From the Department of Biochemistry, Vanderbilt
University School of Medicine, and the § Department of
Molecular Biology, Vanderbilt University,
Nashville, Tennessee 37232-0146
Received for publication, June 26, 2000, and in revised form, October 3, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The crystal structure of the heterodimer formed
by the basic leucine zipper (bZIP) domains of activating transcription
factor-4 (ATF4) and CCAAT box/enhancer-binding protein Protein-protein interactions are involved in a number of different
cellular processes, including the regulation of transcription. Many, if
not most, transcription factors, including the
bZIP1 proteins, bind their
cognate DNA elements as dimers. Dimerization between transcription
factors from the same or different families can generate considerable
functional diversity using only a relatively small number of
components. By binding with different specificity to distinct DNA sites
within promoters, heterodimeric transcription factors facilitate the
positioning and orientation of proteins on DNA, thus providing distinct
surfaces for interaction with other transcriptional regulatory proteins
bound to adjacent DNA sites.
Among coiled-coil proteins, bZIP family members form dimers via their
characteristic leucine zipper helices, which consist of seven-residue
(heptad) repeats, (abcdefg)n. Dimer formation by
bZIP proteins is selective: some form only homodimers, some form only
heterodimers, and others form both homo- and heterodimers. Hydrophobic
interactions by leucine and other hydrophobic amino acids in positions
3 and 4 in the helix (the a and d positions of
the helical wheel) form the hydrophobic core of the bZIP dimer. Leucine
residues occupy most of the d positions in the leucine zipper helix. At the same time, hydrophilic amino acids located just
outside of the hydrophobic core (the e and g
positions of the helical wheel) also participate in stabilizing the
dimer. A highly conserved asparagine amino acid in the a
position in the middle of the leucine zipper helix has been shown to
destabilize oligomer formation in several bZIP dimers (1). Thus,
depending on the precise distribution of hydrophilic and hydrophobic
residues in the bZIP helix, only selective pairs of bZIP transcription factors are able to form stable dimers, which in turn lead to distinct
DNA binding propensity.
The basic DNA-binding region of the bZIP domain adopts a stable
structure upon association with DNA. Binding to the cognate DNA induces
a coil-to-helix transition of the basic DNA-binding region (2-4). NMR
and CD studies of GCN4 and C/EBP bZIP domains have shown that in the
absence of the DNA target, the basic region has some residual helical
character and has been described as an ensemble of transiently formed
helical structures (4-6). The folding transition involved in the DNA
binding of the GCN4 basic region results in an unfavorable contribution
to the overall free energy from the loss of conformational entropy
(6).
The bZIP transcription factor family, one of many dimeric protein
families, participates in a number of different transcriptional activities. The DNA-binding motif, consisting of basic and leucine zipper regions, is one of the simplest DNA-binding domains. However, the bZIP transcription factors are capable of recognizing a broad range
of DNA sequences, yet at the same time, discriminate sufficiently to
regulate the transcription of a diverse range of genes in different promoters. Based on DNA-binding activity, the bZIP transcription factors in mammals are divided into three major families: (i) activator
protein-1 (AP-1), (ii) cAMP response element-binding protein/activating
transcription factor (CREB/ATF), and (iii) C/EBP. AP-1 family members
have been the most heavily studied. In particular, structural analyses
of bZIP domains of AP-1 proteins such as Fos/Jun and GCN4 in the
absence and presence of the AP-1 DNA target (5'-TGAGTCA-3') and cAMP
response element (CRE; 5'-TGACGTCA-3') have led to a general
understanding of bZIP DNA binding.
Among bZIP transcription factors, the CREB/ATF family
appears to have a higher selectivity for DNA target sites. As
homodimers, CREB/ATF proteins recognize CRE, a palindrome of two
inverted identical half-sites (TGAC·GTCA), which differs from the
AP-1 target site by 1 base pair in the center. The AP-1 target is also a palindrome consisting of two inverted identical half-sites with 1 overlapping base pair in the middle (TGACTCA) (7, 8). CREB/ATF proteins in heterodimers formed between CREB/ATF proteins and
AP-1 (9-13) or C/EBP proteins (14-16) retain their affinity to bind
to the CRE half-site; at the same time, they lead their partner to bind
to CRE or CRE-related half-sites with high affinity. Members of the
C/EBP family bind to a relatively broad range of DNA sequences that
satisfy the (A/G)TTGCG(C/T)AA(C/T) consensus CCAAT box (17).
Although a number of three-dimensional structures of bZIP domains in
the presence of DNA targets have been studied (18-20), the
three-dimensional structure of a bZIP homo- or heterodimer containing
the basic regions has not been studied in the absence of a DNA target.
To obtain a better understanding of dimerization properties and the
conformational changes to the dimer upon DNA binding, we determined the
crystal structure of the bZIP heterodimer formed between ATF4 and
C/EBP Protein Purification--
The NdeI-BamHI
fragments containing the coding sequences for residues 280-341 of
human ATF4 (8) and residues 224-285 of mouse C/EBP
The ATF4 bZIP domain was purified from the soluble fraction. The cells
were frozen and thawed in 20 mM Tris (pH 8.0), 0.1 M NaCl, 1 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 5 mM Oligonucleotides and Oligopeptides--
Oligonucleotides were
synthesized at the Vanderbilt Core Facility. Oligopeptides were
synthesized by Alpha Diagnostic International Inc. (San Antonio, TX).
Circular Dichroism Spectroscopy--
CD spectra were recorded at
20 °C using a Jasco-720 spectropolarimeter. All spectra were run
using a 1-mm path length quartz cuvette. Protein samples contained 10 µM protein, 10 mM Tris (pH 7.5), 100 mM NaCl, 5 mM MgCl2, and 0-50
µM DNA. The synthetic peptide samples contained 50 µM peptide, 10 mM Tris (pH 7.5), 100 mM NaCl, 5 mM MgCl2, and 0-80%
2,2,2-trifluoroethanol (TFE). The CD spectra were averaged from three
wavelength scans, blanked, smoothed, and analyzed using the
manufacturer's software. The concentrations of the proteins and
synthetic peptides used for experiments were determined by amino acid analysis.
NMR Spectroscopy--
Lyophilized proteins were dissolved in 0.5 ml of 90:10 (v/v) H2O/D2O at 1.5 mM. The pH was adjusted to 6.5 without correction for
deuterium isotope effect. A series of homonuclear two-dimensional NMR
experiments were recorded at 15, 25, 35, 45, and 55 °C. Experiments were done on a DRX600 (14.2 T) instrument (Bruker) operating at 600.13 MHz. Sweep widths were adjusted to 7183 Hz. The size of acquired
free induction decay was 2048 complex points. The carrier frequency was
set on the H2O. Thirty-two transients were acquired for
each of 1024 t1 values. In NOESY experiments
(22), time proportional phase incrementation was used (23). Total
correlation spectroscopy spectra (24) were recorded with a DIPSI-II
mixing pulse sequence (50 ms) (25) using the States-TPPI method
(26). Pulsed field gradient water suppression was used in all
experiments (27).
DNA Binding Assay--
The DNA-binding activities of the bZIP
domain of C/EBP Crystallization, Data Collection, and Processing--
Equimolar
quantities of purified, concentrated bZIP domains of C/EBP Structure Determination, Refinement, and Model Quality--
The
molecular replacement solution was found using X-PLOR (29). The
polyalanine model of the Fos·Jun complex in the crystal structure of
the Fos·Jun·AP-1 site DNA complex (Protein Data Bank code 1FOS) was
used as a search model. The electron density map phased by the
molecular replacement model allowed us to build the Structure of the C/EBP
The helical conformation of the bZIP domain is maintained in part by
several intrahelical interactions (Fig. 1, B and
C). In particular, intrahelical interactions between
ATF4 Tyr-295 and Lys-299 as well as between ATF4 Gln-297 and Arg-300
contribute to the stability of the
Ala-291, Ala-292, Arg-294, and Tyr-295 in the basic region of ATF4
participate in crystal lattice interactions by making a backbone-backbone contact with the same residues in the symmetrically related ATF4 molecule (ATF4*) (Fig. 2A). The successive
alanine residues in these regions of the helices allow the main chain atoms to approach at the distance of 3.0-3.5 Å to make strong van der
Waals contacts, resulting in two ATF4 helices packing at an angle of
~80°. These lattice contacts may contribute to the stability of the
Dimerization of C/EBP
When the bZIP domain of ATF4 was titrated with the CRE-containing DNA,
two negative peaks at 208 and 222 nm appeared in the CD spectrum, which
are characteristic of an
Proteolytic digestion of the bZIP domains confirmed our CD observation
(Fig. 4). Proteolysis by endoproteinases, V8 (Glu-C) and trypsin
showed that the basic region of the
C/EBP
NMR experiments performed for ATF4 and C/EBP
The bZIP domains of ATF4 and C/EBP DNA-binding Activity--
The functional activity of the
recombinant bZIP domains of ATF4 and C/EBP Redox and C/EBP Formation of a Stable bZIP Dimer--
This study on the bZIP
domains of ATF4 and C/EBP
With the ordered Cross-family bZIP Heterodimer--
Most transcription factors are
dimeric. A dimeric DNA-binding domain provides significant advantages
over its monomeric counterparts for accurate regulation of
transcription. A heterodimer provides a unique advantage: a small
number of components can be used to generate new transcription factors
that bind to distinct DNA sites with different specificity and that
interact with different proteins. They can regulate transcription of
different sets of genes by binding to different sites in different
promoters. Estes et al. (42) showed that ATF4 and C/EBP (C/EBP
),
from two different bZIP transcription factor families, has been
determined and refined to 2.6 Å. The structure shows that the
heterodimer forms an asymmetric coiled-coil. Even in the absence
of DNA, the basic region of ATF4 forms a continuous
-helix, but the
basic region of C/EBP
is disordered. Proteolysis, electrophoretic
mobility shift assay, circular dichroism, and NMR analyses indicated
that (i) the bZIP domain of ATF4 is a disordered monomer and
forms a homodimer upon binding to the DNA target; (ii) the bZIP domain of ATF4 forms a heterodimer with the bZIP domain of C/EBP
that binds
the cAMP response element, but not CCAAT box DNA, with high affinity; and (iii) the basic region of ATF4 has a higher
-helical propensity than that of C/EBP
. These results suggest that the degree
of ordering of the basic region and the fork and the dimerization properties of the leucine zipper combine to distinguish the
structurally similar bZIP domains of ATF4 and C/EBP
with respect to
DNA target sequence. This study provides insight into the mechanism by
which dimeric bZIP transcription factors discriminate between closely related but distinct DNA targets.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. Furthermore, we used circular dichroism and NMR to show that
the bZIP domain of ATF4 does not form homodimers, but does form
heterodimers with C/EBP
, a member of the C/EBP family. The
heterodimer does not bind to the CCAAT box, but binds to CRE with
high affinity. Unexpectedly, the crystal structure of the
C/EBP
·ATF4 heterodimer revealed that the basic region of
the bZIP domain of the ATF4 subunit is an
-helix, whereas the basic
region of C/EBP
is disordered. These results suggest that a degree
of ordering of the DNA-binding region and the dimerization properties
of the bZIP domains are important in DNA recognition by bZIP
transcription factors.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(21) were
generated by polymerase chain reaction from the full-length cDNAs
and cloned into a pET11a plasmid. Both proteins were separately
overexpressed in Escherichia coli. The bZIP domain of
C/EBP
was purified from the insoluble fraction. The inclusion bodies
were solubilized in Buffer A (20 mM Tris (pH 7.5), 1 mM EDTA, and 20 mM
-mercaptoethanol)
containing 6 M urea and applied to an SP-Sepharose column
(Amersham Pharmacia Biotech). The column was washed with Buffer A
containing 6 M urea (10 column volumes) and then with
Buffer A (10 column volumes). Renatured C/EBP
bZIP domain was eluted
with a gradient of 0-1 M NaCl in Buffer A. Protein was
precipitated on ice and pelleted by centrifugation. The resulting
pellet was dissolved in water and used for crystallization trials.
-mercaptoethanol, and 10 mM Na2S2O3. The
soluble fraction was incubated for 20 min at 70 °C; the precipitate
was removed by centrifugation, whereas the supernatant was applied to a
Q-Sepharose column (Amersham Pharmacia Biotech). The protein was eluted
with a linear gradient of 20-800 mM NaCl in Buffer A. The
fractions containing the ATF4 bZIP domain were diluted 5-fold and
applied to an SP-Sepharose column. The protein was eluted with a linear
gradient of 20 mM to 1 M NaCl in Buffer A. The
protein peak was pooled and concentrated further in a Centricon-10
concentrator (Amicon, Inc.). For CD and NMR spectroscopy
studies, Cys-310 of ATF4 was substituted with Ser to prevent the
cysteine oxidation and formation of high molecular mass aggregates.
and the bZIP domain of ATF4 were examined by
electrophoresis mobility shift assay using oligonucleotides containing
CRE or the CCAAT box. bZIP proteins in a micromolar concentration range
were mixed with [32P]DNA (5 nM final
concentration) in 10 µl of binding buffer (40 mM Tris (pH
7.5), 5 mM MgCl2, 100 mM NaCl,
0.01% Nonidet P-40, 1 mM dithiothreitol, and 20 mg/ml
poly(dA-dT)/poly(dG-dC) (1:1, w/w)). The reaction mixtures were
incubated for 30 min on ice and analyzed by native electrophoresis on
20% polyacrylamide gel (Phast system, Amersham Pharmacia Biotech). The
positions of radiolabeled DNA on the gel were determined by autoradiography.
and ATF4
were mixed to form the heterodimer. Crystals were obtained by vapor
diffusion at 15 °C from a solution containing 0.3 mM
C/EBP
·ATF4 heterodimer, 1 M ammonium sulfate, 50 mM sodium cacodylate, and 5% dioxane. Hanging drops were
equilibrated over well solutions containing twice the concentration of
salts and dioxane. Plate-like crystals grew to final dimensions of
1.0 × 0.3 × 0.1 mm over the course of 3 weeks. The crystals
belong to the orthorhombic P2221 space group with unit cell
dimensions of a = 72.628, b = 81.152, and c = 35.317 Å, with one heterodimer in one
asymmetric unit. Using 30% glycerol as a cryoprotectant, a data set
was collected at
172 °C from one crystal to a resolution of 2.6 Å on an R-AXIS II mounted on a Rigaku RU-200 x-ray generator. The data
set was indexed, processed, and scaled using the programs DENZO and
SCALEPACK from the HKL package (28) (Table
I).
Data collection, refinement, and model quality statistics
-helical
backbone of the protein: residues 289-339 for ATF4 and residues
239-284 for C/EBP
. Cycles of rebuilding using the program O (30)
alternating with rigid body refinement, simulated annealing, and
positional refinement against 12 to 3.0-Å data with X-PLOR allowed
most of the side chains to be modeled and reduced the free R
factor to 36.2% and the crystallographic R factor to
26.5%. The model that included residues 286-341 for ATF4 and residues
239-284 for C/EBP
was further refined by torsion angle molecular
dynamics alternating with conjugate gradient minimization refinement
and individual B factor refinement against 8 to 2.6 Å data
using the program CNS (31). The final model contains residues 286-341
for ATF4 and residues 239-285 for C/EBP
. Since the electron density
for side chains of ATF4 Glu-286, Gln-287, and Lys-289, and C/EBP
Lys-242 was not apparent in a 1.5
2Fo
Fc map, these side chains were excluded from the
model. The
-mercaptoethanol moiety, three Fe3+ ions, and
86 water molecules were included at the final steps of the refinement.
Although no iron-containing compounds were used for crystallization,
they were included in refinement as possible contaminants of ammonium
sulfate and dioxane. The final refinement converged to an
R factor of 21.7% and an
Rfree of 27.3% and good stereochemistry judged
by the program PROCHECK (32) (Table I).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
·ATF4 Complex--
The
three-dimensional structure of the C/EBP
·ATF4 bZIP heterodimer
solved at 2.6 Å resolution is similar to those of other bZIP dimers
determined in the complex with DNA targets. The heterodimeric coiled-coil is formed by the more curved C/EBP
helix wrapping around
the approximately straight ATF4 helix (Fig.
1A). The most prominent
structural feature of the C/EBP
·ATF4 heterodimer is that most of
the basic region of the ATF4 bZIP domain is a continuous
-helix. The
-helix starts at Asn-288 of ATF4 in the N terminus, which is
conserved among bZIP domains. In the three bZIP domain·DNA complex
structures determined (18, 20, 33) this asparagine residue has
been shown to contact DNA. In contrast, the entire basic region of
C/EBP
is disordered.
View larger version (43K):
[in a new window]
Fig. 1.
Structure and interface of the
C/EBP ·ATF4 heterodimer.
A, the curved C/EBP
helix wraps around the
straight ATF4 helix. The structure of the heterodimer is shown in two
views, related by 90° rotation about the axis perpendicular to the
left-hand superhelical axis in the plane of drawing. The locations of
the basic region, fork, and leucine zipper region are indicated.
B, sequence alignment of the C/EBP
and ATF4 bZIP domains.
The residue numbers are as in the full-length proteins. The residues
making direct contacts with DNA bases are shown in
boldface. The pairwise intra- and interhelical
interactions are summarized. The residues that make interactions are
connected by solid lines. The distances between interacting
intrahelical atoms are indicated above each line. The distances for
residues making electrostatic interactions are
boxed. C, a helical wheel diagram of the
C/EBP
·ATF4 coiled-coil. Amino acids in the
-helices are
indicated in single-letter code. The coiled-coil sequence is read from
N to C termini outwards from the wheel. Solid arrows
indicate electrostatic interactions between the e and
g positions. Dashed arrows indicate hydrogen bond
interactions. The distances between interacting interhelical atoms are
indicated above the arrows. D, potential
interactions on homodimer interfaces of C/EBP
and ATF4.
Electrostatic attractive interactions are indicated by solid
arrows; electrostatic repulsive interactions are indicated by
solid lines; and hydrogen bond interactions are indicated by
dashed arrows.
-helical conformation in the fork
and the basic region of ATF4. Both pairs of residues are bridged by well ordered water molecules (Figs. 1B and
2A). Interestingly, the
Gln-297 and Arg-300 pair in ATF4 have been shown to be important for
the discrimination between CRE and AP-1 sites by AP-1 and CREB/ATF
proteins. The GCN4 point mutant containing only these two substitutions
from ATF1 has been shown to have a preference for CREB/ATF sites over
the AP-1 site (35). The second pair of interaction, Tyr-295 and
Lys-299, appears to be unique to ATF4 because position 295 in other
bZIP domains is usually occupied by serine, cysteine, or phenylalanine.
ATF4 Arg-296 is conserved in the basic region and the crystal
structures of GCN4 bound to either an AP-1 site or a CRE DNA target
show that ATF4 Arg-296 interacts differently with the central base
pair(s) of the DNA target (18, 19). In our structure of the
non-complexed bZIP dimer, ATF4 Arg-296 is sandwiched between Tyr-295
and Lys-299 in the same turn of the
-helix and is unable to adopt
many alternative conformations. These four residues are involved in
important intrahelical interactions that may increase a degree of
ordering of the basic region and the fork of ATF4.
View larger version (40K):
[in a new window]
Fig. 2.
bZIP -helices.
A, electron density of the ATF4 basic region from the
2Fo
Fc map contoured at
1.0
. Intrahelical hydrogen bonds in the ATF4 fork-basic region
junction formed between ATF4 Tyr295 and Lys299
as well as between ATF4 Gln297 and Arg300
contribute to the stability of the
-helical conformation in the fork
and the basic region. Dashed lines indicate the distances
between water molecules and respective atoms of the side chains. The
symmetry-related ATF4* molecule is shown in red.
B, comparison of
-helices. The ideal (gray)
-helix was generated using insightII. Each bZIP domain was
superimposed on the ideal
-helix. The ATF4 bZIP domain is
indicated in red, the C/EBP
bZIP domain in the
heterodimer in green, and the C/EBP
bZIP domain in the
C/EBP
·DNA complex in yellow-green. C, the
potential disulfide bond in the ATF4 bZIP homodimer. The second
subunit of the ATF4 bZIP dimer was generated by superimposing the
bZIP domain of ATF4 in the heterodimer on the bZIP domain of C/EBP
in the same heterodimer structure.
-helical conformation of the basic region of ATF4 in the crystal
structure. The crystallization of the heterodimer in this particular
conformation reflects that the
-helical propensity of the ATF4 basic
region is higher than that of C/EBP
. Evidence that the basic region
of ATF4 may have some
-helical propensity also comes from circular
dichroism studies performed on two synthetic 26-residue peptides
comprising both the basic region and the fork of ATF4 or C/EBP
. In
aqueous solution, both peptides lack helicity. The
-helical content
increased upon addition of TFE (Fig.
3C), which is known to
stabilize the helical conformation of a peptide and is used as a
quantitative probe for helical tendency in polypeptides (36). In both
peptides, the mean residue ellipticity ([
]222) exhibited sigmoidal dependence on TFE concentration and showed a sharp
transition as TFE concentration was increased above 10%. For the ATF4
peptide, [
]222 was higher than for the
C/EBP
-derived peptide at TFE concentrations above 20% and
approached
31,800 degrees cm2
dmol
1, which corresponds to ~89% helicity
assuming a value of
35,800 degrees cm2
dmol
1 for 100% helicity of a 26-residue
peptide at 20 °C (37). For the C/EBP
peptide,
[
]222 approached
24,700 degrees cm2
dmol
1, which corresponds to 69% helicity and
is 20% below the value shown by the ATF4 peptide. Under these
conditions, the basic region of ATF4 has a significantly greater
helical propensity than the same region of C/EBP
and lends support
to the notion that the
-helical basic region of ATF4 seen in our
structure is not just reflection of crystal packing forces.
View larger version (32K):
[in a new window]
Fig. 3.
CD spectra of C/EBP
and ATF4. A and B, the CD
spectra of the bZIP domains of C/EBP
and ATF4, respectively, at
different DNA target concentrations. A, the CD spectrum of
the bZIP domain of C/EBP
without DNA (
) and the CD spectrum of
the 1:1 complex of the bZIP domain of C/EBP
and the CCAAT box (
).
The lines between with no symbols represent the CD spectra
of the bZIP domain of C/EBP
with various concentrations (0-10
µM) of the CCAAT box. DNA concentrations are indicated.
The concentration of the bZIP domain of C/EBP
was 10 µM. B, the CD spectra of the bZIP
domain of ATF4 without DNA (
)) and the CD spectra of the 1:1 complex
of the bZIP domain of ATF4 and CRE (
). The lines between
with no symbols represent the CD spectra of the bZIP domain of ATF4
with various concentrations (0-10 µM) of CRE. DNA
concentrations are indicated. The concentration of the bZIP domain of
ATF4 was 10 µM. C, the CD spectra of
the bZIP domains of ATF4, C/EBP
, and the C/EBP
·ATF4 bZIP
heterodimer. The contribution of the bZIP domain of ATF4 to the
spectrum of the heterodimer was estimated as the difference between the
spectra of the heterodimer and the bZIP domain of C/EBP
alone
(thin line with no symbols). D,
-helix
induction in C/EBP
and ATF4 peptides constituting both the basic
region and the fork of C/EBP
and ATF4, respectively, upon increasing
concentrations of TFE. deg, degrees.
and ATF4--
As expected, the CD spectra
of C/EBP
showed two negative peak at 208 and 222 nm, indicating the
presence of
-helix in the leucine zipper region, and a negative peak
near 200 nm, indicating a random conformation (38) in the basic region
(Fig. 3A). The CD spectra of the bZIP domain of ATF4,
however, revealed a negative peak near 200 nm and no strong negative
peak at 208 and 222 nm. This indicates not only that the basic region
is a random coil, but also that the leucine zipper region is also
disordered, which suggests that the bZIP domain of ATF4 may not form a
coiled-coil or a typical stable bZIP homodimer in the absence of DNA.
-helical structure. The
-helical content
is estimated by [
]222. The end point of the titration
does not exceed
18,200 degrees cm2
dmol
1, which corresponds to 48% helicity and
suggests that the leucine zipper region of ATF4 may not necessarily
form a stable coiled-coil even in the presence of the DNA target. In
contrast, the CD spectra for the bZIP domain of C/EBP
showed the
helical content of ~50%, which increased to 81% upon binding to
CCAAT box DNA (Fig. 3A). When equal amounts of the bZIP
domains of C/EBP
and ATF4 were mixed together, the CD spectrum
showed two strong negative bands at 206 and 222 nm (Fig.
3B), leading to an estimation of the helical content of
~85%. This exceeds not only that of the bZIP domain of ATF4 in the
presence of specific DNA (CRE), but also that of the bZIP domain of
C/EBP
without DNA. In fact, it is comparable to that of the bZIP
domain of C/EBP
in the presence of the DNA target (CCAAT box). This
indicates that the formation of the heterodimer between the bZIP
domains of ATF and C/EBP
induces an
-helix in the bZIP
domain of ATF4 even in the absence of specific DNA.
bZIP domain is digested in a short period time (<10 min under
the condition we used), whereas the leucine zipper region remained
uncut for a longer period of time. However, the basic region of the
ATF4 bZIP domain is more stable than that of C/EBP
, with only about one-third of the basic region from the N terminus digested in the same
period time. Surprisingly, there was an additional cleavage at the
middle of the leucine zipper region of the ATF4 bZIP domain, suggesting
that the leucine zipper of ATF4 may not be able to maintain an
-helical conformation in the absence of specific DNA.
View larger version (57K):
[in a new window]
Fig. 4.
Proteolysis of C/EBP
and ATF4. A, protease V8 (
) and trypsin (
)
cleavage sites in C/EBP
and ATF4. The sites of cleavage, determined
by N-terminal sequencing of proteolytic fragments, are marked on the
schematic representations of the structural elements of the bZIP
domains.
-Helix regions in the C/EBP
·ATF4 structure are shown
by empty boxes, and disordered regions are indicated by
solid lines. B, protease V8 (lanes
1-6) and trypsin (lanes 7-11) cleavage patterns of
C/EBP
and ATF4. Numbers above each lane refer to the time
the reaction was allowed to proceed. Arrows a-d indicate
the N-terminal amino acids of the cleaved fragments determined by
N-terminal sequencing. GluC 230, C/EBP
Glu230; GluA 286, ATF4
Glu286. Intact indicates that there is no cut in
the N terminus.
at protein
concentration of 1.5 mM at different temperatures are in
good agreement with the results of the CD experiments. Fragments of
NOESY spectra showing HN-HN cross-peaks
indicative of the potential
-helical conformation for both proteins
are shown in Fig. 5. C/EBP
shows a
significantly larger variation of chemical shifts. The dispersion
observed for ATF4 is rather typical for a marginally structured
polypeptide. At 35 °C, very few non-intra-residue cross-peaks were
observed for ATF4. In contrast, the C/EBP
spectra showed a number of
broad cross-peaks in the HN-HN area at 15 °C
that narrowed significantly on increasing the temperature to 35 °C.
A number of other non-intra-residue nuclear Overhauser effect
cross-peaks were found for C/EBP
, although many of them were broad.
This behavior is consistent with a 15.2-kDa dimer with an aspect ratio
of ~4:1. As the temperature was further increased, the intensity of
these cross-peaks diminished, albeit at a much slower rate than in the
case of ATF4, most likely due to the increased rate of exchange with
solvent. The abundance of non-intra-residue HN-H
cross-peaks is characteristic of an
-helical conformation (data not shown) (39). The comparison of total
correlation spectroscopy and NOESY spectra at 35 °C (data not shown)
revealed no non-intra-residue HN-H
cross-peaks for ATF4. The same comparison for C/EBP
showed ~30 cross-peaks.
View larger version (48K):
[in a new window]
Fig. 5.
NOESY spectra showing
HN-HN cross-peaks for C/EBP
and ATF4. The NOESY spectra of HN-HN
cross-peaks at different temperatures indicate
-helical conformation
of the bZIP domain structures. The temperatures used are indicated in
each panel.
are compared with an ideal
-helix in Fig. 2B. The bZIP domain of ATF4 is nearly a
perfect (root mean square for C-
of 0.8 Å)
-helix, whereas the
bZIP domains of C/EBP
in the C/EBP
·ATF4 heterodimer and in the
C/EBP
complexed with the CCAAT box are significantly curved.
Interestingly, the C/EBP
in the heterodimer is more curved than that
in the C/EBP
·CCAAT box complex. This suggests that the bZIP domain
of C/EBP
is flexible and that, depending on the partner molecule, it
may adapt readily to form a stable coiled-coil. The flexibility of the
bZIP domain of ATF4 awaits the structural analysis of the ATF4 in the
homodimer complexed with the DNA target (CRE). The fact that ATF4 does
not form a stable homodimer (it is monomeric in solution) and that,
even in the presence of the DNA target, it forms a relatively weak
homodimer suggests that ATF4 may maintain its straight
-helical
conformation, i.e. it fails to adapt the intertwining
conformation necessary for the establishment of a stable coiled-coil.
Our recent CD data (data not shown) indicate that when ATF4 (bZIP
domain) is oxidized, it has a higher
-helical content in the
presence of CRE than the unoxidized form or the Cys-310
Ser mutant.
The covalent disulfide bond may hold two bZIP domains of ATF4 together,
maintaining the more stable bZIP dimer, which subsequently binds to the
DNA target with higher affinity.
was also examined by an
electrophoretic gel shift assay using CRE- or CCAAT box-containing DNA
probes (Fig. 6). Both proteins bound
their specific DNA elements (ATF4 to CRE and C/EBP
to the CCAAT box)
with high affinity, resulting in formation of protein·DNA complexes
with distinct mobility on native polyacrylamide gel. As expected, the
ATF4 bZIP domain did not bind to CCAAT box DNA (compare lanes
3 and 9), whereas the C/EBP
bZIP domain bound to
both CRE and CCAAT box probes with comparable affinity (compare lanes 2 and 8), indicating a broad range of
sequence specificity. The addition of higher concentrations of the ATF4
bZIP domain did not affect the binding of C/EBP
to the CCAAT box
(lanes 4-6). Only a small amount of the CCAAT box-bound
C/EBP
·ATF4 heterodimer appeared in the gel as a minor band with
higher mobility. In contrast, the addition of even a small amount of
the ATF4 bZIP domain to the C/EBP
bZIP domain resulted in the
preferential formation of the heterodimeric C/EBP
·ATF4·CRE
complex (lanes 10-12). These results clearly indicate that
the ATF4 bZIP domain has a preference for binding to CRE, whereas the
C/EBP
bZIP domain accommodates both binding sites with comparable
affinity. While the CCAAT box DNA failed to attract the ATF4 bZIP
domain even in the presence of the C/EBP
bZIP domain, CRE was able
to accommodate both bZIP domains (lanes 4-6). Thus, the
heterodimer formed between C/EBP
and ATF4 displays the same
DNA-binding preferences as ATF4 does. Apparently, the heterodimer forms
a more stable complex on CRE than the ATF4 homodimer does.
View larger version (40K):
[in a new window]
Fig. 6.
DNA-binding activity of
C/EBP and ATF4. The DNA probes used are
duplexes of the CCAAT box (5'-GCAGATTGCGCAATCTGC-3') and
CRE (5'-GCAGATGACGTCATCTGC-3'). The concentration of each
DNA probe was 5 nM. The concentrations of the proteins
indicated above each lane are multiples of
10
6 M. Arrows a-d
indicate the mobilities of free DNA and protein·DNA complexes.
·ATF4--
It has been shown that some bZIP
transcription factors are regulated by redox tension (40, 41). Most of
the bZIP domains contain a cysteine amino acid in the basic region,
which functionally interferes with DNA-binding activity when it is
oxidized. The bZIP domain of ATF4 does not contain the cysteine residue
in the basic region; instead, it contains one cysteine amino acid in the leucine zipper region, which is not conserved among other bZIP
domains. The cysteine residue in the ATF4 bZIP domain is in the
a position (in the helical wheel), which is in position to
be a part of the hydrophobic core of the bZIP dimer. The crystal structure of the C/EBP
·ATF4 bZIP heterodimer indicates that this cysteine residue in the ATF4 bZIP domain is positioned to make a
disulfide bond with the same cysteine residue from the other subunit of
the ATF4 bZIP domain when ATF4 forms a homodimer, presumably in the
presence of the DNA target (see Fig. 2C). This
cysteine residue, when oxidized, may stabilize the ATF4 homodimer by
forming the covalent disulfide bond. Our study indicated that ATF4 is monomeric in the absence of the DNA target, but in the presence of the
DNA target, it forms a relatively less stable homodimer on DNA. On the
other hand, ATF4 forms a more stable heterodimer with C/EBP
even in
the absence of the DNA target. The heterodimer binds to CRE with high
affinity, but not to the C/EBP site. Depending on the oxidation state
(due to the cellular oxygen level), ATF4 forms either a stable
homodimer via the intersubunit disulfide bond or a more stable
heterodimer, such as C/EBP
·ATF4. As a heterodimer, it binds CRE or
a CRE-related DNA element and regulates transcription of distinct
genes. It has been reported (42) that both ATF4 and C/EBP
are
induced to a higher level in anoxic tension. Although there is no
direct evidence, perhaps in a low oxygen state, ATF4 may form a stable
heterodimer with C/EBP
, rather than a less stable ATF4 homodimer
without the disulfide bond, and may activate the transcription of genes
involved in the recovery process from anoxic stress.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and the heterodimer formed by these
proteins addresses the formation a stable cross-family bZIP heterodimer
and the interaction with the DNA target. It has been generally
understood that the hydrophobic core of the bZIP dimer is formed by
hydrophobic amino acids occurring at every 4-3 position of the
helix (or the a and d positions in the helical
wheel) with a leucine in every seventh position. In fact, these
residues are conserved throughout bZIP proteins. Nonetheless, some bZIP
dimers are more stable than others, and this difference in stability
depends on the distribution of polar amino acid side chains that are
partially exposed (particularly in the g and e positions). These amino acids potentially present attraction or repulsion interactions within the same
-helix and between two parallel
-helices. A number of structural studies including ours have confirmed this notion. As indicated above, ATF4 does not form a
stable homodimer, but C/EBP
does. From the potential
attraction-repulsion interaction scheme based on the distribution of
polar side chains on the surface of the helices of the C/EBP
homodimer and the C/EBP
·ATF4 heterodimer, a slight preference for
heterodimer may be inferred. The exclusive formation of the heterodimer
from the 1:1 mixture of the two proteins was not expected. In the
crystal structure of the heterodimer, there is a clear asymmetry of the
-helices, the nearly straight ATF4
-helix and the more curved C/EBP
-helix, which wraps around the former. To form a stable coiled-coil, two parallel or antiparallel helices need to be curved enough to intertwine in order to maintain the optimal orientation of
main chain atoms necessary for side chains to participate in the
maximum number of attractive interhelical interactions. Although it
remains to be seen, ATF4 may maintain its straight
-helical conformation, preventing it from intertwining to form the stable homodimer. However, C/EBP
appears to be more flexible and allows necessary intertwining for the formation of a stable homodimer. In the
heterodimer, the more flexible C/EBP
compensates for the lack of
flexibility in ATF4 by wrapping around it to maintain the maximum
attractive interaction between the two helices.
-Helical Basic Region of ATF4--
The most prominent feature
of the crystal structure of the C/EBP
·ATF4 bZIP heterodimer is the
-helix in the basic region of the ATF4 bZIP domain, which orients
most of the conserved residues in this region in position for specific
DNA (CRE) binding; in contrast, the basic region of the C/EBP
bZIP
domain is disordered. It appears that the
-helix in the basic region
of the ATF4 bZIP domain is stabilized in part by the crystal lattice
interaction between the two symmetrically related basic regions of the
ATF4 bZIP domains. Although, other than the crystal structure analyzed in this study, there is no direct evidence indicating the
-helix in
the basic region of the ATF4 bZIP domain, there are critical data
complementing the crystal structure result. The proteolysis by trypsin
and Glu-C suggested that the basic region of the ATF4 bZIP domain is
more stable than that of the C/EBP
bZIP domain (Fig. 4). The CD
spectra of the mixture of the ATF4 bZIP domain and C/EBP
also
support that the
-helix in the basic region of the ATF4 bZIP domain
shown in the crystal structure of the C/EBP
·ATF4 heterodimer may
not be a mere artifact resulting from crystallization. The CD spectra
indicate that, in solution, the
-helix content of the bZIP
heterodimer consisting of the ATF4 and C/EBP
bZIP domains in the
absence of specific DNA (CRE) (Fig. 3B) is comparable to, if
not higher than, that of the C/EBP
bZIP domain in the presence of
specific DNA (CCAAT box) (Fig. 3A). In addition, the CD
spectra (Fig. 3C) obtained from the 26-amino acid peptides comprising both the basic region and the fork of ATF4 (ATF4-bf) or
C/EBP
(C/EBP
-bf) in the presence of the helix-stabilizing agent
TFE suggest a higher
-helix propensity for ATF4-bf than for
C/EBP
-bf. Although it contains a leucine zipper region, our NMR and
CD studies indicated that the entire bZIP domain of ATF4 is in
equilibrium between an
-helix and a random coil, existing mostly as
a random coil in the absence of DNA. The addition of specific DNA (CRE)
shifts the equilibrium toward an
-helix by inducing an
-helix in
the bZIP domain including the basic region. The heterodimerization of
the ATF4 bZIP domain with the C/EBP
bZIP domain may also induce an
-helix in the ATF4 bZIP domain including the basic region, but
not in the basic region of the C/EBP
bZIP domain probably because of
its lower
-helix propensity. Once the
-helix is induced in the
leucine zipper region of the ATF4 bZIP domain by the formation of the
heterodimer with the C/EBP
bZIP domain, the intrahelical
water-mediated hydrogen bonds found in the basic and fork regions of
the ATF4 bZIP domain (Figs. 1B and 2A) may help
extend stabilizing
-helical conformation from the leucine zipper
region to the basic region.
-helical conformation in the basic DNA-binding
region, ATF4 may be more selective in binding to the DNA target via an
entropy-controlled manner. With its disordered basic region, C/EBP
may be able to adapt to have a broad range of DNA-binding specificities
and to accommodate CRE with comparable affinity to its canonical DNA
site (CCAAT box). As a result, the C/EBP
·ATF4 heterodimer has a
DNA-binding preference similar to that of the ATF4 homodimer, but forms
a more stable complex on CRE than the ATF4 homodimer does. Whether the
preformed C/EBP
·ATF4 heterodimer interacts with CRE or the binding
of each bZIP monomer to DNA precedes the dimerization cannot be
concluded from available data. In both possible pathways, the
interaction of the ATF4 basic region with the CRE half-site apparently
plays a decisive role in selection of target DNA. Many of the CREB/ATF
proteins form heterodimers with AP-1 (9-13) or C/EBP (14-16) family
members that bind CRE or composite sites containing at least one CRE
half-site. The data suggest that other CREB/ATF proteins may interact
with DNA targets and dimerization partners using a mechanism similar to that discussed here for ATF4.
levels increase upon exposure of cells to anoxic conditions and that
anoxic tension leads to enhanced binding of ATF4 to a CCAAT
box/CRE (or AP-1 site) composite site. This suggests that ATF4 may form
a heterodimer with C/EBP
and participate in a unique role in this
condition of stress. More recently, Fawcett et al. (34)
demonstrated that CREB/ATF family members form heterodimeric complexes
with C/EBP
on a CCAAT box/CRE composite site that resides in the
Gadd153 promoter. Gadd153, also known as CHOP and a
member of the C/EBP transcription factor family, is transcriptionally
activated by cellular stress signals, resulting in growth
arrest or DNA damage (GADD). In
response to arsenic stress, ATF4 activates Gadd153 transcription by
forming the complex with C/EBP
, whereas ATF3 represses Gadd153
transcription from the CCAAT box/CRE composite site.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank T. Hai for providing human ATF4
cDNA and L. Sealy for providing rat C/EBP cDNA; E. Howard,
K. Tesh, and K. Balasubramanian for excellent technical assistance; and
M. Newcomer, M. Waterman, D. Gewirth, and G. Vanduyane for critical
reading of the manuscript and helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by a startup fund and Institutional Grant P60 DK20593 from the Diabetes Research and Training Center (to Y. K.) and by Institutional Grant IRG IN-25-36 from the American Cancer Society (to Y. K.). Color figures were prepared using the Vanderbilt University Medical Center Cell Imaging Resource, which is supported by National Institutes of Health Grants CA68485 and DK20593.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 1ci6) 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 should be addressed. Tel.: 630-252-3962; Fax: 630-252-6126/5517; E-mail: ykim@lhmrba.hh.vanderbilt.edu or ykim{at}anl.gov.
Published, JBC Papers in Press, October 3, 2000, DOI 10.1074/jbc.M005594200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: bZIP, basic leucine zipper region; C/EBP, CCAAT box/enhancer-binding protein; AP-1, activator protein-1; CREB, cAMP response element-binding protein; ATF, activating transcription factor; CRE, cAMP response element; TFE, 2,2,2-trifluoroethanol; NOESY, nuclear Overhauser effect correlation spectroscopy.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Harbury, P. B., Zhang, T., Kim, P. S., and Alber, T. (1993) Science 262, 1401-1407[Medline] [Order article via Infotrieve] |
2. | Patel, L., Abate, C., and Curran, T. (1990) Nature 347, 572-575[CrossRef][Medline] [Order article via Infotrieve] |
3. | Weiss, M. A., Ellenberger, T., Wobbe, C. R., Lee, J. P., Harrison, S. C., and Struhl, K. (1990) Nature 347, 575-578[CrossRef][Medline] [Order article via Infotrieve] |
4. | O'Neil, K. T., Shuman, J. D., Ampe, C., and DeGrado, W. F. (1991) Biochemistry 30, 9030-9034[Medline] [Order article via Infotrieve] |
5. | Weiss, M. A. (1990) Biochemistry 29, 8020-8024[Medline] [Order article via Infotrieve] |
6. | Bracken, C., Carr, P. A., Cavanagh, J., and Palmer, A. G. (1999) J. Mol. Biol. 285, 2133-2146[CrossRef][Medline] [Order article via Infotrieve] |
7. | Andrisani, O. M., Pot, D. A., Zhu, Z., and Dixon, J. I. (1988) Mol. Cell. Biol. 8, 1947-1956[Medline] [Order article via Infotrieve] |
8. | Hai, T., Allegretto, E. A., Karin, M., and Green, M. R. (1989) Genes Dev. 3, 2083-2090[Abstract] |
9. | Benbrook, D. M., and Jones, N. C. (1990) Oncogene 5, 295-302[Medline] [Order article via Infotrieve] |
10. | Macgregor, P. F., Abate, C., and Curran, T. (1990) Oncogene 5, 451-458[Medline] [Order article via Infotrieve] |
11. | Hai, T., and Curran, T. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3720-3724[Abstract] |
12. | Konradi, C., Kobierski, L. A., Nguyen, T. V., Heckers, S., and Hyman, S. E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7005-7009[Abstract] |
13. | Chatton, B., Bocco, J. L., Goetz, J., Gaire, M., Lutz, Y., and Kedinger, C. (1994) Oncogene 9, 375-385[Medline] [Order article via Infotrieve] |
14. | Vinson, C. R., Hai, T., and Boyd, S. M. (1993) Genes Dev. 7, 1047-1058[Abstract] |
15. | Vallejo, M., Ron, D., Miller, C. P., and Habener, J. F. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4679-4683[Abstract] |
16. |
Shuman, J. D.,
Cheong, J.,
and Coligan, J. E.
(1997)
J. Biol. Chem.
272,
12793-12800 |
17. |
Osada, S.,
Yamamoto, H.,
Nishihara, T.,
and Imagawa, M.
(1996)
J. Biol. Chem.
271,
3891-3896 |
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. | Glover, J. N., and Harrison, S. C. (1995) Nature 373, 257-261[CrossRef][Medline] [Order article via Infotrieve] |
21. | Cao, Z., Umek, R. M., and McKnight, S. L. (1991) Genes Dev. 5, 1538-1552[Abstract] |
22. | Kumar, A., Ernst, R. R., and Wuthrich, K. (1980) Biochem. Biophys. Res. Commun. 95, 1-6[Medline] [Order article via Infotrieve] |
23. | Marion, D., and Wuthrich, K. (1983) Biochem. Biophys. Res. Commun. 113, 967-974[Medline] [Order article via Infotrieve] |
24. | Braunschweiler, L., and Ernst, R. R. (1983) J. Magn. Reson. 53, 521-528 |
25. | Shaka, A. J., and Freeman, R. (1983) J. Magn. Reson. 51, 169-173 |
26. | Marion, D., Ikura, M., Tschudin, R., and Bax, A. (1989) J. Magn. Reson. 85, 393-399 |
27. | Piotto, M., Saudek, V., and Sklenar, V. (1992) J. Biomol. NMR 2, 661-665[Medline] [Order article via Infotrieve] |
28. | Otwinowski, Z., and Minor, W. (1993) in Data Collection and Processing (Sawyer, L. , Isaacs, N. I. , and Bailey, S., eds) , pp. 59-62, CLRC Daresbury Laboratory, Warrington, United Kingdom |
29. | Brunger, A. T. (1992) X-PLOR Version 3.1: A System for X-Ray Crystallography and NMR , Yale University Press, New Haven, CT |
30. | 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] |
31. | Brunger, A. T., Adams, P. D., Clore, G. M., Delano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J.-S., Kuszewski, J., Nilges, M., and Pannu, N. S. (1998) Acta Crystallogr. Sect. D Biol. Crystallogr. 54, 905-921[CrossRef][Medline] [Order article via Infotrieve] |
32. | Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. Crystallogr. 26, 283-291[CrossRef] |
33. | Keller, W., Konig, P., and Richmond, T. J. (1995) J. Mol. Biol. 254, 657-667[CrossRef][Medline] [Order article via Infotrieve] |
34. | Fawcett, T. W., Martindale, J. L., Guyton, K. Z., Hai, T., and Holbrook, N. J. (1999) Biochem. J. 339, 135-141[CrossRef][Medline] [Order article via Infotrieve] |
35. | Kim, J., and Struhl, K. (1995) Nucleic Acids Res. 23, 2531-2537[Abstract] |
36. | Jasanoff, A., and Fersht, A. R. (1994) Biochemistry 33, 2129-2135[Medline] [Order article via Infotrieve] |
37. | Scholtz, J. M., Qian, H., York, E. J., Stewart, J. M., and Baldwin, R. L. (1991) Biopolymers 31, 1463-1470[Medline] [Order article via Infotrieve] |
38. | Woody, R. W. (1992) Advances in Biophysical Chemistry , Vol. 2 , pp. 37-79, JAI Press, Greenwich, CT |
39. | Wuthrich, K. (1986) NMR of Proteins and Nucleic Acids , John Wiley & Sons, Inc., New York |
40. | Bannister, A. J., Cook, A., and Kouzarides, T. (1991) Oncogene 6, 1243-1250[Medline] [Order article via Infotrieve] |
41. |
Santiago-Rivera, Z. I.,
Williams, J. S.,
Gorenstein, D. G.,
and Andrisani, O. M.
(1993)
Protein Sci.
2,
1461-1471 |
42. | Estes, S. D., Stoler, D. L., and Anderson, G. R. (1995) Exp. Cell Res. 220, 47-54[CrossRef][Medline] [Order article via Infotrieve] |