(Received for publication, August 26, 1994; and in revised form, December 16, 1994)
From the
The ETS domain family of transcription factors is comprised of
several important proteins that are involved in controlling key
cellular events such as proliferation, differentiation, and
development. One such protein, Elk-1, regulates the activity of the
c-fos promoter in response to extracellular stimuli. Elk-1 is
representative of a subgroup of ETS domain proteins that utilize a
bipartite recognition mechanism that is mediated by both protein-DNA
and protein-protein interactions. In this study, we have overexpressed,
purified, and characterized the ETS DNA-binding domain of Elk-1
(Elk-93). Elk-93 was expressed in Escherichia coli as a fusion
protein with glutathione S-transferase and purified to
homogeneity from both the soluble and insoluble fractions using a
two-column protocol. A combination of CD, NMR, and fluorescence
spectroscopy demonstrates that Elk-93 represents an independently
folded domain of mixed /
structure in which the three
conserved tryptophans appear to contribute to the hydrophobic core of
the protein. Moreover, DNA binding studies demonstrate that Elk-93
binds DNA with both high affinity (K
0.85
10
M) and specificity.
Circular permutation analysis indicates that DNA binding by Elk-93 does
not induce significant bending of the DNA. Our results are discussed
with respect to predictive models for the structure of the ETS
DNA-binding domain.
Families of transcription factors are defined on the basis of the primary sequence homology within their DNA-binding domains. Notable examples for which three-dimensional structures have been resolved include the helix-turn-helix (for review, see (1) and (2) ), the zinc fingers (for review, see (3) ), the helix-loop-helix(4, 5, 6) , and the b-Zip proteins(7) . However, structural features of a representative member from the ETS domain transcription factor family remain to be elucidated.
Members of the ETS domain family of eukaryotic
DNA-binding proteins were originally identified by their primary
sequence homology with the protein product of the ets-1
proto-oncogene(8) . A homologous sequence of approximately 86
amino acids comprising the ETS domain has since been identified within
more than 30 proteins, many of which are transcription factors (for
review, see (9) and (10) ). It has been demonstrated
for several proteins that the ETS domain corresponds to the DNA-binding
domain (for review, see Refs. 9 and 10). Indeed, several ETS domain
proteins have been shown to bind DNA sequences of approximately 10 base
pairs containing the core consensus sequence (C/A)GGA(A/T) known as the
ets motif (for review, see (9) and (10) ). One
particular feature, which is diagnostic of almost all ETS domains, is
the presence of 3 conserved tryptophans spaced 18 residues apart.
Secondary structure predictions by several standard methods (11, 12) suggest that the ETS domain shows no strong
homology to any known DNA-binding motifs, although certain regions are
predicted to be -helical. However, others have used the predictive
method of hydrophobic cluster analysis, which suggests that the
structure of the ETS domain may be related to that of the DNA-binding
domains of Myb and HMG(13) .
Members of the ETS family of DNA-binding proteins are expressed throughout the Metazoa in a variety of cell types(14, 15) . ETS domain proteins are involved in a range of important biological processes, including cell growth control, differentiation, and embryonic development. For example, the protooncogene products Ets-1 and Ets-2 are transcriptional activators implicated in T-cell activation and development(16) . Moreover, the ETS domain proteins Pok/Yan and PntP2 are transcription factors of the sevenless pathway involved in regulation of photorecepter cells during Drosophila eye development(17, 18, 19) .
The
transcription factor Elk-1 (20) is an ETS domain protein that
is involved in the transcriptional regulation of the immediate-early
gene c-fos (for review, see (21) ). Elk-1 forms a
ternary nucleoprotein complex with the serum response factor (SRF) ()and the c-fos serum response element
(SRE)(22) . Three domains within Elk-1 were initially defined
on the basis of amino acid homology (23) within a subgroup of
ETS domain proteins that also includes SAP-1 (23) and ERP (24) (Fig. 1A). Subsequently, biochemical
functions have been ascribed to each of these domains. The C-terminal
region of Elk-1 contains a transcriptional activation domain regulated
by growth factor-induced mitogen-activated protein
kinases(25, 26, 27) . The Bdomain is a short
internal region of 20 amino acids that is necessary for ternary complex
formation(23, 28) . This domain mediates direct,
specific, protein-protein interactions between Elk-1 and the
DNA-binding domain of SRF(29) . The A-domain of Elk-1 comprises
the ETS DNA-binding domain and is located at the N-terminal end of the
protein. When in complex with SRF, the ETS domain of Elk-1 makes direct
contact with the ets binding motif within the SRE; autonomous DNA
binding of Elk-1 to the ets site within the SRE does not occur (28) . (
)However, in the absence of SRF, Elk-1
specifically binds to the Drosophila E74 ets site (28) (
)and other artificial sites (30) in vitro.
Figure 1:
Purification of the ETS DNA-binding
domain of Elk-1. A, schematic representation of the domain
structure of Elk-1. The location of the A-domain (the ETS DNA-binding
domain; amino acids 1-90), the B-domain (SRF interaction domain;
amino acids 148-168), and C-domain (mitogen-activated protein
kinase target/transcriptional activation domain; 352-399) are
indicated. The fragment of Elk-1 representing the ETS DNA-binding
domain of Elk-1 utilized in this study (amino acids 1-93; Elk-93)
is shown below the full-length protein. B, SDS-polyacrylamide gel electrophoresis analysis of Elk-93 samples
at various stages of purification; soluble and insoluble extracts from
uninduced E. coli. harboring pAS74 (lanes2 and 3), soluble and insoluble extracts from E. coli. harboring pAS74 after 3 h of
isopropyl-1-thio--D-galactopyranoside induction (lanes4 and 5), soluble and insoluble
protein samples after incubation of the insoluble induced extract shown
in lane5 with 500 mM NaCl (lanes6 and 7), glutathione S-transferase-Elk-93 fusion protein after glutathione affinity
chromatography (lane8), glutathione S-transferase (lane9), and Elk-93 (lane 10)
after thrombin cleavage. Molecular mass markers are shown in lane1 with sizes in kDa indicated on the left. C, elution profile of Elk-93 after fast protein liquid
chromatography heparin affinity chromatography. Elk-93 elutes as a
single peak between 350 and 500 mM NaCl (fractions
9-12). 5-µl samples from 1-ml fractions 8-13 were
analyzed by SDS-polyacrylamide gel electrophoresis (inset).
Elk-93 in fractions 9-12 migrates as a single band with molecular
mass
11 kDa.
In this study, we have overexpressed
the Elk-1 ETS domain as a fusion with glutathione S-transferase. A purification protocol has been developed to
obtain homogenous preparations of the isolated ETS domain from both the
soluble and insoluble cell fractions. A combination of biochemical and
biophysical techniques has been used to establish that the purified
isolated ETS domain is a functional, monomeric, DNA-binding protein
with a dissociation constant of 0.85
10
M. We also demonstrate that the Elk-1 ETS domain binds
DNA specifically and unlike many DNA-binding domains, does not appear
to induce significant DNA bending as judged by the circular permutation
assay. Circular dichroism (CD) and nuclear magnetic resonance (NMR)
studies show that the ETS domain is a mixed
/
structure.
Furthermore, fluorescence spectroscopy indicates that the repeating
tryptophan residues are buried within the interior of the protein. We
discuss our results in relation to the various predictive structural
analyses that have been carried out on the ETS DNA-binding domain.
All steps in the purification, unless
otherwise stated, were performed at 4 °C. Cells were pelleted by
centrifugation, resuspended in 5 ml of PBS (16 mM NaHPO
, 4 mM NaH
PO
, 150 mM NaCl, pH 7.4)
containing 1 mM phenylmethylsulfonyl fluoride and lysed by 6
30-s rounds of sonication on a MSE Soniprep 150 sonicator at an
amplitude setting of 18 µm. The expressed fusion protein,
glutathione S-transferase-Elk-93, was found in both the
soluble and insoluble phases. The soluble protein was purified
essentially as described by Smith and Johnson (31) with an
additional high salt wash to selectively remove nucleic acid.
Specifically, glutathione S-transferase-Elk-93 was bound to a
1-ml reduced glutathione-agarose column (Sigma) and washed with 10
column volumes of PBS to remove unbound protein. A second wash
procedure with 20 column volumes of PBS containing 750 mM NaCl
was then performed to specifically remove nucleic acids from the
column. The presence of nucleic acids in the eluant was detected by
spotting aliquots onto agarose plates containing 10 mg/ml ethidium
bromide.
The insoluble protein was extracted from the insoluble pellet by resuspending the pellet in 10 ml of PBS containing 500 mM NaCl and incubating on a rotating drum for 1 hr. The protein suspension was centrifuged at 18,000 rpm for 20 min to remove insoluble material. The supernatant containing solubilized glutathione S-transferase-Elk-93 was then applied to a reduced glutathione-agarose column, and purification continued as described for the soluble fusion protein.
After thrombin cleavage (1 unit/ml; Sigma) the Elk-1 ETS domain (Elk-93) was eluted from the reduced glutathione-agarose column in 10 ml of TN buffer (50 mM Tris, pH 7.5, 150 mM NaCl). A 5-ml Econo-pac heparin cartridge (Bio-Rad) linked to fast protein liquid chromatography (Pharmacia) was used for the final purification step. The column was equilibrated with TN buffer prior to loading 10 ml of Elk-93 at a flow rate of 0.5 ml/min. The column was washed with 20 ml of TN buffer followed by a 20-ml linear gradient of NaCl (0.15-1 M) in 50 mM Tris, pH 7.5, at a flow rate of 1 ml/min. Column fractions of 1 ml were collected. Elk-93 eluted as a single peak between 350 and 500 mM NaCl.
The concentrations of protein samples were
routinely estimated using the Bio-Rad protein assay reagent. A standard
curve was derived using known concentrations of bovine serum albumin as
recommended by the reagent supplier. Alternatively, sample
concentrations were determined by ultraviolet absorption at 280 nm
using the theoretical extinction coefficient of 24,750 M derived from the the Elk-93 primary amino
acid sequence(32) . N-terminal protein sequencing was performed
on an Applied Biosystems Inc., model 473A automated sequencer, at the
Krebs Institute, Sheffield, UK. Samples were concentrated using
Centricon microconcentrators (Amicon) with a molecular mass cut-off of
10 kDa according to the manufacturer's instructions.
Competition experiments were
performed by the addition of either unlabeled E74B oligonucleotide or
unlabeled mutant E74 oligonucleotide mE74B (synthesised by annealing
ADS125 (5`-CTAGAGCTGAATAACCGCAAGTAACTCAT-3`) and ADS126
(5`-CTAGATGAGTTACTTGCGGTTATTCAGCT-3`) in the reaction mixture
prior to the addition of P-labeled E74B probe; reactions
were allowed to proceed for an additional 20 min prior to loading onto
the gel.
Gels were dried, and DNA-protein complexes were visualized by autoradiography. Quantification of DNA-protein complexes was achieved by phosphorimaging (425 S PhosphorImager and ImageQuant software; Molecular Dynamics).
DNA bending analysis was performed on
circularly permuted 145-bp DNA fragments containing the E74B binding
site obtained from the plasmid pAS75. Fragments were routinely labeled
with P using the kinase exchange reaction (35) or
alternatively synthesized by polymerase chain reaction in the presence
of [
-
P]dCTP.
The apparent
dissociation constant (K) for Elk-93 was obtained
using gel retardation analysis in which the protein concentration was
held constant (2.3
10
M) while the
DNA concentration was varied (0.15-1.04
10
M). Under these conditions, the dissociation constant
can be calculated graphically by subjecting the data to Scatchard
analysis. K
is defined from the gradient of the
resulting graph representing the equation; [PD] = -K
[PD]/[D]
+ [P
], where [D] is the
concentration of free DNA, [PD] is the concentration of
protein:DNA complexes, and [P
] is the
concentration of total active protein. DNA binding reactions were set
up under standard conditions except that they were carried out at 125
mM KCl in the absence of poly(dI
dC) competitior.
Dilutions of Elk-93 were carried out in SDB buffer (1
PBS, 10%
glycerol, 1 mg/ml bovine serum albumin).
The solubilized
high salt fractions were pooled and bound to reduced
glutathione-agarose beads; binding to the beads was not prevented by
the high salt concentration (Fig. 1B, lane8). After unbound proteins were removed by washing, an
additional step was introduced to selectively remove nucleic acids
associated with the fusion protein. PBS containing 750 mM NaCl
was used to wash the column in order to selectively elute nucleic acids
from the column while retaining the protein. After thrombin cleavage,
the ETS domain was eluted in 2 column volumes of TN buffer (Fig. 1B, lane10). In order to
remove the thrombin and achieve further purification, the Elk-93 ETS
domain was applied to a heparin column and eluted with a linear NaCl
gradient (Fig. 1C). Elk-93 elutes between 350 and 500
mM NaCl. This two-column purification yielded 0.6 mg of
pure Elk-93/liter of bacterial culture. SDS-polyacrylamide gel
electrophoresis analysis of the purified protein demonstrates that the
recombinant purified Elk-93 ETS domain migrates as a single band of
molecular mass
11 kDa; the preparation was judged to be homogeneous
(>98%) by Coomassie staining. Elk-93 from the original soluble
fraction was bound to reduced glutathione-agarose beads and purified by
the same protocol after thrombin cleavage
and yielded
0.2 mg/liter. The total yield from the soluble and insoluble phases
was
0.8 mg/liter. N-terminal sequencing of the purified polypeptide
demonstrated that the first 2 residues were glycine and serine from the
glutathione S-transferase moiety; this is expected after
thrombin cleavage. The next 10 amino acid residues were identical to
the predicted sequence for the Elk-1 ETS domain.
Figure 2:
DNA binding properties of Elk-93. A, DNA competition assay by gel retardation analysis
demonstrating specific DNA binding of Elk-93 to the E-74 site. Elk-93
was incubated with P-labeled E74B oligonucleotide in the
presence of increasing amounts of either mutant (mE74B; lanes2-7) or wild-type (E74B; lanes8-13) oligonucleotides. The amounts of competitor
DNA included in the binding reactions are; 0 ng (lane1), 0.2 ng (lanes2 and 8),
2.5 ng (lanes3 and 9), 5 ng (lanes4 and 10), 10 ng (lanes5 and 11), 20 ng (lanes6 and 12), and 40
ng (lanes7 and 13). The increase in
competitor concentration is indicated schematically above each series
of lanes. B, circular permutation analysis of Elk-93 binding
to the E74B site. The location of the E74B binding site (represented by
a hatchedrectangle) within the 145-base pair
circularly permuted probes is shown schematically on the right. Probes were made by cutting pAS75 with the following
restriction enzymes; BamHI (Ba), PvuII (Pv), MluII (Ml). The result of the gel
retardation experiment is shown on the left. DNA binding sites
were; Ba (lane1), Pv (lane2), Ml (lane3). The location of
protein:DNA complexes (PD) and free DNA (D) are
indicated. C, gel retardation analysis of Elk-93 binding to
the E74A oligonucleotide in the presence of increasing concentrations
of NaCl. The percentage of maximal DNA binding by Elk-93 at each NaCl
concentration was determined by phosphorimaging and is shown
graphically. The inset shows the results of the gel
retardation experiment. NaCl concentrations in each binding reaction
are; 0 mM (lane1), 42 mM (lane2), 83 mM (lane3), 167 mM (lane4), 333 mM (lane5), 500 mM (lane6), 667
mM (lane7), and 833 mM (lane8). The increase in NaCl concentration in each reaction
is indicated schematically above the gel.
It has
been demonstrated that several transcription factors induce bending in
the DNA sequence upon
binding(43, 44, 45, 46) . Since SRF
induces bending in the SRE(47) we investigated
whether Elk-93 was also capable of bending DNA. Such bending may
contribute to a possible mechanism of transcriptional regulation at
Elk-1 targets such as the c-fos SRE. DNA bending can be
analyzed using the circular permutation assay(48) . This assay
is based on the observation that the mobility of a DNA fragment through
a nondenaturing polyacrylamide gel is dependent upon the extent to
which the fragment is bent. Bending at the center of a DNA fragment
results in a lower mobility than if the bend occurs at one end. Three
circularly permuted DNA fragments of identical size were produced that
contain the E74B binding site located either at the center or toward
the ends of the fragment (Fig. 2B). All three fragments
when bound by Elk-93-produced complexes that migrated with virtually
identical mobilities in a 5% nondenaturing polyacrylamide gel (Fig. 2B, lanes13). These results
indicate that the isolated ETS domain of Elk-1 does not significantly
bend the E74B DNA sequence.
The effect of increasing salt concentrations on the DNA-binding activity of Elk-93 was also investigated. Elk-93 binds the E74A site in the absence of NaCl and remains bound at all NaCl concentrations tested between 42 mM and 833 mM (Fig. 2C). DNA binding appears to be relatively insensitive to high NaCl concentrations, indicating that the protein is stable in high ionic strength solutions.
Elk-93
also binds the E74 site with high affinity. Gel retardation experiments
were carried out in which the concentration of protein was held
constant while the concentration of the DNA substrate was varied. The
data from this experiment were subjected to Scatchard analysis (Fig. 3). An apparent dissociation constant, K, for Elk-93 of 0.85
10
M was calculated from the gradient of the resulting
graph. The value of the y axis intercept indicates that less
than 10% of the protein was active in DNA binding. Such fractional
binding activity is a poorly understood phenomenon(49) .
However, Elk-93 purified from either the soluble or insoluble fractions
binds with equivalent affinities and are defined spectroscopically as
homogenous populations.
Figure 3:
DNA binding affinity of Elk-93. Inset, results of gel retardation analysis in which the
concentration of E74A oligonucleotide was titrated against a constant
concentration of Elk-93 (2.3 10
M).
DNA concentrations were 0.15 nM (lane1),
0.3 nM (lane2), 0.45 nM (lane3), 0.6 nM (lane4), 0.75
nM (lane5), 0.9 nM (lane6), 1.04 nM (lane7). The
increase in input DNA concentration ([D
]) is
represented schematically above the gel. The locations of protein:DNA
complexes (PD) and free DNA (D) are
indicated. The results are represented by a Scatchard plot. The
apparent dissociation constant for Elk-93 binding to the E74 binding
site was calculated from the gradient of the resulting graph as 0.85
10
M.
Figure 4:
Circular dichroism spectra of the
expressed domain. Elk-93 was measured at a protein concentration of 77
µM in a 0.1-mm cell. 20 scans were accumulated between 195
and 250 nm. The spectrum was corrected by subtraction of a blank
containing only buffer. The abscissa is represented in units
of (M
cm
) using the
molar concentration of amino acids present.
The one-dimensional
NMR spectrum of Elk-93 (Fig. 5A) shows resonance line
widths characteristic of a monomeric 11-kDa protein that is free from
aggregation effects. The considerable dispersion of resonances
indicates the independence of this domain as a folded protein. The
two-dimensional TOCSY spectrum of Elk-93 (Fig. 5B)
showing the ``fingerprint region,'' confirms the folded
nature of the peptide backbone. This region of the TOCSY spectrum shows
a high degree of dispersion including, in particular, a number of
resonances with both downfield amide and proton chemical shifts
typically observed from residues in
-sheets of proteins. A
statistical analysis of
proton chemical shifts in proteins
reveals that those of residues in coil conformations normally resonate
in the region 4.1-4.6 ppm, whereas those of residues within
-sheets resonate between 4.6 and 5.1 ppm(52) . In Elk-93,
23 residues have
proton resonances at frequencies lower than 4.6
ppm, and from this an estimate of 25 ± 7%
-sheet can be
derived. The size of the error reflects an estimate of other potential
influences that may be occurring on the chemical shift of these
resonances as seen in a wide range of proteins (for review, see (53) ). Although an analogous estimate of the helix content is
less accurate, owing to the greater overlap of the chemical shift range
with that of residues in coil conformations(52) , from the
number of alpha protons resonating above 4.0 ppm it is likely that the
ETS domain has a mixed
/
-structure. These data are entirely
consistent with results obtained using CD spectroscopy, which strongly
predicts a high proportion of
-helical content and also indicates
the presence of a significant proportion of
-sheet.
Figure 5:
H NMR spectra of Elk-93. A, one-dimensional spectrum recorded with presaturation and
removal of the residual water resonance with a time-domain convolution
difference filter. B, fingerprint region of a 30-ms TOCSY
spectrum showing the high degree of dispersion of the amide-
cross-peaks.
The fluorescence emission spectra of native and denatured Elk-93 were compared with that of the model compound N-acetyltryptophanamide (NATA) (Fig. 6), which has a completely solvent-exposed indole side chain in aqueous solutions. The position of the peak in the NATA spectrum arises from the effects of solvent relaxation in which excited state energy is transferred to the aqueous phase prior to fluorescence emission. The position of the emission peak of Elk-93 is blue shifted relative to that of NATA. This indicates shielding of the tryptophan indole rings from general solvent effects by the bulk structure of the protein and is typical of a protein with buried tryptophan residues.
Figure 6: Corrected fluorescence emission spectra of Elk-93 and NATA. The excitation wavelength was 295 nm. Both native and denatured Elk-93 spectra are shown for comparison with the NATA spectrum. The native Elk-93 spectrum has a peak at 332 nm compared with the denatured Elk-93 and NATA spectra which peak at 350 and 352 nm respectively.
The emission spectrum of denatured Elk-93 is similar to that of NATA as expected for tryptophan residues that are fully exposed to the solvent (54) . Taken together, these data suggest that the tryptophan residues in the native Elk-1 ETS domain are buried within the interior of the protein and are likely to play a key structural role.
The structure of the DNA-binding domains of several eukaryotic transcription factors have recently been elucidated both in isolation and in DNA-bound complexes. The molecular interactions of transcription factors with particular DNA sites are of paramount importance to an understanding of the complex mechanisms of transcriptional regulation.
The ETS family of transcription factors is a large and important group of proteins that regulates the transcription of a wide range of genes. Both protein-DNA and protein-protein interactions are important in imparting the specificity of interactions of ETS domain proteins with their target promoters (for review, see (9) and (10) ). However, to date, there have been no structural studies on the DNA-binding domain of a representative member of the ETS family. Such information would provide the basis to our understanding of the specificity determination by protein-DNA interactions that are mediated by the ETS domain. We have therefore overexpressed and purified the ETS DNA-binding domain of Elk-1 (Elk-93) and subsequently characterized this domain using a plethora of spectroscopic and biochemical assays.
A simple
two-column purification of the ETS domain has been developed that
yields highly purified preparations of Elk-93. The purification scheme
incorporates steps that solubilize the insoluble glutathione S-transferase-Elk-93 fusion protein by eluting in a high salt
buffer. Such a strategy has previously been used to purify the DNA
methyltransferase M.HhaI(42) . In addition, we have
demonstrated that contaminating DNA, which is bound with the protein on
the reduced glutathione-agarose column, can be specifically removed by
a 750 mM NaCl wash. Purified proteins from both the soluble
and insoluble fractions were identical as determined functionally by
gel retardation analyses and spectroscopically by CD and NMR
analyses. Our protocol for purifying insoluble protein may
be applicable to other DNA-binding domains expressed as fusion proteins
with glutathione S-transferase.
The purified isolated Elk-1
ETS domain, Elk-93, binds the E-74 site with both high specificity and
affinity with an apparent dissociation constant (K) of 0.85
10
M. This dissociation constant is within the same range
as observed for a truncated version of Ets-1 containing the ETS domain
(
N322; (55) ). In this case, K
values
varied between 0.4 and 4
10
M depending on the DNA binding site. Moreover, this binding is
stable in concentrations of NaCl up to 833 mM. This indicates
that the function of Elk-93 is not greatly affected by the presence of
NaCl and as such we conclude that our structural data obtained in high
NaCl concentrations is likely to reflect the native conformation of the
ETS domain. The salt insensitivity of DNA binding by Elk-93 is in
marked contrast to that of other DNA binding proteins that lose their
ability to bind DNA with high affinity and specificity at high salt
concentrations. Notable examples include two members of the MADS family
of transcription factors, SRF and RSRFC4/MEF2A, whose DNA binding is
severely compromised in 250 mM KCl (56) and the
estrogen receptor that shows greatly reduced DNA binding in 250 mM NaCl(57) . This may reflect a novel type of interaction
between the ETS domain and DNA, which presumably is not highly
dependent on salt bridges to the phosphate backbone that would be
disrupted under high ionic strength conditions(58) .
Several
DNA-binding proteins that interact with DNA via -helices have been
shown to bend DNA. Examples include CAP(48) ,
Fos/Jun(59) , the POU domain(43) , and more recently
Myb(46) . Circular permutation analysis demonstrates that the
ETS domain of Elk-1, while capable of specific binding to the E74 site,
does not induce significant bending in this DNA sequence. The lowest
limit to the sensitivity of the circular permutation assay is not
known, although bend angles between 12 and 21° have been
reported(60) . It is therefore possible that Elk-93 may
introduce a low degree of bending into DNA that is not detectable by
this assay. However, the apparent lack of DNA bending by Elk-93
detected by circular permutation analysis is in contrast to results
using the same assay that demonstrates that the Myb DNA-binding domain
induces significant (110-116°) DNA bending(46) . Myb
contains 3 tryptophan residues with similar spacing to those found in
the ETS domain. Based on the presence of these 3 conserved tryptophan
residues, it has been postulated that the Myb and ETS DNA-binding
domains are structurally related(13) . However, as the ETS
domain is incapable of bending DNA, and indeed recognizes completely
different sites than Myb, the mechanism of DNA binding is likely to be
fundamentally different. Therefore, it is likely that the two domains
will show a high degree of structural divergence. However, our results
suggest that the 3 conserved tryptophan residues may play a similar
role in the two DNA-binding domains. In Myb, these tryptophans are
buried within the interior of the protein and form a hydrophobic core
around which
-helices are packed(61) . Our fluorescence
studies on the ETS domain containing similarly spaced tryptophans also
indicate that these residues are within the interior of the domain.
These highly conserved residues within the ETS domain may therefore
play a structural role similar to that observed in Myb. Due to their
buried nature, it is unlikely that these residues make direct contacts
with DNA. Moreover, no change in fluorescence quenching by specific DNA
is observed, (
)further suggesting that these residues are
not involved directly in DNA binding.
CD and NMR analysis have been
used in this study to determine the nature of the secondary structure
within the ETS domain. Our results are consistent with the ETS domain
being an independently folded monomeric polypeptide with approximately
40% -helix and a significant quantity of
-sheet. The Elk-1
ETS domain is therefore of a mixed
/
structure. Given the
degree of conservation amongst other family members, it is likely that
all ETS domains contain an
/
DNA-binding motif. Structural
predictions suggest that the ETS domain represents a novel structure
with no obvious strong homology to any known DNA binding
motifs(11, 12) . Several regions within the ETS domain
are predicted to be
-helical(13, 62, 63) , which is
consistent with our observation of the presence of significant amounts
of
-helix. However, our detection of a significant proportion of
-sheet within the ETS domain rules out the possibility that the
ETS domain is predominantly
-helical as observed for the
DNA-binding domains of the b-Zip (7) and bHLH
proteins(4, 5, 6) . Moreover our results are
consistent with the Elk-1 ETS domain being a correctly folded, single
domain, polypeptide.
In summary, we have overexpressed, purified and characterized the ETS DNA-binding domain of Elk-1. By several functional and spectroscopic criteria, this domain is fully folded and functional. This work provides the framework around which further structural information about the ETS domain can be obtained. The determination of a high resolution three-dimensional structure for the ETS DNA-binding domain of Elk-1 should now be possible both in the presence and absence of its cognate binding site.
Note added in proof-While this manuscript was under review, two separate papers were published on the secondary structures of the ETS domains of Ets-1 (Donaldson, L. W., Peterson, J. M., Graves, B. J., and McIntosh, L. P.(1994) Biochemistry33, 13509-13516) and Fli-1 (Liang, H., Olejniczak, E. T., Mao, X., Nettesheim, D. G., Yu, L., Thompson, C. B., and Fesik, S. W.(1994) Proc. Natl. Acad. Sci. U. S. A.91, 11655-11659). Our results on the ETS-domain of Elk-1 are supported by the conclusions in these two papers.