1 S)
with a minor species at 12.1 S (Sw,20 = 20.0 S) that corresponds
to calculated masses of 1.34 MDa
[(CCAN)2–Cenp-ANuc] and 896 kDa [possibly
(CCAN)1–Cenp-ANuc] respectively with a
fitted value of 1.761 for the frictional ratio; g, for
uncross-linked samples, the major species is resolved into two species that
sediment at 14.3 S (Sw,20 = 22.6 S) and 15.7 S (Sw,20
= 24.9 S) with a minor species at 12.3 S (Sw,20 = 19.4 S) which
gave masses of 1.32 MDa [(CCAN)2–Cenp-ANuc] and
1.15 MDa [(CCAN)2] for the major species and 716 kDa
[(CCAN)1–Cenp-ANuc] for the minor species.
The experiments shown in d-g were performed independently in
triplicate with similar results. h, Examples of two 2D class
averages showing the dimeric CCAN–Cenp-ANuc particles
viewed in the plane of the C2 symmetry axis (red outline) (data from Extended Data Fig. 2c) and the 2D
reprojections of a modelled dimeric CCAN–Cenp-ANuc based
on the CCAN–Cenp-ANuc cryo-EM reconstruction (yellow
outline) (Extended Data Fig. 10i).
There is a close correspondence in shape and dimensions between the
calculated reprojections and the observed 2D classes. The two-fold symmetry
axes of the dimeric CCAN-Cenp-ANuc complex are shown as dashed
arrows. i, j, Two alternative models for how CCAN assembled
onto a Cenp-A nucleosome would interact with the outer
kinetochore–microtubule interface (Supplementary Video
2). i, In scenario (1), CCAN interacts with the outer
kinetochore from the same side as the DNA-binding surface. Microtubules
attached to the outer kinetochore would hoist CCAN from below the over-lying
nucleosome and out-stretched DNA. j, In scenario (2), the
microtubule-outer kinetochore interface contacts CCAN from the opposite side
to the CCAN-DNA binding surface. Outer-KT (outer-kinetochore): KMN network
and microtubule attachment complexes: Dam1/DASH (budding yeast) and Ska
proteins of vertebrates. The combined dimension of dimeric
CCAN–Cenp-ANuc (32 nm) matches that of the hub at the
centre of the yeast kinetochore [63].
Table of CCAN subunits.
Details of structured regions of CCAN subunits built into the
cryo-EM density maps are indicated, including regions built as polyAla. The
calculated molecular masses for CCAN and Cenp-ANuc complexes are
(i) CCAN: 543.3 kDa, (ii) CCAN dimer: 1.09 MDa, (iii) Cenp-ANuc:
223 kDa, (iv) (CCAN)1−Cenp-ANuc: 0.766 MDa and
(v) (CCAN)2−Cenp-ANuc 1.31 MDa.
Authors: Kara L McKinley; Nikolina Sekulic; Lucie Y Guo; Tonia Tsinman; Ben E Black; Iain M Cheeseman Journal: Mol Cell Date: 2015-11-19 Impact factor: 17.970
Authors: Raymond Camahort; Manjunatha Shivaraju; Mark Mattingly; Bing Li; Shima Nakanishi; Dongxiao Zhu; Ali Shilatifard; Jerry L Workman; Jennifer L Gerton Journal: Mol Cell Date: 2009-09-24 Impact factor: 17.970
Authors: M Winey; C L Mamay; E T O'Toole; D N Mastronarde; T H Giddings; K L McDonald; J R McIntosh Journal: J Cell Biol Date: 1995-06 Impact factor: 10.539
Authors: Oleg Klykov; Mykhailo Kopylov; Bridget Carragher; Albert J R Heck; Alex J Noble; Richard A Scheltema Journal: Mol Cell Date: 2022-01-20 Impact factor: 17.970
Extended Data Figure 1
Extended Data Figure 2
Extended Data Figure 3
Figure 1
Extended Data Figure 4
Figure 2
Extended Data Figure 5
Extended Data Figure 6
Figure 3
Extended Data Figure 7
Figure 4
Extended Data Figure 8
Extended Data Figure 9
Extended Data Figure 10