From the Protein Structure Section, Macromolecular
Crystallography Laboratory, and the ¶ Regulation of Cell Growth
Laboratory, National Cancer Institute-Frederick, Frederick, Maryland
21702-1201 and
Synchrotron Radiation Research Section,
Macromolecular Crystallography Laboratory, National Cancer Institute,
and National Synchrotron Light Source, Brookhaven National
Laboratory, Upton, New York 11973
Received for publication, January 14, 2003
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ABSTRACT |
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CCAAT/enhancer-binding proteins (C/EBPs) are
basic region leucine zipper (bZIP) transcription factors that regulate
cell differentiation, growth, survival, and inflammation. To understand
the molecular basis of DNA recognition by the C/EBP family we
determined the x-ray structure of a C/EBP The transcription factor
C/EBP bZIP proteins function as transcriptional regulators in most or all
eukaryotes (13) and can be arranged into several subfamilies, each
recognizing a unique palindromic DNA motif (13) (Fig. 1). The canonical
bZIP DNA-binding domain consists of a basic region juxtaposed to a
sequence of heptad leucine repeats (the leucine zipper) (1). bZIP basic
regions show a high degree of sequence similarity and contain two
invariant residues, Asn and Arg (Fig. 1). The bZIP-DNA complex consists
of two To date, x-ray structures of bZIP domain peptides bound to DNA have
been determined for GCN4 (bound either to an AP-1 site (18) or to a CRE
site (19, 20)), the Jun/Fos heterodimer (21), CREB (22), and PAP1 (23).
These structures show that bZIPs recognize specific DNA sites through
base contacts made by five residues within the basic region motif
characteristic for each subfamily. These five positions are well
conserved among all bZIP proteins and include the invariant Asn and Arg
residues (Fig. 1). The invariant Asn
contacts bases C3 and T bZIP polypeptide bound to
its cognate DNA site
(A
5T
4T
3G
2C
1G1C2A3A4T5)
and characterized several basic region mutants. Binding specificity is
provided by interactions of basic region residues
Arg289, Asn292, Ala295,
Val296, Ser299, and Arg300 with DNA
bases. A striking feature of the C/EBP
protein-DNA interface that
distinguishes it from known bZIP-DNA complexes is the central role of
Arg289, which is hydrogen-bonded to base A3,
phosphate, Asn292 (invariant in bZIPs), and
Asn293. The conformation of Arg289 is also
restricted by Tyr285. In accordance with the structural
model, mutation of Arg289 or a pair of its interacting
partners (Tyr285 and Asn293) abolished C/EBP
binding activity. Val296 (Ala in most other bZIPs)
contributes to C/EBP
specificity by discriminating against purines
at position
3 and imposing steric restraints on the invariant
Arg300. Mutating Val296 to Ala strongly
enhanced C/EBP
binding to cAMP response element (CRE) sites while
retaining affinity for C/EBP sites. Thus, Arg289 is
essential for formation of the complementary protein-DNA interface, whereas Val296 functions primarily to restrict interactions
with related sequences such as CRE sites rather than specifying binding
to C/EBP sites. Our studies also help to explain the phenotypes
of mice carrying targeted mutations in the C/EBP
bZIP region.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 is the founding
member of the bZIP class of DNA-binding proteins (1). C/EBP
was
originally identified as a DNA binding activity in rat liver nuclear
extracts (2-4) and has since been shown to regulate terminal
differentiation of several cell types including adipocytes (5-7) and
neutrophilic granulocytes (reviewed in Ref. 8). Consistent with these
functions, mice carrying a homozygous deletion of the C/EBP
gene die
at birth due to energy imbalance caused by impaired glycogen storage in
the liver and lack lipid accumulation in their adipose tissues (9).
C/EBP
-deficient embyros also display a complete absence of mature
neutrophils and increased numbers of immature myeloid progenitor cells
(10). Furthermore, mutations in the C/EBP
gene have been found in a
subclass of human myeloid leukemias (11, 12). It has been proposed that
loss of C/EBP
can contribute to the development of myeloid
neoplasias by impairing the normal program of cellular differentiation
and mitotic arrest (11, 12).
-helices lying perpendicular to the DNA, associated in a
coiled-coil structure with each basic region contacting a half-site in
the DNA major groove (1, 14). A unique aspect of DNA recognition by
bZIP proteins is that the helical fold of the basic region, which
creates a surface complementary to the target DNA sequence, is induced
upon association with the DNA ligand (15-17).
4 in the complexes of
GCN4, Fos/Jun, and CREB with DNA
(G1T2C3A4) (18,
20-22), whereas in PAP1 bound to a PAR site
(G1T2A3A4) this residue
adopts a different conformation and makes a direct contact with base
A4 (23). Thus, the bZIP structures have revealed functional
variability of conserved residues in DNA recognition.
View larger version (29K):
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Fig. 1.
Basic region amino acid sequences and DNA
binding sites for representatives of bZIP subfamilies.
Shown here are proteins for which crystal structures have been
determined (except D box-binding protein (DBP)). The
invariant Asn and Arg are depicted in red, conserved basic
residues in blue, and the first residue of the leucine
zipper in green. The core sequences of the DNA half-sites
are underlined.
Here we present the crystal structure of a C/EBP bZIP polypeptide
bound to its cognate DNA site. The structure reveals that two residues
(Tyr285 and Arg289) located amino-terminally to
the signature bZIP basic region motif are integral components of the
protein-DNA interface. The functional significance of these residues
was confirmed by analysis of substitution mutants. Arg289
is conserved in AP-1 proteins but adopts a very different conformation; therefore, its importance for DNA binding in C/EBP proteins could not
have been predicted from structures of other bZIP proteins nor was it
indicated by previous mutagenesis studies. Finally, our data clarify
the key role of Val296 in determining C/EBP binding
specificity. This study thus provides the first detailed description of
DNA recognition by a C/EBP family member.
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EXPERIMENTAL PROCEDURES |
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Protein Production and Crystallization--
A protein containing
residues 281-340 of rat C/EBP was over-produced in
Escherichia coli and purified as described previously (15).
Cloning appended the sequence Met-Gly-Ser to the amino terminus of the
polypeptide; the terminal Met residue was removed in E. coli, resulting in a 62-amino acid product (Fig. 2A).
DNA oligomers (Yale Keck Laboratory) were gel-purified.
The crystallization strategy included trials with DNA duplexes of varying length and 5' overhanging bases. The best diffracting cocrystals were obtained with a 21-mer DNA duplex (Fig. 2B). Crystals were grown at 4 °C in hanging drops prepared by mixing equal volumes of the protein-DNA complex (0.75 mM bZIP monomer and 0.5 mM DNA duplex in 50 mM NaCl, 100 mM MES, pH 5.7) and the precipitant solution (100 mM MES, pH 5.7, 100 mM NaCl, 30 mM MgCl2 ,18% polyethylene glycol 400, 10% glycerol) and were harvested for flash freezing directly from the mother liquor. Crystals belong to the P21212 space group with unit cell dimension a = 140.89 Å, b = 53.09 Å, and c = 67.41 Å. The solvent content of the crystals was estimated as 73% (24).
Crystallographic Procedures--
X-ray data were collected at
100 K, 1.07-Å wavelength on beamline X9B, National Synchrotron
Light Source, Brookhaven National Laboratory, using the ADSC CCD
Quantum-4 detector, and were processed with HKL2000 (25) (Table I). The
structure was solved by molecular replacement using the AMoRe package
(26) with the truncated (residues 286 to 339 from chains A and C, 10
to +10 from chains B and D) polyalanine model derived from the
CREB bZIP-DNA complex (22) (Protein Data Bank code: 1DH3). The
asymmetric unit contains one protein-DNA complex. The DNA segments form
a pseudo-continuous helix with base pair interactions between
complementary 5'-overhanging bases from the adjacent complexes. The
maximum likelihood target refinement was carried out with CNS_1.0 (27)
initially against data from the 10-2.8-Å resolution shell. The
solution was first refined as three rigid groups: the coiled-coiled
region and the basic region of each monomer with its DNA half-site.
Subsequent cycles of simulated annealing refinement and model
rebuilding with O, version 8.0 (28) allowed localization of all
missing residues (except the two derived from the expression vector)
and fitting of most of the protein side chains and the DNA sequence. The orientation of the DNA backbone was determined based on the electron density corresponding to the asymmetric ends flanking the
central palindromic sequence. Further refinement against all data with
the correction for bulk solvent was followed by restrained thermal
parameter refinement. Water molecules were added based on peak heights
(
3
) in the difference Fourier map and proper hydrogen bond
distance. Several simulated annealing OMIT maps were calculated during
the course of refinement to verify the model. The geometrical
properties of the model were analyzed with the programs PROCHECK (29)
and 3DNA (30). The refinement statistics are summarized in Table
I. The mean B-factor of 91.6 Å2 of the final model correlates well with an estimate of
Wilson plot B-factors.
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Mutagenesis and Reporter Assays--
Specific amino acid changes
were introduced into full-length rat C/EBP or C/EBP
(in
pcDNA3.1) with the QuikChange mutagenesis kit (Stratagene). For
transactivation assays, 8 × 104 L929 cells were
transfected with 1 µg of 2× C/EBP Luc or 2× CRE Luc reporter
plasmids and 25 ng of C/EBP
or C/EBP
expression plasmids using
Fugene6 (Roche Molecular Biochemicals). A Renilla luciferase
vector (pRLTK, Promega) was included to correct for transfection
efficiency. After 48 h the cells were harvested, and lysates were
prepared and assayed for reporter expression using the Dual Luciferase
System (Promega). Firefly luciferase activity was normalized to
Renilla luciferase, and the ratio for reporter alone was set
to 1. Three experiments were averaged, and the data were graphed as the
mean ± S.E.
In Vitro Protein Expression and EMSA--
Proteins were
expressed using the TnT Quick transcription/translation system
(Promega). For Western blot analysis, 1 µl of each protein extract
was separated on 12% SDS-PAGE, transferred to nitrocellulose, and
probed using C/EBP or C/EBP
antibodies (C-14 and C-19,
respectively; Santa Cruz Biotechnology). Secondary goat anti
rabbit-horseradish peroxidase was used to visualize antigen-antibody
with chemiluminescence (ECL, Pierce). DNA binding reactions contained
20 mM Hepes, pH 7.5, 5% Ficoll, 1 mM EDTA, 200 mM NaCl, 1 mM dithiothreitol, 0.01% Nonidet
P-40, 400 ng/µl bovine serum albumin, and 40 ng/µl poly(dI-dC). The
following probes (binding sites indicated in bold type) were used for
EMSA assays:
C/EBP probe: | 5'-GATCCATATCCCTGATTGCGCAATAGGCTCAAAA |
GTATAGGGACTAACGCGTTATCCGAGTTTTCTAG-5' | |
CRE probe: | 5'-GATCCATATCCCTGATGACGTCATAGGCTCAAAA |
GTATAGGGACTACTGCAGTATCCGAGTTTTCTAG-5' | |
PAR probe: | 5'-GATCCATATCCCTGATTACGTAATAGGCTCAAAA |
GTATAGGGACTAATGCATTATCCGAGTTTTCTAG-5' |
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RESULTS |
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Overview of the Complex--
We crystallized a C/EBP bZIP
polypeptide bound to a 21-mer DNA duplex and determined the structure
at 2.8 Å resolution (Fig. 2). The
complex consists of two polypeptides (residues 281-340) that form a
dimer of
-helices associated with DNA in the well known fork-like
structure (14, 18). Residues 285-300 from each subunit comprise the
recognition helix located in the major groove of each DNA half-site.
The DNA duplex adopts a nearly straight B-type helix with an average
rise per nucleotide of 3.3 Å. Residues 281-284 from the "extended
basic region" characteristic of the C/EBP family (Fig. 2) do not
participate in any intramolecular or DNA contacts and are highly
mobile.
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The first contact between the two protein subunits is made via
electrostatic interactions between the side chains of their Asn307 residues. The C/EBP leucine zipper contains two
hydrophilic residues, Thr310 and Asn321, at the
d and a positions of the heptad repeat (Fig.
2A), buried in the hydrophobic core of the coiled-coil
dimer. The side chain hydroxyl oxygen of Thr310 from each
chain can potentially form a hydrogen bond to Gln311 from
the other subunit (Gln311'). Asn321 is
conserved among many bZIP transcription factors and, as observed in
GCN4 (18, 19) and CREB (22), its side chains form an inter-helical
hydrogen bond. The dimer is stabilized by two salt links formed by the
interaction of Asp320 with Arg325' and
Glu334 with Arg339'. Formation of equivalent
salt bridges between the reciprocal pairs of subunits is prevented by
crystal contact interactions.
The two protein chains are bent by crystal packing forces. The complete
chains superimpose with an r.m.s.d. of 2.03 Å for 118 C atom pairs,
whereas for the leucine zippers the r.m.s.d. is 1.06 Å for
superposition of 272 backbone atoms. The two basic regions immersed in
the DNA major groove are very similar, the r.m.s.d. for superposition
of the backbones of recognition helices together with all atoms of the
corresponding DNA half-sites is 0.46 Å for 538 common atoms. Thus, the
protein-DNA interface is not significantly affected by crystal packing.
Protein-DNA Interface--
The boundaries of the C/EBP-DNA
interface are defined by Tyr285 and Arg300.
Twelve amino acids from each recognition helix contact the DNA half-sites (Fig. 3A)
essentially in a symmetrical manner. Eight of the side chains are
potential proton donors in hydrogen bonds with phosphate oxygen atoms
from the DNA backbone. Direct contacts with DNA bases are made by
Arg289, Asn292, Ala295,
Val296, Ser299, and Arg300 (Fig.
3B). Details of the C/EBP
-DNA interface are shown in
Figs. 4 and 5. The invariant
Asn292 is positioned to make side chain hydrogen bond
interactions with the O-4 atom of base T
4 and the
N-6 amino group of base A3.
Adenine at position 3 is also specified by Arg289, which
contacts the N-7 atom of A3. C
atoms of
Ala295 and Ser299 as well as C
2
of Val296 make van der Waals contacts with the methyl group
of T
4. An apolar environment for the 5-methyl group of
T
3 is formed by atoms from the side chain of
Val296 and the aliphatic portion of Arg300
(Fig. 5A). The guanidinium group of Arg300 makes
electrostatic interactions with G1 and G
2.
Arg300 was modeled in one conformation in both subunits.
However, weak electron density in this region of the second subunit
(Arg300') indicates that Arg300' is perhaps
disordered with a low occupancy in this conformation. Asymmetric
contacts of the invariant Arg were also observed in the structure of
CREB bound to a symmetric CRE site (22).
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The protein-DNA interface is stabilized by interactions between side
chains of basic region amino acids. Particularly important in this
respect is the buried side chain of Arg289, which can make
bidentate hydrogen bonds with carboxylate oxygens from
Asn292 and Asn293. In addition, the aliphatic
portion of the Arg289 side chain makes extensive van der
Waals contacts with the phenolic ring of Tyr285,
whereas Val296 contacts the C and
C
atoms of Arg300 (Figs. 4A and
5B).
The conformation and functional roles in DNA recognition of
Asn292, Ala295, and Ser299,
residues that are conserved among bZIP proteins, are similar to those
observed in GCN4 and CREB bound to CRE sites (Fig. 4B). In
contrast, the ability of the invariant Arg300 residue to
adopt the conformation seen in GCN4/CREB, where it contacts base
G1 and DNA phosphate oxygen, is precluded by the proximity
of Val296 (Fig. 5B). In this manner,
Val296 enhances the preference for G2 in the
consensus CEBP binding site. However, the most striking difference
between the C/EBP
and GCN4 structures is the conformation of
Arg289 (Fig. 4). In AP-1 proteins, including Jun/Fos, this
residue does not participate in specific base recognition (18, 19, 21, 22). Furthermore, the cluster of polar residues involving
Arg289, Asn292, and Asn293 pushes
base C2 away from the position occupied by T2
in the GCN4 (Fig. 5C) and CREB (not shown) complexes and
enforces movement of the DNA backbone away from the protein in this
region. Because of this displacement, Val296 does not
interact with base C2 and would accept thymine at position
2 of the half-site (see below). In AP-1 and CREB proteins, Ala occupies
the position analogous to Val296 and specifies thymine at
position 2 by hydrophobic contact with its methyl group (Fig.
5C).
Members of the PAP bZIP family recognize a half-site sequence
(G1T2A3A4) that differs
from the consensus C/EBP site only at position 2. In the structure of
PAP1 bound to its cognate DNA, the invariant Asn adopts a different
conformation from that seen in C/EBP and GCN4 (Fig. 4B)
and does not contact base A3 directly (23). PAP1 recognizes
the A3·T
3 base pair in a very different
manner than C/EBP
because of dissimilarities in their signature
basic region sequences (Fig. 1).
Basic Region Mutants--
To test their functional roles, we
mutated several basic region amino acids predicted from the structure
to be important for C/EBP DNA recognition (Fig.
6A). Tyr285,
Arg289, and Val296 were converted to Ala and
Asn293 was mutated to Arg, the corresponding residue in
most AP-1 family members (Fig. 1). The mutations were introduced into
full-length C/EBP individually or in combination. Mutant proteins
were first tested for their ability to activate transcription from a
synthetic C/EBP-dependent reporter gene (2× C/EBP-luc) in
L929 fibroblasts (Fig. 6B, left panel). Although wt C/EBP
stimulated 2× C/EBP-luc by ~15-fold, most of the basic region
mutations severely reduced or eliminated transactivation. The
activities of Y285A, N293R, and Y285A/N293R were 5-6-fold lower
than wt, whereas R289A was essentially inactive. Mutating the residue
analogous to Tyr285 in C/EBP
(Y226A) also severely
reduced transactivation (Fig. 6B), indicating that the Tyr
residue has an important functional role within the C/EBP family.
Interestingly, changing Val296 to Ala did not impair
transactivation of the C/EBP reporter and in fact caused a modest
increase in activity. Similarly, the Y285A/N293R/V296A mutant was
~4-fold more active than Y285A/N293R.
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Several of the substitution mutants contain amino acids that occur at
the equivalent positions in AP-1 or CREB proteins (see Fig. 1).
Therefore, we tested their ability to transactivate a CRE-dependent reporter gene, 2× CRE-luc (Fig. 6B,
right panel). wt C/EBP weakly activated 2× CRE-luc
(~3-fold), consistent with its low affinity for CRE sites. The Y285A,
R289A, N293R, and Y285A/N293R mutants showed reduced activity. In
marked contrast, V296A and Y285A/N293R/V296A activated the reporter by
9-fold and 7-fold, respectively. Thus, the V296A substitution
significantly enhances the ability of C/EBP
to transactivate a
CRE-driven promoter.
We next examined the binding specificities of recombinant C/EBP
proteins using the electrophoretic mobility shift assay (EMSA) (Fig.
6C). wt C/EBP (lane 2) bound efficiently to
the C/EBP probe but interacted very weakly with the CRE. R289A
(lane 4) did not bind appreciably to either probe.
Interaction of C/EBP
Y285A (lane 3) and N293R (lane
5) with both probes was also significantly reduced, as was
C/EBP
Y226A (lane 10). Y285A/N293R and Y285A/N293R/V296A bound poorly to the C/EBP site, whereas V296A activity was similar to
wt C/EBP
. Strikingly, both mutants bearing the V296A substitution associated very efficiently with the CRE probe (lanes 6 and
8). EMSA quantitation showed that the V296A substitution
enhanced binding to the CRE site by 7-8-fold (Fig. 6C).
Thus, Val296 inhibits the interaction of C/EBP
with CRE
sites, whereas Ala strongly enhances binding to CREs but does not
impair binding to C/EBP elements. We also examined affinity of the
mutant proteins for a PAR site (Fig. 6C). Although the
overall pattern of binding was similar to that for the C/EBP probe, the
V296A mutant displayed increased binding to the PAR probe (1.4-fold
greater than the wt protein). In addition, the Y285A/N293R and
Y285A/N293R/V296A mutants maintained weak binding to the PAR site, in
contrast to their severe effects on binding to C/EBP sites. Thus, with
the exception of Y285A and R289A, the C/EBP
mutations were less
detrimental to PAR site binding than to interaction with the C/EBP element.
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DISCUSSION |
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Binding Specificity of C/EBP Proteins--
The data
presented here reveal the structural basis of C/EBP binding to a
consensus (i.e. highest affinity) C/EBP recognition element.
However, C/EBP
frequently binds in vivo to promoters containing non-canonical C/EBP sites (see Ref. 31 for examples). Binding site selection experiments have established the preference of
C/EBP family members for the sequence RTTGCGYAAY (where R and Y denote
purines and pyrimidines, respectively); some tolerance for different
bases at the first position of the half-site was also observed in these
studies (32, 33). Allowed substitutions usually do not occur
symmetrically, and the modified sites tend to be
pseudo-palindromic.
The strict requirement for adenine at half-site positions 3 and 4 is
clearly explained by the structure of the C/EBP bound to consensus
DNA site. The base pair A4·T
4 is defined by
a very tight hydrophobic pocket for the methyl group of base
T
4 and hydrogen bond of its carbonyl to
Asn292. Adenine, and not cytosine, is preferred at base 3 as a proton donor in hydrogen bond to the Asn292
carboxylate oxygen, because its N-7 atom can interact with
Arg289. More importantly, preference for its base pair
partner (T
3) is dictated by the bulky side chain of
Val296, which provides the proper environment for the
T
3 methyl group and effectively discriminates against
guanine at this position. On the other hand, base pairs
G1·C
1 and C2·G
2
are specified only by interactions of the guanine moieties with Arg300, in which the flexible side chain is partially
exposed to solvent (Fig. 5B). To accommodate substitution of
base pair C2·G
2 with
T2·A
2, the guanidinium group of
Arg300 must move away from DNA into the solvent channel
between the two protein chains emerging from the major groove. Thymine
can be accepted at position 2 without changes in the DNA backbone (Fig.
5C). This substitution will result in hydrophobic contact of
the T2 methyl group with Val296, possibly
compensating for the loss of the interaction between G
2
and Arg300. However, because of steric hindrance imposed by
the Val296 side chain (see "Results"), movement of the
Arg300 side chain is possible only toward the molecular
dyad of the complex. Such a displacement of Arg300 on both
subunits would place their charged groups too close to each other,
which may explain why the T2 substitution is usually
tolerated on only one of the two half-sites.
Role of Val296 in DNA Recognition--
The C/EBP
structure is apparently the first example in which Val plays a key role
in DNA recognition. Replacing Val296 with Ala had little
effect on the binding of C/EBP
to its consensus site (GCAAT), while
greatly increasing affinity for CREB sites (GTCAT). An earlier study
(34) found that a C/EBP
mutant containing the analogous
Val
Ala substitution displayed reduced binding to consensus C/EBP
sites but retained its affinity for PAR sites (GTAAT) and concluded
that Val is responsible for the relaxed sequence specificity (binding
promiscuity) displayed by C/EBP proteins. In contrast, we observe that
replacing Val296 with Ala actually diminishes
C/EBP
selectivity by increasing its affinity for CREB and PAR sites.
Our findings can be rationalized in structural terms. The V296A
substitution releases restriction on the Arg300 guanidinium
group, allowing it to occupy a position similar to that seen in GCN4
and CREB complexes on both subunits. This may account for the ability
of V296A to recognize PAR sites. Simultaneously, Ala will tolerate
guanine at position 3, permitting binding of the mutant protein to
CREB sites. The V296A substitution reduces the area of hydrophobic
interaction between the C/EBP site T
3 methyl group and
its environment (Fig. 5A). However, the resulting loss of
free energy of binding may be offset by increased entropy due to
Arg300 gaining a second energetically favorable
conformation and thereby maintaining affinity of the mutant protein for
C/EBP sites. Val296 thus appears to affect C/EBP
specificity by discriminating against guanine at position
3 and by
restricting the conformation of Arg300.
Importance of Side Chain Interactions in the Protein-DNA
Interface--
The side chain conformations of Tyr285,
Arg289, Asn292, and Asn293, which
form part of the protein surface conforming to the cognate DNA, depend
on an extended network of interactions (Fig. 4A). The
results of mutating Tyr285, Arg289, and
Asn293 underscore the importance of these stabilizing
interactions for DNA binding affinity. Mutation of Arg289
to Ala abolished C/EBP binding to C/EBP, CRE, and PAR sites, demonstrating the critical role played by this residue in the formation
of the complementary protein-DNA interface. The precise conformation of
Arg289 is maintained by interactions with
Tyr285 and Asn293, residues that are specific
for the C/EBP family. In accordance with the structure, single
mutations at either of these positions reduced C/EBP
binding to a
consensus C/EBP site, whereas the double mutation Y285A/N293R nearly
abolished binding. On the other hand, the N293R substitution had very
little effect on C/EBP
binding to CRE or PAR sites that have T at
position 2, the methyl group of which can contact Val296
(Fig. 5C).
Biological Implications--
Several groups recently postulated
that C/EBP can cause cell growth arrest by suppressing E2F-mediated
transcription and have suggested that C/EBP
associates with and
inhibits E2F in a DNA-binding independent manner (35-37). Porse
et al. (36) proposed that C/EBP
repression of E2F
function requires a trio of basic region residues, Tyr285,
Ile294, and Arg297, which were predicted to
face away from the DNA and thus could interact with proteins such as
E2F. Mutation of these amino acids diminished the ability of C/EBP
to repress E2F-dependent reporter genes in transfected
cells, and "knock-in" mice carrying either the Y285A or I294A/R297A
mutations displayed defects in adipogenesis and granulopoiesis. It was
concluded that C/EBP
promotes growth arrest and differentiation in
part by making inhibitory interactions with E2F via residues
Tyr285, Ile294, and Arg297.
Our findings provide an alternative interpretation for the phenotypes
of mice carrying C/EBP basic region mutations. Tyr285
contributes to a stable protein-DNA interface, and its replacement with
Ala severely impairs C/EBP
binding to C/EBP sites in addition to
transactivation of a C/EBP reporter gene (Fig. 6). Thus, the defects in
mice carrying the Y285A mutation can be explained most simply by the
reduced affinity of the mutant protein for C/EBP sites. Suckow et
al. (38) also reported that an Ile
Glu substitution at the
residue corresponding to Ile294 in GCN4 (see Fig. 1) conferred some
C/EBP-like characteristics to GCN4, indicating that Ile294 may play a
role in determining C/EBP
binding selectivity by influencing the
conformation of neighboring side chains. In addition, the structure of
the GCN4-CREB site complex (20) suggests that interaction between the
analogous pair of GCN4 residues, Glu237 and
Arg240, is an important determinant of binding specificity
by directing the Arg240 side chain to contact the phosphate
groups of G1 and C
2. In C/EBP
Arg297 interacts only with the phosphate group of
G1. Thus, it is possible that changes in C/EBP
DNA-binding activity and/or specificity also account for the biological
defects seen in mice carrying the I294A/R297A mutation. Clearly,
further studies are necessary to enhance understanding of the
mechanisms by which C/EBP
regulates cell growth arrest and
differentiation. The availability of the C/EBP
bZIP structure should
facilitate these investigations.
Concluding Remarks--
In this paper we present the first
detailed description of DNA recognition by a C/EBP family member.
Tahirov et al. (39, 40) have reported structures of
multiprotein complexes that include the bZIP domain of C/EBP in
association with DNA ligands containing non-consensus C/EBP sites;
however, the interaction of C/EBP
with DNA was not described in
these studies. We examined the C/EBP
structure (Protein Data Bank
code: 1HJB) and found that it recognizes DNA in a manner consistent
with our model. This is not surprising in view of the nearly identical
basic region sequences of the C/EBP
and C/EBP
proteins.
The C/EBP DNA-binding interface contains both similarities and
dissimilarities with other bZIP structures. The distinct functional roles for conserved residues such as Arg289 underscore the
notion that mechanisms of specific DNA recognition must be elucidated
independently for each bZIP family.
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ACKNOWLEDGEMENTS |
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We appreciate the encouragement and advice of A. Wlodawer and thank him and C. Vinson for critical reading of the manuscript. We acknowledge the help of colleagues in the Macromolecular Crystallography laboratory, particularly N. Nandhagopal. We also thank V. Pett for help with the initial crystallization experiments. Finally, we recognize the important contributions of S. McKnight and the late P. Sigler during early stages of this work.
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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.
The atomic coordinates and the structure factors (code 1NWQ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ To whom correspondence for structure-related issues should be addressed: Macromolecular Crystallography Laboratory, NCI-Frederick, Frederick, MD 21702-1201. Tel.: 301-846-5342; Fax: 301-846-7101; E-mail: millerm@ncifcrf.gov.
** To whom correspondence for all other issues should be addressed: Regulation of Cell Growth Laboratory, NCI-Frederick, Frederick, MD 21702-1201. Tel.: 301-846-1627; Fax: 301-846-5991; E-mail: johnsopf@ncifcrf.gov.
Published, JBC Papers in Press, February 10, 2003, DOI 10.1074/jbc.M300417200
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ABBREVIATIONS |
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The abbreviations used are: C/EBP, CCAAT/enhancer-binding protein; bZIP, basic region leucine zipper; CRE, cyclic AMP response element; CREB, cAMP-response element-binding protein; r.m.s.d., root mean square deviation; wt, wild type; EMSA, electrophoretic mobility shift assay; MES, 4-morpholineethanesulfonic acid; GCN, general control of amino acid biosynthesis; AP-1, activating protein 1; PAR, proline and acidic amino acid-rich; E2F, E2 promoter binding factor.
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REFERENCES |
---|
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---|
1. | Landschulz, W. H., Johnson, P. F., and McKnight, S. L. (1988) Science 240, 1759-1764[Medline] [Order article via Infotrieve] |
2. | Graves, B. J., Johnson, P. F., and McKnight, S. L. (1986) Cell 44, 565-576[Medline] [Order article via Infotrieve] |
3. | Johnson, P. F., Landschulz, W. H., Graves, B. J., and McKnight, S. L. (1987) Genes Dev. 1, 133-146[Abstract] |
4. | Landschulz, W. H., Johnson, P. F., Adashi, E. Y., Graves, B. J., and McKnight, S. L. (1988) Genes Dev. 2, 786-800[Abstract] |
5. | Lin, F. T., and Lane, M. D. (1992) Genes Dev. 6, 533-544[Abstract] |
6. | Freytag, S. O., Paielli, D. L., and Gilbert, J. D. (1994) Genes Dev. 8, 1654-1663[Abstract] |
7. | Freytag, S. O., and Geddes, T. J. (1992) Science 256, 379-382[Medline] [Order article via Infotrieve] |
8. |
Tenen, D. G.,
Hromas, R.,
Licht, J. D.,
and Zhang, D. E.
(1997)
Blood
90,
489-519 |
9. | Wang, N. D., Finegold, M. J., Bradley, A., Ou, C. N., Abdelsayed, S. V., Wilde, M. D., Taylor, L. R., Wilson, D. R., and Darlington, G. J. (1995) Science 269, 1108-1112[Medline] [Order article via Infotrieve] |
10. |
Zhang, D. E.,
Zhang, P.,
Wang, N. D.,
Hetherington, C. J.,
Darlington, G. J.,
and Tenen, D. G.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
569-574 |
11. | Pabst, T., Mueller, B. U., Zhang, P., Radomska, H. S., Narravula, S., Schnittger, S., Behre, G., Hiddemann, W., and Tenen, D. G. (2001) Nat. Genet. 27, 263-270[CrossRef][Medline] [Order article via Infotrieve] |
12. | Tenen, D. G. (2001) Leukemia 15, 688-689[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Vinson, C.,
Myakishev, M.,
Acharya, A.,
Mir, A. A.,
Moll, J. R.,
and Bonovich, M.
(2002)
Mol. Cell. Biol.
22,
6321-6335 |
14. | Vinson, C. R., Sigler, P. B., and McKnight, S. L. (1989) Science 246, 911-916[Medline] [Order article via Infotrieve] |
15. | Shuman, J. D., Vinson, C. R., and McKnight, S. L. (1990) Science 249, 771-774[Medline] [Order article via Infotrieve] |
16. | Patel, L., Abate, C., and Curran, T. (1990) Nature 347, 572-575[CrossRef][Medline] [Order article via Infotrieve] |
17. | O'Neil, K. T., Shuman, J. D., Ampe, C., and DeGrado, W. F. (1991) Biochemistry 30, 9030-9034[Medline] [Order article via Infotrieve] |
18. | Ellenberger, T. E., Brandl, C. J., Struhl, K., and Harrison, S. C. (1992) Cell 71, 1223-1237[Medline] [Order article via Infotrieve] |
19. | Konig, P., and Richmond, T. J. (1993) J. Mol. Biol. 233, 139-154[CrossRef][Medline] [Order article via Infotrieve] |
20. | Keller, W., Konig, P., and Richmond, T. J. (1995) J. Mol. Biol. 254, 657-667[CrossRef][Medline] [Order article via Infotrieve] |
21. | Glover, J. N., and Harrison, S. C. (1995) Nature 373, 257-261[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Schumacher, M. A.,
Goodman, R. H.,
and Brennan, R. G.
(2000)
J. Biol. Chem.
275,
35242-35247 |
23. | Fujii, Y., Shimizu, T., Toda, T., Yanagida, M., and Hakoshima, T. (2000) Nat. Struct. Biol. 7, 889-893[CrossRef][Medline] [Order article via Infotrieve] |
24. | Matthews, B. W. (1968) J. Mol. Biol. 33, 491-497[Medline] [Order article via Infotrieve] |
25. | Otwinowski, Z., and Minor, W. (1997) Methods Enzymol. 276, 307-326 |
26. | Navaza, J. (1994) Acta Crystallogr. Sect. A 50, 157-163[CrossRef] |
27. | Brünger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. Sect. D Biol. Crystallogr. 54, 905-921[CrossRef][Medline] [Order article via Infotrieve] |
28. | Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta Crystallogr. Sect. A 47, 110-119[CrossRef][Medline] [Order article via Infotrieve] |
29. | Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. Crystallogr. 26, 283-291[CrossRef] |
30. | Lu, X. J., Shakked, Z., and Olson, W. K. (2000) J. Mol. Biol. 300, 819-840[CrossRef][Medline] [Order article via Infotrieve] |
31. | Johnson, P., and Williams, S. C. (1994) in Liver Gene Expression (Yaniv, M. , and Tronche, F., eds) , pp. 231-258, R. G. Landes Company, Austin, TX |
32. |
Osada, S.,
Yamamoto, H.,
Nishihara, T.,
and Imagawa, M.
(1996)
J. Biol. Chem.
271,
3891-3896 |
33. | Johnson, P. F. (1993) Mol. Cell. Biol. 13, 6919-6930[Abstract] |
34. | Falvey, E., Marcacci, L., and Schibler, U. (1996) Biol. Chem. 377, 797-809[Medline] [Order article via Infotrieve] |
35. |
Slomiany, B. A.,
D'Arigo, K. L.,
Kelly, M. M.,
and Kurtz, D. T.
(2000)
Mol. Cell. Biol.
20,
5986-5997 |
36. | Porse, B. T., Pedersen, T. A., Xu, X., Lindberg, B., Wewer, U. M., Friis-Hansen, L., and Nerlov, C. (2001) Cell 107, 247-258[Medline] [Order article via Infotrieve] |
37. |
Johansen, L. M.,
Iwama, A.,
Lodie, T. A.,
Sasaki, K.,
Felsher, D. W.,
Golub, T. R.,
and Tenen, D. G.
(2001)
Mol. Cell. Biol.
21,
3789-3806 |
38. | Suckow, M., von Wilcken-Bergmann, B., and Muller-Hill, B. (1993) EMBO J. 12, 1193-1200[Abstract] |
39. | Tahirov, T. H., Inoue-Bungo, T., Morii, H., Fujikawa, A., Sasaki, M., Kimura, K., Shiina, M., Sato, K., Kumasaka, T., Yamamoto, M., Ishii, S., and Ogata, K. (2001) Cell 104, 755-767[Medline] [Order article via Infotrieve] |
40. | Tahirov, T. H., Sato, K., Ichikawa-Iwata, E., Sasaki, M., Inoue-Bungo, T., Shiina, M., Kimura, K., Takata, S., Fujikawa, A., Morii, H., Kumasaka, T., Yamamoto, M., Ishii, S., and Ogata, K. (2002) Cell 108, 57-70[Medline] [Order article via Infotrieve] |