¹H, ¹³C and ¹⁵N resonance assignments of the kinetochore localisation domain of BUBR1, a central component of the spindle assembly checkpoint.

Abstract

Human BUBR1 is a 120 kDa protein that plays a central role in the spindle assembly checkpoint (SAC), the evolutionary conserved and self-regulatory system of higher organisms that monitors and repairs defects in chromosome segregation in mitotic cells. BUBR1 is organised into several domains, with an N-terminal region responsible for its localisation into the kinetochore, the multi-component proteinaceous network that assembles onto chromosomes upon mitotic entry. We have expressed and purified uniformly-(15)N/(13)C N-terminal BUBR1 and assigned backbone and side-chain resonances bound to an unlabelled peptide from the protein Blinkin, an element essential for recruitment of BUBR1 to the kinetochore. These assignments provide insights on the Blinkin interaction interface and form the basis of the three-dimensional structure determination of a BUBR1-Blinkin complex.

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ARTICLE
1H,13C and15N resonance assignments of the kinetochore
localisation domain of BUBR1, a central component of the
spindle assembly checkpoint
Peter J. Simpson•Ernesto Cota•
Victor M. Bolanos-Garcia
Received: 3 October 2011/Accepted: 14 December 2011/Published online: 27 December 2011
? Springer Science+Business Media B.V. 2011
Abstract
central role in the spindle assembly checkpoint (SAC), the
evolutionary conserved and self-regulatory system of higher
organisms that monitors and repairs defects in chromosome
segregation in mitotic cells. BUBR1 is organised into several
domains, with an N-terminal region responsible for its locali-
sation into the kinetochore, the multi-component proteina-
ceousnetworkthatassemblesontochromosomesuponmitotic
entry. We have expressed and purified uniformly-15N/13C
N-terminal BUBR1 and assigned backbone and side-chain
resonances bound to an unlabelled peptide from the protein
Blinkin, an element essential for recruitment of BUBR1 to the
kinetochore.TheseassignmentsprovideinsightsontheBlinkin
interaction interface and form the basis of the three-dimen-
sional structure determination of a BUBR1-Blinkin complex.
Human BUBR1 is a 120 kDa protein that plays a
Keywords
Chromosome segregation defects ? NMR
BUBR1 ? KNL1 ? Mitotic checkpoint ?
Biological context
Maintenance of genome stability is a sine qua non condi-
tion to ensure the accurate and timely transmission of the
genetic material to progeny during multiple rounds of cell
division.Prematureseparationofsisterchromatidsresultsin
the loss or gain of chromosomes in daughter cells. This
condition,commonlyreferredtoasaneuploidy,constitutesa
prevalentformofgenetic instabilitythathasbeen associated
with various classes of human cancer (Kops et al. 2005;
Bolanos-Garcia and Blundell 2011). Such an adverse situa-
tion is prevented by activation of the spindle assembly
checkpoint (SAC), which monitors the proper assembly of
the mitotic spindle and delays the onset of anaphase until all
chromosomesarestablyattachedtokinetochores.BUBR1is
amulti-domainproteinthatcontainsaconservedN-terminal
domain, a middle non-conserved domain harbouring the
bindingregionforothermitoticcheckpointcomponents,and
a C-terminal Ser/Thr kinase domain that plays central roles
in the mitotic checkpoint response. In mice, BUBR1 insuf-
ficiency causes infertility and promotes early aging (Baker
etal.2004)whileinyeastBUBR1iscommonlyreferredtoas
MAD3 and lacks the C-terminal catalytic domain. Despite
the lack of the kinase domain, both MAD3 and BUBR1 are
substratesoftheanaphase-promotingcomplexorcyclosome
(APC/C), an interaction that plays a critical role in CDC20
turnoverinmitosis(Kingetal.2007).TheN-terminalregion
is essential for BUBR1 association with unattached/incor-
rectly attached kinetochores in a process that involves
BUBR1 binding to the central kinetochore protein Blinkin
(Kiyomitsu et al. 2007; D’Arcy et al. 2010). Human Blinkin
(also known as AF15Q14, CASC5, KNL1, and Spc105) is a
large (*262 kDa) protein containing multiple regions of
predicted low structural complexity, that acts as a molecular
scaffoldtodockotherproteinstothekinetochore(Przewloka
and Glover 2009). A small motif within Blinkin is sufficient
for the interaction with the N-terminal tetratricopeptide
(TPR)-containing domain of BUBR1, for which the struc-
ture has been solved by X-ray crystallography in the
P. J. Simpson ? E. Cota (&)
Division of Molecular Biosciences, Department of Life Sciences,
Imperial College London, Exhibition Rd.,
South Kensington, London SW7 2AZ, UK
e-mail: e.cota@imperial.ac.uk
V. M. Bolanos-Garcia (&)
Group of Crystallography and Bioinformatics, Department of
Biochemistry, University of Cambridge, 80 Tennis Court Road,
Cambridge CB2 1GA, UK
e-mail: victor@cryst.bioc.cam.ac.uk
123
Biomol NMR Assign (2012) 6:115–118
DOI 10.1007/s12104-011-9355-9

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unbound form (D’Arcy et al. 2010). We have thus sort to
obtaintheNMRassignmentsofthisdomainincomplexwith
aBlinkinpeptidetolocalisetheinteractionsiteandassistthe
structure determination of a BUBR1-Blinkin complex
(Bolanos-Garcia et al. 2011).
Experimental procedures
Production of13C,15N-BUBR1
A human bubr1 gene fragment encoding for the N-terminal
region,residues57-220,wasPCRamplifiedfromcDNAand
cloned into a pGST plasmid (GE Healthcare) under the
control of the T7 promoter. The resulting expression vector
(pGST-BUBR1) encodes a 45 kDa fusion protein that con-
tains an N-terminal GST-tag. The (pGST-BUBR1) plasmid
was transformed into an Escherichia coli BL21 (DE3)
expression strain. Recombinant protein was prepared from
cells grown to an OD600 of 0.5–0.6 at 20?C in M9 medium
containing0.1%15NH4Cl,0.2%13C-Glucoseand100 lg/ml
ampicillin. Protein expression was then induced by addition
of 1 mM isopropyl-1-thio-b-D-galactopyranoside (IPTG),
and the culture was further grown for 3 h at 20?C. The cells
were harvested by centrifugation (8,000g for 30 min), re-
suspended in 50 mM TBS buffer (40 mM Tris–HCl,
200 mM NaCl and 1 mM 1,4-dithiothreitol, pH 8.0) with
protease inhibitor cocktail (Roche) and lysed with Bug-
Buster lysis solution (Novagen). The soluble fraction enri-
ched with the N-terminal BUBR1 protein was loaded onto a
GST chromatography column (GE Healthcare) and purified
using a conventional procedure after GST tag cleavage and
removal.Asecondstepofpurificationwasperformedusinga
Superdex 75 chromatographic column (GE Healthcare)
previously equilibrated in PS (20 mM sodium phosphate,
100 mM NaCl, pH 6.0) buffer solution. 5 ml fractions were
collected and concentrated by ultrafiltration using a 10-kDa
cutoff membrane (Millipore) prior to NMR analyses.
NMR experiments
For NMR spectroscopy, samples were buffer-exchanged
by dilution/re-concentration into 50 mM pH 7.0 sodium
Fig. 1
of BUBR1 bound to Blinkin
peptide. Backbone amide
assignments are indicated as
residue numbers (N.B. Residue
numbering corresponds to the
construct used in the study,
hence residue 1 would be 54 in
the BUBR1 full-length
sequence). An expansion of the
boxed area is shown on the
right-hand side and a region of
the spectrum between 10 and
10.6 ppm containing two Trp
NHesignals is shown bottom
right. The majority of sidechain
amide signals were unassigned
and putative pairs are indicated
by horizontal lines (Asn35 Hds
indicated). A number of Arg
NHesignals identified based on
their Nefrequency in a wide
spectral width HSQC are
indicated as ‘‘Re’’. The asterisk
for Leu133 indicates its aliased
position with the15N spectral
width used
1H-15N HSQC spectrum
116P. J. Simpson et al.
123

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phosphatebuffer,200 mMNaCl,containing1 mMNaN3and
1 mM tris(2-carboxyethyl)phosphine (TCEP). NMR experi-
ments were performed on samples of *200 lM uniformly
15N,13C-labelled protein in either 3 mm (Cortec) or 5 mm
(Sigma-Aldrich) Shigemi tubes at 30?C (free BUBR1 titra-
tion)and37?C(assignmentoftheBUBR1-Blinkincomplex).
To form the complex, titration of BUBR1 with up to 2 molar
equivalentsofBlinkinpeptide,NH2-KIDFNDFIKRLKTGK-
CO2(Designer Bioscience, Cambridge, UK) was monitored
using 2D1H-15N SOFAST-HMQC spectra (Schanda et al.
2005). Backbone atom assignments were completed with
standard experiments (Sattler et al. 1999) and extended
into aliphatic sidechains using a combination of HBHA
(CBCACO)NH, H(C)CH-TOCSY, (H)CCH-TOCSY and
amide-detected (H)C(CCO)NH- and H(CCCO)NH-TOCSY
spectra. Aromatic ring assignments were made from a
13C-NOESY-HMQC spectrum in conjunction with the
TROSY-1H,13C-aromatic HSQC (Pervushin et al. 1998).
Data were collected on Bruker AvanceIII (600 MHz) and
AvanceII (800 MHz) spectrometers equipped with TCI and
TXIcryoprobes,respectively,controlledbyTopspin3(Bruker
BiospinLtd).Data wereprocessedusingNMRPipe(Delaglio
etal.1995)andanalysedinNMRView(OneMoonScientific).
Assignment was aided by NMRView modules which pro-
videdrapidinputforMARSautomatedassignment(Jungand
Zweckstetter 2004) and facile handling of sidechain data
(Marchant et al. 2008).
Assignments and data deposition
Backbone assignments could be completed for the majority
of the BUBR1 domain (Fig. 1); exceptions were residues at
the extreme N-terminus and additionally K41 (K94 in the
full-length BUBR1 sequence). Amide signals from resi-
dues neighbouring K41 are broadened, suggesting the
presence of ms–ls timescale dynamics in this region and
consistent with the lack of electron density for this loop in
the structure of the free protein (D’Arcy et al. 2010). The
backbone assignments for the 3rd and 4th Glu residues in
the unusual penta-glutamate sequence near the C-terminus
(Fig. 2) are also ambiguous due to chemical shift degen-
eracy and may be interchangeable. This motif is, unsur-
prisingly, highly degenerate in the chemical shifts of most
nuclei; whilst the 1stand 5thGlu residues in the sequence
were chemically distinct, presumably because of their
differing neighbours, the 2nd Glu was only distinguishable
from the 3rd and 4th due to a slight difference in
frequency of the preceding residue. The 3rd and 4th
glutamates are degenerate in all shifts. The chemical shift
13C’
Fig. 2 Chemical shift index
(CSI) plot of BUBR1-Blinkin
peptide complex and
comparison with the free state.
Red and blue lollipops denote
a-helical and b-strand-like
deviations from random coil,
respectively, for Ha, Caand C’
nuclei. The amino acid
sequence is coloured red for
residues whose amide could be
assigned, black for missing
residues or prolines. The
consensus CSI secondary
structure is shown below as a
cartoon and is compared with
the secondary structure taken
from the X-ray structure of
BUBR1 in the unbound state
(PDB code: 2WVI). The CSI
was generated in NMRView
A central component of the spindle assembly checkpoint 117
123

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index (CSI; Wishart and Sykes 1994) indicates BUBR1 in
the bound state comprises 8 a-helices (Fig. 2), which are
essentially identical to those observed in the crystal struc-
ture of the free form. An exception is the length of the
C-terminal a-helix which is extended significantly (ca.
three turns) in the crystal structure, encompassing the
penta-Glu sequence. Although the a-helical propensity of
poly-Glu at acidic pH has been established (Olander and
Holtzer. 1968; Grigoryev et al. 1971), the conformation of
this a-helix at pH 7.0 may result from co-ordination of two
praseodymium(III) ions by the penta-Glu sequence, and
therefore an artefact of the crystallisation conditions, as
suggested (D’Arcy et al. 2010). Indeed, amide linewidths
in this region suggest it is highly mobile on the ns-ps
timescale both in the free and bound state (data not shown).
Aliphatic sidechain assignments were completed to
approximately 90%, missing nuclei arising mainly from
shift degeneracy in extended hydrophilic sidechains. One
in three residues in BUBR1 are of this nature (13 Arg, 22
Gln, 22 Glu, 7 Lys) and thus the dispersion afforded by the
NH signal in amide-detected TOCSY experiments proved
essential. However, many correlations were found to be
absent in these experiments due to their inherently lower
sensitivity. 78% of the 24 aromatic sidechain resonances
could be assigned, with most of the missing assignments
arising from overlap in the Phe e and f positions. With the
exception of N35, no assignments could be made for Arg
NHe and Asn/Gln sidechain amides, due to a complete
absence of NOEs (both intra-residue and longer-range) in
NOESY spectra, presumably the result of rapid proton
exchange experienced in solvent-exposed residues, coupled
with the elevated intrinsic exchange rate at the pH and
temperature required for NMR analysis (pH 7.0 and 37?C).
An early indication that the tertiary structure of BUBR1
remains essentially unchanged upon Blinkin interaction is
provided by a number of ring-current shifted signals, as they
are in good qualitative agreement with their position in the
crystal structure of the free state. Of note, the sidechain NHe
signal of W72 (W125) is highly shifted from random coil
(dH= 6.97, dN= 123.7 ppm; BioMagResBank mean for
diamagnetic proteins: 10.08 ± 0.65, 129.3 ± 2.1). In the
structure of the free state this NH group points towards the
centre of the aromatic ring of F68 (F121), where it may form
an NH-aromatic hydrogen bond (Levitt and Perutz 1988;
To ´th et al. 2001). Additionally, one Arg sidechain has a
highly unusual HNefrequency (9.60 ppm), but the lack of
assignment precluded further analysis. The full set of chem-
ical shifts has been deposited in the BioMagResBank data-
base (http://www.bmrb.wisc.edu), accession number 17960.
Acknowledgments
Marchant (Imperial College London) for provision of the assignment
modules used in the study and continuing valued assistance with
PJS and EC would like to thank Dr. Jan
software. VMB-G thanks Prof. Tom L. Blundell for the continuous
support and the stimulating discussions.
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