From the Department of Chemistry, Dartmouth College, Hanover, New
Hampshire 03755, the Department of Biochemistry,
Dartmouth Medical School, Hanover, New Hampshire 03755, and the
¶ Department of Biochemistry, University of New Hampshire,
Durham, New Hampshire 03824
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ABSTRACT |
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Core-binding factors (CBF) are heteromeric
transcription factors essential for several developmental
processes, including hematopoiesis. CBFs contain a DNA-binding
CBF subunit and a non-DNA binding CBF
subunit that increases the
affinity of CBF
for DNA. We have developed a procedure for
overexpressing and purifying full-length CBF
as well as a truncated
form containing the N-terminal 141 amino acids using a novel
glutaredoxin fusion expression system. Substantial quantities of the
CBF
proteins can be produced in this manner allowing for their
biophysical characterization. We show that the full-length and
truncated forms of CBF
bind to a CBF
·DNA complex with very
similar affinities. Sedimentation equilibrium measurements show these
proteins to be monomeric. Circular dichroism spectroscopy demonstrates
that CBF
is a mixed
/
protein and NMR spectroscopy shows that
the truncated and full-length proteins are structurally similar
and suitable for structure determination by NMR spectroscopy.
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INTRODUCTION |
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The core-binding factor subunit
(CBF
)1 is the non-DNA
binding subunit of the heterodimeric transcription factor complex called core binding factor (CBF) (1, 2). CBF binds to target DNA sites
with the consensus sequence PyGPyGGT (3-6). CBF subunits are encoded
by four genes in mammals. CBFA1, CBFA2
(AML1), and CBFA3 encode DNA-binding CBF
subunits, and CBFB encodes the common CBF
subunit (1, 2,
7-12). The CBF
subunit can bind DNA in the absence of CBF
in vitro but with 5-10-fold lower affinity than the
CBF
/
complex (1, 2). CBF
modulates the affinity of the CBF
subunit for DNA without establishing additional contacts on the DNA or
changing the magnitude of DNA bending (2, 13).
The amino acid sequence of CBF yields few clues to its tertiary
structure or the mechanism by which it modulates the affinity of the
CBF
subunit for DNA. The heterodimerization domain in CBF
has
been localized to its N-terminal 135 amino acids, which corresponds to
a region of significant homology between CBF
and its two
Drosophila homologs, Brother (Bro) and Big Brother (Bgb) (13, 14). A truncated CBF
protein containing amino acids 1-141
(CBF
(141)), which includes the region of homology to Bro and Bgb,
stably associates with the CBF
subunit in vitro (1, 14,
15). Further truncation of the C terminus to amino acid 133 results in
a protein that weakly associates with CBF
, and C-terminal truncation
to amino acid 117 disrupts stable heterodimerization with CBF
(1,
15).
The CBF subunit is essential for the in vivo function of
at least one of the CBF
subunits that were encoded by the
CBFA2 gene (also known as the acute myeloid leukemia 1 or
AML1 gene). Homozygous disruption of either the
Cbfa2 or the Cbfb genes in mice results in
identical phenotypes: midgestation embryonic lethality accompanied by
extensive hemorrhaging and a profound block at the fetal liver stage of
hematopoiesis (15-19). In humans, chromosomal rearrangements that
disrupt the CBFA2 and CBFB genes are associated with a variety of leukemias, including de novo acute myeloid
leukemias t(8;21)(q22;q22), inv(16)(p13;q22), t(16;16), and
del(16)(q22), acute lymphocytic leukemias t(12;21)(p13;q22), and
therapy-related leukemias and myelodysplasias t(3;21)(q26;q22) (10-11,
20-22). All of these translocations result in the synthesis of
chimeric proteins, two of which have been directly demonstrated to
block CBF function in a transdominant manner (23, 24).
The primary structures of CBF and its Drosophila homologs
are not similar to those of any other proteins, and the mechanism by
which CBF
stabilizes the CBF
·DNA complex is unusual. The CBF
subunit is an essential component of the CBF complex and is mutated in
a significant percentage of human leukemias, making it both an
interesting and important target for biophysical and structural
analyses. In this study, we describe a novel system for expressing the
CBF
subunit in bacteria and a purification protocol with which we
can obtain large amounts of homogeneous CBF
protein. We confirm that
a truncated form of CBF
, CBF
(141), demonstrated previously to
contain an intact heterodimerization domain (1), binds to CBF
with
an affinity very similar to that of the full-length CBF
(187)
protein. The isolated heterodimerization domain in CBF
(141) also
assumes a folded structure indistinguishable from that which it assumes
in the context of the full-length CBF
(187) protein. We also
demonstrate that both the full-length CBF
(187) and truncated
CBF
(141) proteins are monomeric and contain a mixture of
-helical
and
-strand secondary structural elements.
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EXPERIMENTAL PROCEDURES |
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Plasmid Construction--
The pGRXCBF141 plasmid encoding the
glutaredoxin-CBF
(141) fusion protein was constructed in the
following manner. DNA sequence corresponding to Escherichia
coli glutaredoxin-1 was polymerase chain reaction amplified from
the plasmid pMG524-GRX (25) using the following primers:
5
-CGGAATTCGGTTAAACCTACTTTCAGCG-3
(S-GRX) and
5
-CGGGATCCCTTGTCATCGTCATCGGCGTCCAGATTTTCTTTCACC-3
(AS-GRX). The
sense primer (S-GRX) contains the recognition site for EcoRI at its 5
end. The AS-GRX primer contains a restriction site for BamHI, followed by sequences encoding a cleavage site for
the protease enterokinase (DDDDK). DNA encoding CBF
(141) was
amplified from the plasmid CBF
(p21.5) (2) using the primers
5
-AGACGGATCCATGCCGCCGTCGTCCCGGAC-3
(S-CBF
141) and
5
-GGCCCAAGCTTTCACTGTTGTGCTAATGCATCTTCC-3
(AS-CBF
141). A
restriction site for BamHI was included at the 5
end of the sense primer (S-CBF
141), and a HindIII site followed by a
translational stop codon was added to the antisense primer
(AS-CBF
141). The polymerase chain reaction amplified fragments were
digested with the appropriate restriction enzymes and subcloned between
the EcoRI and HindIII sites of pMG524. The
resultant plasmid was transformed into the bacterial strain N99cI for
the purpose of DNA characterization and sequencing and into strain
N4830 or AR58 for protein expression.
Expression of CBF(141) and CBF
(187)--
E.
coli AR58 were transformed with the pGRXCBF
141 plasmid, and a
single colony was used to inoculate 10 ml of terrific broth (Sigma)
containing 100 µg/ml carbenicillin. After overnight shaking at 200 rpm at a temperature of 29 °C, the culture was used to inoculate 1 liter of terrific broth (100 µg/ml carbenicillin). This culture was
grown to an A600 of 1.5 and then induced by
raising the temperature to 40 °C and maintaining at this temperature
for 4 h. Cells were collected by centrifugation, resuspended in an equal weight of 10% sucrose, 50 mM Tris (pH 7.5), and
frozen by dripping into liquid nitrogen. The frozen cells were stored
at
70 °C. For expression of full-length CBF
, pGRXCBF
187 was
transformed into E. coli AR58, and cells were grown in the
identical manner as for CBF
(141).
Expression of 15N-Labeled CBF(141) and
CBF
(187)--
For expression of 15N-labeled CBF
(141)
or CBF
(187), E. coli AR58 transformed with the
appropriate plasmid was grown in minimal medium containing
15(NH4)2SO4 (Isotec,
Inc.) as the sole nitrogen source.
Purification of CBF(141)--
All operations were carried out
at 4 °C. Pefabloc (1 mM, Boehringer Mannheim), EDTA (1 mM), and lysozyme (0.4 mg/ml) were added to the thawed
bacterial cell pellet from a 1-liter culture. The solution was stirred
gently for 30 min, and the cells were then further lysed by four
passages through a French press at 18,000 p.s.i. The lysate was
clarified by centrifugation at 11,000 × g for 10 min.
The resulting supernatant was loaded onto a 2.5 × 25-cm column of
DEAE-Sephacel (Pharmacia Biotech Inc.) equilibrated in 25 mM Tris-Cl (pH 7.5), 1 mM EDTA, 1 mM DTT at 1 ml/min and eluted from the column with a
1-liter linear gradient of 0-500 mM NaCl in the same
buffer. Fractions containing the fusion protein were identified by
hydroxyethyl disulfide assay, which detects the enzymatic activity of
the glutaredoxin system as described by Holmgren (26). The active
fractions were pooled and concentrated by ultrafiltration on a YM3
membrane (Amicon, Inc.) to 15 ml. The DEAE pool was loaded onto a
2.5 × 113-cm column of Sephacryl S-100 (Pharmacia) in 25 mM Tris-Cl (pH 7.5), 1 mM EDTA, 75 mM NaCl and eluted with the same buffer at 0.6 ml/min. The
active fractions were again identified by 2-hydroxyethyl disulfide
assay, pooled, and concentrated by ultrafiltration to 15 ml. The
resulting homogeneous fusion protein was cleaved by treatment with 90 units of enterokinase (Biozyme, San Diego)/A280
unit of fusion protein with addition of DTT to 0.5 mM and
incubation at 30 °C for 20 h. The cleavage reaction was halted
by addition of 0.5 mM Pefabloc. The resulting cleaved
protein was exchanged into a lower ionic strength buffer (25 mM NaCl), loaded onto a 2.5 × 10-cm column of
Q-Sepharose (Pharmacia) in 25 mM Tris-Cl (pH 7.5), 1 mM EDTA, 25 mM NaCl, 1 mM DTT, and
eluted with a 500-ml linear gradient of 25-500 mM NaCl in
the same buffer at 0.8 ml/min. The CBF
fractions were identified by
SDS-PAGE, pooled, and concentrated to 10 ml by ultrafiltration. From 1 liter of culture, a yield of 20 mg is typically obtained. For the
15N-labeled protein used for NMR studies, the Q-Sepharose
pool was loaded onto a 2.5 × 36-cm column of Sephacryl S-100 in
25 mM potassium phosphate (pH 6.5), 0.1 mM
EDTA, 1 mM DTT, 0.1% NaN3 and eluted at 0.8 ml/min. The protein was concentrated by ultrafiltration.
Purification of CBF(187)--
All operations were carried out
at 4 °C. The Grx-CBF
(187) fusion protein was purified in the same
manner as described above for the Grx-CBF
(141) fusion protein.
Following fractionation on DEAE-Sephacel and Sephacryl S-100, the
resulting homogeneous Grx-CBF
187 fusion protein was cleaved by
treatment with 3.5 units of Factor Xa
(Pharmacia)/A280 unit of fusion protein in a
buffer containing 25 mM Tris-Cl (pH 7.5), 1 mM
EDTA, 75 mM NaCl, 1 mM CaCl2, and
no DTT. Secondary cleavage at an undesired Factor Xa-sensitive site was
minimized by carrying out the reaction for 6 h at 22 °C. The
cleavage reaction was halted by addition of 4 mM Pefabloc with a subsequent 40-min incubation at 37 °C. The cleaved protein was exchanged into a lower ionic strength buffer (25 mM
NaCl) by ultrafiltration, loaded onto a 2.5 × 10-cm column of
Q-Sepharose in 25 mM Tris-Cl (pH 7.5), 1 mM
EDTA, 25 mM NaCl, 1 mM DTT, and eluted with a
500-ml linear gradient from 25 to 500 mM NaCl in the same
buffer at 0.8 ml/min. The CBF
(187) fractions were identified by
SDS-PAGE, pooled, and concentrated to 4 ml by ultrafiltration. The
concentrated protein was loaded onto a 2.5 × 113-cm column of
Sephacryl S-100 (Pharmacia) in 25 mM Tris (pH 7.5), 1 mM EDTA, 1 mM DTT, 75 mM NaCl and
eluted with the same buffer to separate the full-length protein from
the product of the secondary cleavage reaction.
SDS-Polyacrylamide and Isoelectric Focusing Gel Electrophoresis-- SDS-polyacrylamide gel electrophoresis was carried out using 15% Ready Gels from Bio-Rad with Coomassie staining as described by the manufacturer. Isoelectric focusing gel electrophoresis was carried out on a Pharmacia Phast System using IEF 3-9 gels, and Coomassie staining was performed as described by the manufacturer.
Molar Extinction Coefficients--
The molar extinction
coefficients for the CBF(141) and CBF
(187) proteins were
calculated on the basis of the tryptophan, tyrosine, and cystine
content of the proteins as described by Pace et al. (27). A
value of 18,500 M
1 cm
1 was
calculated for both proteins.
MALDI Mass Spectrometry-- MALDI mass spectrometry was carried out by Dr. Gary Siuzdak at the mass spectrometry laboratory at the Beckman Center for Chemical Sciences, Scripps Research Institute. Samples were dialyzed into 20 mM ammonium acetate (pH 7), 0.5 mM DTT prior to analysis.
N-terminal Sequencing-- To confirm the site of cleavage by enterokinase, N-terminal sequencing of the first five residues was carried out at the Biotechnology Resource Laboratory located at Yale University. A sequence of Xaa-Xaa-Met-Pro-Arg was obtained showing that cleavage had occurred at the correct site; however, the sequence of the first two amino acids was not determined with certainty.
Measurement of Dissociation Constants--
The affinities of
CBF(141) and CBF
(187) for a CBF
·DNA complex were measured by
electrophoretic mobility shift assay. The source of CBF
2 was an
41-214-amino acid fragment from the murine CBF
2 protein
encompassing the DNA-binding Runt domain, expressed in bacteria and
purified as described previously (28). Basically, binding conditions
were chosen such that the concentration of DNA was >10-fold above the
Kd of the CBF
2 Runt domain for the DNA site
(~1 × 10
8 M DNA), and the
concentration of active CBF
2 Runt domain was >10-fold below the
Kd of CBF
for the CBF
2·DNA complex [5 × 10
10 M CBF
2]. A fixed amount of DNA
and the Runt domain, along with various amounts of CBF
proteins,
were used in binding reactions, and the complexes were resolved by
electrophoretic mobility shift assay. Assay conditions and methods for
quantitation were the same as described previously (15). The data are
plotted as the percentages of CBF
2·CBF
·DNA complex
versus the concentration of CBF
, and the
Kd is defined as the concentration of CBF
at 50%
saturation (Kaleidagraph, Synergy Software). 100% ternary complex was
defined as the point of saturation, and 0% ternary complex is taken
from the background at the position in the absence of added CBF
.
Sedimentation Equilibrium--
High speed (29) sedimentation
equilibrium experiments were conducted at 20 °C in a Beckman XL-l
ultracentrifuge at rotor speeds of 15,000, 20,000, 25,000, and 30,000 rpm (CBF(141)) or 10,000, 15,000, 20,000, and 25,000 rpm
(CBF
(187)) using absorbance detection. For each sample, three cell
loading concentrations were examined, from 0.25 to ~1 mg/ml in 25 mM Tris-Cl (pH 7.5), 150 mM KCl, 1 mM DTT. Data were fit using NONLIN (30). Partial specific
volumes and solvent density (1.00960 g/ml) were calculated as described
(31). For CBF
(141), the calculated partial specific volume (0.7225 ml/g) was adjusted for the peptide charge (
3 at pH 7.5) by decreasing
the molar volume 25 ml/mol charge to 0.7185 ml/g. An additional
uncertainty of 0.004 ml/g in the partial specific volume was included
when calculating the molecular weights (32, 33). For CBF
(187), the
calculated partial specific volume (0.7161 ml/g) also was reduced for
its expected charge (
4 at pH 7.5) by 0.004 ml/g, and the same
additional uncertainty was included in the molecular weight
calculations.
Circular Dichroism Spectroscopy--
Circular dichroism spectra
were collected at 20 °C on a Jasco 715 spectrometer calibrated using
10-camphorsulfonic acid. Mean amide values were calculated using
the known protein sequence. The protein solutions were dialyzed
extensively against 25 mM potassium phosphate (pH 7.5), 0.1 mM EDTA, 0.5 mM DTT prior to CD measurements.
Quartz cells of 0.05 mm were used for measurements in the far
ultraviolet (176-260 nm). The data were corrected by subtraction of a
spectrum of the buffer alone. A total of eight scans were recorded at 1 nm resolution from 265 to 175 nm for both protein and buffer at a rate
of 10 nm/min with a 16-s response time. The resulting data for 178-260
nm were fit using the variable selection protocol of Johnson and
co-worker (34, 35) using software provided by Dr. Johnson. Three
proteins at a time were removed from the 33-protein data base, and the
resulting 5456 combinations were examined for total percentage of
secondary structure and root mean square error. Eleven combinations
were finally selected, all of which had root mean square error values
less than 0.20 (CBF
(141)) and 0.25 (CBF
(187)).
NMR Spectroscopy-- All measurements were performed on a Varian UnityPlus 500 NMR spectrometer equipped with an actively shielded triple-resonance probe from Nalorac Corporation and pulsed field gradients. Measurements were carried out at 20 °C with solutions of the proteins in 25 mM potassium phosphate (pH 6.5), 0.1 mM EDTA, 0.1% sodium azide, 5 mM DTT. 15N-1H HSQC spectra were recorded using the gradient sensitivity-enhanced HSQC sequence (36). The number of complex points and acquisition times for these experiments were 15N (F1), 128 points, 79.3 ms and 1H (F2), 1024 points, 157 ms.
For measurement of 15N T1 and T2 values, the pulse sequences of Farrow et al. (37) optimized for minimal saturation of water were employed. A recycle delay of 1.0 s was used between acquisitions to ensure sufficient recovery of NH magnetization (38). 15N T1 values were measured from spectra recorded with eight different durations of the delay: 44.4, 111.1, 222.2, 333.2, 555.4, 777.6, 999.7, and 1444.0 ms for CBF
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(Eq. 1) |
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RESULTS |
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Construction of Glutaredoxin Fusion Protein Vector--
The
biophysical and structural studies we wish to carry out to characterize
CBF require large quantities of homogeneous protein. Attempts to
overexpress CBF
in a number of different vectors including
glutathione S-transferase fusion vectors met with limited success. To overcome these difficulties and provide the milligram quantities of CBF
necessary for biophysical and structural studies, we have engineered a novel fusion vector based on E. coli
glutaredoxin. Based on the high level of overexpression obtained for
E. coli glutaredoxin (25), we reasoned that a fusion vector
with CBF
fused to the C-terminal end of glutaredoxin should also
give very high levels of expression. Fig.
1A illustrates the construct
we have employed in which the coding sequence for E. coli
glutaredoxin-1 has been fused to that of CBF
(141) via a linker
containing the DDDDK recognition sequence for enterokinase. The DDDDK
sequence permits the release of CBF
(141) from the fusion protein via
proteolysis with enterokinase. Two additional amino acids are retained
at the N-terminal end (Gly-Ser) due to the BamHI restriction
site used for subcloning purposes. A similar fusion vector has been constructed with the related protein thioredoxin (44); however, this
vector employs tryptophan for induction, making it unsuitable for
labeling proteins with 13C and 15N for NMR
spectroscopy. Our Grx fusion vector employs a heat-inducible induction
based on the heat stability of a mutant
repressor (25), thus
avoiding these difficulties.
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Purification of CBF(141) and CBF
(187)--
Utilizing the
pGRXCBF
141 fusion vector, we obtain high levels of expression (Fig.
2, lane 3) of protein that is
virtually completely soluble (Fig. 2, lanes 4 and
5). Fig. 2 illustrates a typical purification of CBF
(141)
starting with cells from a 1-liter bacterial culture. After lysis of
the cells and clarification by centrifugation, the Grx-CBF
(141)
fusion protein can be purified close to homogeneity in two steps via
ion-exchange chromatography on DEAE-Sephacel and size exclusion
chromatography on Sephacryl S-100 (Fig. 2, lanes 6 and
7). The fusion protein-containing fractions were readily
identified using the 2-hydroxyethyl disulfide assay specific for
glutaredoxin (26), pooled, and concentrated by ultrafiltration.
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CBF Binding Properties of CBF
(141) and Full-length
CBF
(187)--
Previous studies have demonstrated that the first 141 amino acids of CBF
were sufficient for interaction with CBF
(1, 15, 16). To determine whether CBF
(141) and the full-length CBF
(187) have similar affinities for CBF
, we performed
electrophoretic mobility shift assays to measure the
Kd of both proteins for a CBF
·DNA complex. The
source of CBF
used in these experiments consists of a 175-amino acid
fragment containing the DNA binding Runt domain from the murine CBF
2
(AML1) protein, which was expressed and purified as described
previously (28). The supershift of the Runt domain-DNA complex
associated with binding of CBF
has been employed to obtain
equilibrium dissociation constants for the binding of CBF
to a Runt
domain-DNA complex (15). Fig. 4 shows gel
shift data obtained from these measurements, and the data employed for
the calculation of the Kds.
Kd values of (4.5 ± 0.5) × 10
8
M and (1.8 ± 0.1) × 10
8 M
(measured at 4 °C) were obtained for the binding of CBF
(141) and
CBF
(187) to a Runt domain-DNA complex, respectively. These values
are very similar, demonstrating that virtually all the determinants
necessary for binding to the Runt domain reside in the N-terminal 141 amino acids. This agrees well with the observed homology between
mammalian CBF
and its Drosophila homologs where there is
a high degree of homology in the N-terminal 137 amino acids but very
low homology in the C-terminal region of the proteins. The exact
function of the C-terminal region is unknown at this time.
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Sedimentation Equilibrium--
To assess the solution
oligomerization state of CBF(141) and CBF
(187), sedimentation
equilibrium measurements have been carried out. Both CBF
(141) and
CBF
(187) sediment as single ideal species with apparent molecular
weights of 16,600 ± 1200 (root mean square of the fit = 0.008 Å) and 25,700 ± 1000 (root mean square of the fit = 0.0011 Å). These values are in good agreement with the calculated
molecular masses of the proteins (16.7 and 22.1 kDa); thus we conclude
that both proteins are monomeric under these conditions. Because the
two proteins do not aggregate to any observable extent up to the
concentrations employed for the sedimentation equilibrium measurements,
both proteins should be good candidates for structure determination by
NMR spectroscopy.
CD Analysis--
The far ultraviolet CD spectra for the two
proteins have been analyzed to obtain estimates of the percentages of
various secondary structures (Fig. 5)
using the variable selection procedure of Johnson (34) and Manavalan
and Johnson (35). Both CBF(141) and CBF
(187) are mixed
/
proteins with CBF
(141) having 21%
-helix (~30 residues) and
27%
-sheet (~39 residues) content, whereas CBF
(187) displays
34%
-helix (~64 residues) and 21%
-sheet (~39 residues)
content. The remainder of the residues in both proteins are partitioned
between turn and random coil conformations with both proteins having a
similar number of residues in random coil conformations, but
CBF
(187) having a larger number of residues in turn conformations.
Based on the similar numbers of residues in
-sheet conformations,
the similar biochemical activity, and the very similar NMR spectra of
the two proteins (see below), it appears that the N-terminal 141 amino
acids retain the same conformation in both CBF
(141) and the
full-length protein. Based on this, the increase in the number of
-helical residues (64 for CBF
(187) versus 30 for
CBF
(141)) must correspond to residues in the C-terminal portion of
CBF
(187). This implies that this nonconserved C-terminal tail is
almost completely
-helical.
|
NMR Spectroscopy--
15N-1H gradient
sensitivity-enhanced HSQC (36) spectra of 15N-labeled
CBF(141) and CBF
(187) have been recorded to assess the structural
similarity of the two proteins and to assess their suitability for
structure determination by NMR spectroscopy. Fig. 6 shows the
15N-1H HSQC spectra of the two proteins. Both
proteins could readily be concentrated to the ~1 mM
concentration necessary for NMR spectroscopy and remained soluble for
extended periods. Both proteins give well dispersed spectra
characteristic of mixed
/
proteins. The spectra show a striking
resemblance to one another in almost all regions, providing additional
confirmation of the retention of the conformation of the N-terminal 141 amino acids in both proteins. In particular, the residues with
downfield NH frequencies that generally correspond to amino acids in
extended conformations in
-sheets (45) display virtual identity to
one another.
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DISCUSSION |
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The core binding factor proteins constitute a small family of
transcription factors containing a DNA-binding subunit and a
non-DNA-binding
subunit (53). The Runt domain of CBF
is responsible for both DNA binding and heterodimerization with CBF
. CBF
binds to the Runt domain and is believed to induce a
conformational change that results in an increase in the affinity of
the Runt domain for DNA of approximately 6-fold
(1-2).2 The
three-dimensional structures of CBF
and the Runt domain are unknown,
and the primary sequences do not show any homology to other DNA-binding
domains or protein-protein interaction domains. Knockouts of the genes
encoding CBF
2 and CBF
in mice are embryonic lethal and lead to a
complete blockage in definitive hematopoiesis (15-19), indicating that
CBF plays a critical role in hematopoietic development. Mutations in
the genes encoding CBF
2 and CBF
are associated with a large
number of leukemias (15, 23). Biophysical characterization and
structure determination of these proteins and their interactions with
each other as well as with DNA should greatly facilitate the
development of novel therapeutic strategies to treat the leukemias
associated with the variant forms of these proteins.
We have developed a procedure for the high level overexpression and
purification of full-length CBF, CBF
(187), as well as a truncated
form containing only the N-terminal 141 amino acids, CBF
(141), using
a novel protein fusion system employing E. coli glutaredoxin
as the fusion partner. The use of the glutaredoxin protein as the
fusion partner results in very high levels of expression of the
proteins in a soluble form. Cleavage of the desired CBF
product from
the fusion protein has been effected via either an enterokinase or
Factor Xa site in the fusion protein. Use of Factor Xa was necessary
for the full-length protein because of the presence of two secondary
sites of cleavage for enterokinase in the C-terminal portion of
CBF
(187). These sites have been identified by MALDI mass
spectrometry of the cleaved products and do show a similarity to the
Asp-Asp-Asp-Asp-Lys recognition sequence of enterokinase. The use of
glutaredoxin fusions for overexpression may be generally applicable to
many other proteins.
We have utilized a number of different methods to characterize the
biophysical properties of CBF(141) and CBF
(187). Electromobility gel shift assays have been utilized to measure the equilibrium binding
constants for the binding of CBF
(141) and CBF
(187) to a Runt
domain-DNA complex. We have shown that the binding of both proteins is
nearly identical, confirming that the determinants for binding to the
Runt domain reside in the N-terminal 141 amino acids. Sedimentation
equilibrium NMR measurements show both proteins to be monomeric species
in solution, thus good candidates for structure determination by NMR
spectroscopy. Circular dichroism spectroscopy shows these proteins are
mixed
/
proteins with the additional C-terminal residues in
CBF
(187) being predominantly helical. This is quite interesting
considering the fact that the fusion protein formed by the chromosomal
inversion that disrupts the CBFB gene, inv(16)(p13;q22),
results in the fusion of the N-terminal 165 amino acids of CBF
to a
smooth muscle myosin heavy chain protein encoded by the gene
MYH11 (10, 46). The portion of the smooth muscle myosin
heavy chain fused to CBF
corresponds to the
-helical coiled coil
domain, resulting in a helical conformation for this portion of the
fusion protein as well. NMR spectroscopy has been conducted on the two
proteins to demonstrate their structural similarity and establish their
suitability for structure determination by NMR spectroscopy. A
structure determination by NMR spectroscopy of CBF
(141) is currently
being pursued.
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ACKNOWLEDGEMENTS |
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We thank Dr. Gary Siuzdak of the Scripps Research Institute for performing the MALDI mass spectrometry measurements and Wayne Casey for maintaining the Varian UnityPlus 500 MHz NMR spectrometer. We also thank Dr. Bill Wickner for use of the French press.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
A Leukemia Society of America Scholar. Supported by U. S.
Public Health Service Grants R01 CA/GM 58343 and R01 CA75611. To whom
correspondence should be addressed. Tel.: 603-650-1159; Fax: 603-650-1128.
** Supported by U. S. Public Health Service Grants K02 AI01481 and R29 AI39536 from the NIAID, National Institutes of Health. To whom correspondence should be addressed. Tel.: 603-646-1567; Fax: 603-646-3946.
1
The abbreviations used are: CBF, core-binding
factor
subunit; CBF
(141), the 143-amino acid protein containing
residues Gly-Ser followed by the N-terminal 141 amino acids of murine
CBF
; CBF
(187), the 189-amino acid protein containing residues
Gly-Ser followed by amino acids encoding the 187-amino acid isoform of CBF
; DTT, dithiothreitol; Grx, E. coli glutaredoxin-1;
HSQC, heteronuclear single quantum coherence; NH, protein backbone
amide NH; PAGE, polyacrylamide gel electrophoresis; MALDI,
matrix-assisted laser desorption ionization.
2 B. E. Crute, X. Huang, J. J. Kelley, N. A. Speck, and J. H. Bushweller, manuscript in preparation.
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