Consensus and Variant cAMP-regulated Enhancers Have Distinct
CREB-binding Properties*
Johanna C.
Craig
,
Maria A.
Schumacher
§,
Steven E.
Mansoor§,
David L.
Farrens§,
Richard G.
Brennan§, and
Richard H.
Goodman
¶
From the
Vollum Institute and § Department
of Biochemistry and Molecular Biology, Oregon Health Sciences
University, Portland, Oregon 97201-3098
Received for publication, November 10, 2000, and in revised form, December 19, 2000
 |
ABSTRACT |
Recent determination of the cAMP response
element-binding protein (CREB) basic leucine zipper (bZIP)
consensus CRE crystal structure revealed key dimerization and
DNA binding features that are conserved among members of the
CREB/CREM/ATF-1 family of transcription factors. Dimerization appeared
to be mediated by a Tyr307-Glu312
interhelical hydrogen bond and a Glu319-Arg314
electrostatic interaction. An unexpected hexahydrated Mg2+
ion was centered above the CRE in the dimer cavity. In the present study, we related these features to CREB dimerization and DNA binding.
A Y307F substitution reduced dimer stability and DNA binding
affinity, whereas a Y307R mutation produced a stabilizing effect.
Mutation of Glu319 to Ala or Lys attenuated dimerization
and DNA binding. Mg2+ ions enhanced the binding affinity of
wild-type CREB to the palindromic CRE by ~20-fold but did not do so
for divergent CREs. Similarly, mutation of Lys304, which
mediates the CREB interaction with the hydrated Mg2+,
blocked CREB binding to the palindromic but not the variant CRE
sequences. The distinct binding characteristics of the K304A mutants to
the consensus and variant CRE sequences indicate that CREB binding to
these elements is differentially regulated by Mg2+ ions. We
suggest that CREB binds the consensus and variant CRE sequences through
fundamentally distinct mechanisms.
 |
INTRODUCTION |
The cAMP response element-binding protein
(CREB)1 is a 43-kDa basic
leucine zipper (bZIP) transcription factor that couples gene activation
to a wide variety of cellular signals (for review, see Ref. 1).
Initially identified as a mediator of the cAMP pathway, CREB is now
recognized to respond to calcium/calmodulin and growth factor pathways
as well (2-4). The prototypical target sequence for CREB is the
palindromic cAMP response element (CRE; 5'-TGACGTCA-3'), first
identified in the neuropeptide somatostatin gene (5). Consensus CRE
sequences, or slight variants of this sequence, have since been
identified in hundreds of cellular and viral genes. In many instances,
functional studies have indicated that these sequences are required for
second messenger-directed transcriptional responses (for review, see
Ref. 6). Whether variations of the CRE from the consensus sequence have
functional consequences has not been determined.
Two general categories of factors can recognize the CRE (1, 7, 8). The
first is composed of factors that can dimerize with CREB. This group
includes the CRE modulator CREM
(and the multitude of splice
variants that contain the CREM
DNA binding domain) and the
activating transcription factor-1 (ATF-1). CREM
and ATF-1 recognize
the CRE as homodimers and as heterodimers with CREB (9-11). The second
category of CRE binding proteins includes factors that cannot
heterodimerize with CREB, such as c-Jun, some of the other ATFs,
and members of the CAAT/enhancer-binding protein gene family
(12, 13). In addition to their inability to heterodimerize with CREB,
these factors appear to be capable of recognizing DNA sequences that
are distinct from the CRE (14). This promiscuity in DNA binding is not
entirely unexpected, because some of these distinct elements, such as
the AP-1 site, differ from the CRE by only a single nucleotide. CREB
family members are notable in that they recognize the CRE exclusively,
or nearly so. One caveat in this characterization, however, is that the defining features of the CRE have not been established unequivocally.
CREB binds as a dimer to the CRE with an affinity of ~1-2
nM (15). Dimerization and DNA binding are mediated by the
adjacent basic and leucine zipper domains (7, 8). At the extreme C-terminal end of the protein is the leucine zipper, characterized by a
conserved heptad repeat of seven residues, denoted a-g. The residues at positions a and d typically form a
hydrophobic interface, with conserved leucines at position
d. As with other bZIP proteins, this configuration in CREB
allows for the formation of a two-stranded parallel coiled-coil and,
along with charged residues at positions e and g,
presents a plane for dimerization specificity (12, 16-19). In
addition, dimerization of the CREB monomers apposes the basic regions
in a parallel orientation to the DNA, allowing the dimer to bind at a
right angle to the DNA helical axis. DNA binding elicits an
-helical
conformation of the basic region that facilitates DNA recognition. The
basic region of CREB abuts the amino terminus of the leucine zipper.
This segment mediates DNA half-site sequence recognition and binding by
passing through the major groove and forming several direct and
indirect protein-DNA contacts.
Insights into the mechanisms underlying CREB dimerization and DNA
binding were obtained from the crystal structure of the CREB bZIP·CRE
complex (20). Two features of this structure were unexpected. First was
a hexahydrated Mg2+ ion centered on the CRE between the two
bZIP monomers at the dimer fork. This hexahydrated Mg2+ ion
is not found in related bZIP-DNA complexes and participates in a
water-mediated contact between Lys304 in the CREB basic
domain and the CRE. Structurally, the hexahydrated Mg2+ ion
was proposed to function by positioning and stabilizing the CREB basic
region in the CRE major groove. It may also impart additional rigidity
to the CREB basic domain, which may prevent binding to shorter or
longer divergent CRE sequences. Fluorescence anisotropy assays revealed
that CREB bZIP binding to the CRE was dramatically regulated by
Mg2+ and other divalent cations. In the absence of divalent
cation, binding decreased by at least 20-fold. The divalent cation
concentration required for half-maximal binding was 340 µM, approximately the concentration of free
Mg2+ ions in many types of cells. Thus, it was proposed
that the concentration of free Mg2+ might regulate the
level of CREB binding to the CRE.
The second unexpected feature of the complex was the mechanism
underlying the specificity of CREB dimerization. In addition to
predicted electrostatic interactions between Glu319 on one
monomer with Arg314 on the other, a critical interhelical
hydrogen bond was found between Tyr307 and
Glu312 (Fig. 1). The combination of
Glu319-Arg314 and
Tyr307-Glu312 is unique to members of the CREB
family and was proposed to be the primary determinant of CREB, CREM
,
and ATF-1 heterodimerization. Of note, although dimerization is
generally attributed to leucine zipper sequences, the
Tyr307 is located at the C-terminal end of the basic region.
Studies in this paper were designed to test the major predictions of
the CREB bZIP·CRE crystal structure. First, we wanted to test the
specificity of the divalent cation effect by determining whether the
interaction of Lys304 with the hexahydrated
Mg2+ ion was essential for CREB binding to DNA. Next, we
asked whether Mg2+ ions regulated the binding of CREB to
consensus and nonconsensus CREs. Finally, we tested the functional
importance of the residues predicted to direct CREB dimerization
specificity. Our studies confirm the predictions of the CREB-consensus
CRE crystal structure and suggest that consensus and nonconsensus CREs
interact with CREB through fundamentally distinct mechanisms.
 |
EXPERIMENTAL PROCEDURES |
Plasmid Construction and Protein Purification--
Construction
of the wild-type full-length and CREB bZIP expression vectors was
described by Richards et al. (15). The three cysteine
residues (Cys300, Cys310, and
Cys337) in the bZIP region of rat CREB (residues 285-341)
were mutated to serine. These mutations improved protein solubility but
did not alter DNA binding, as demonstrated by the similar relative affinities of CREB/Ser and wild-type CREB for the CRE (15). The
following mutations were introduced into CREB bZIP/Ser by site-directed
mutagenesis using oligonucleotide primers from Life Technologies, Inc.:
Y307F, Y307R, E319A, E319K, and K304A. Purification of CREB bZIP/Ser
proteins was described by Schumacher et al. (20). Purification of full-length CREB protein was described by Richards et al. (15). Protein concentrations were determined by
Bradford protein assay (Bio-Rad).
Thermal Stability Measurements--
The stability of each bZIP
protein was assessed in thermal melting assays by monitoring
fluorescence as previously described (21). In brief, the fluorescence
of the two tyrosine residues (positions 307 and 336) in wild-type CREB
bZIP was monitored as a function of temperature. Measurements were
taken on 1.3 ml of a sample containing 5 µM CREB bZIP in
HEPES buffer (pH 7.6; with 50 mM or 150 mM NaCl
in the presence or absence of 10 mM Mg2+) in a
6-mm cuvette. Temperature was monitored using a thermistor element/controller device (Omega Engineering Inc.; model no.
DP25-TH-A), with the thermistor element positioned in the solution
inside the cuvette. Excitation was set at 280 nm, and emission was
monitored at 300 nm using 3-nm band pass settings. The emission data
were collected by ramping the temperature from 4 to 85 °C at
2 °C/min. The temperature was ramped back down to 4 °C, and the
process was repeated for each sample to confirm reversibility of
protein folding. The fluorescence intensity data were fit using a
nonlinear least squares regression analysis (Sigma Plot; Jandel
Scientific). The thermodynamic relationship between the folded and
unfolded states of the protein, represented by the melting temperature (Tm), was used to compare protein stability and is
represented as follows,
|
(Eq. 1)
|
where
Hu is the enthalpy and
Su is the entropy of unfolding (22, 23). Tyrosine
emission (300 nm; y axis) was plotted as a function of
temperature (in degrees Centigrade). In the thermal melting
experiments, the sharp upward slopes represent the transitions between
protein folding and unfolding. Pretransition state fluorescence defines
the fluorescence of the folded state of the protein (represented by the
left tails of the melting curves). Post-transition state fluorescence
defines the fluorescence characteristics of the unfolded state
(represented by the right tails of the melting curves). By measuring
free tyrosine in solution, we confirmed that the downward slopes
observed for the pre- and post-transition states were due to the
temperature sensitivity of tyrosine fluorescence (data not shown).
Assays were performed on the wild-type CREB bZIP, and the mutants
K304A, E319A, E319K, Y307F, and Y307R. Note that in the case of the
Y307F and Y307R mutants, the emission of only one tyrosine was
measured. Although the signal was reduced, the single tyrosine was
adequate to measure the transition state of the mutant proteins.
Selected proteins were also denatured with urea, yielding profiles that
were similar to those produced by thermal melting (data not shown),
indicating that heat is a satisfactory denaturing reagent for measuring
tyrosine emission.
Fluorescence Polarization Measurements--
The binding
affinities of CREB bZIP proteins for DNA were determined in solution
using a PanVera Beacon 2000 fluorescence polarization system.
5'-Fluoresceinated oligonucleotides corresponding to the
somatostatin (5'-CCTGACGTCAGCCCCCTGACGTCAGG-3'), rat c-Fos
(5'-AGTGACGTAGGCCCCCCTACGTCACT-3'),
phosphoenolpyruvate carboxykinase (PEPCK;
5'-CCTTACGTCAGCCCCCTGACGTAAGG-3'), and the
tyrosine aminotransferase (TAT;
5'-TCTGCGTCAGCCCCCTGACGCAGA-3') CREs were
purchased from Life Technologies, Inc. The CRE binding sites are
indicated in boldface type. Each oligonucleotide was designed to form a
hairpin with a 5-nucleotide loop and was self-annealed by heating to
95 °C and snap-cooled on ice. Binding experiments with the
somatostatin CRE were performed with wild-type and mutant CREB bZIP
(residues 283-341) and wild-type full-length CREB (residues 1-341)
proteins. Binding reactions with the PEPCK CRE were performed with
wild-type and K304A bZIP and wild-type full-length CREB, and those with
the c-Fos and TAT CREs were performed with wild-type and K304A CREB
bZIP. Protein was titrated into 0.99-ml solutions that contained 1 nM fluoresceinated oligonucleotide in 25 mM
Tris-HCl, pH 7.6, 50 mM NaCl, 0.5 mM EDTA, 5%
glycerol, 6 µg of bovine serum albumin, 10 µg of poly(dI-dC) in the
presence or absence of 10 mM MgCl2. Binding
reactions were performed at 25 or 37 °C, and samples were incubated
for 1 min to achieve equilibrium. Each sample was excited at 490 nm,
and emission was measured at 530 nm. The binding curves were fit with a
nonlinear least squares regression analysis using Sigma Plot (Jandel
Scientific). In addition to the rectangular hyperbolic binding
function, the equation utilized for curve fit determination included a
nonspecific component to account for the linear increase in
polarization at higher protein concentrations. For a curve that begins
at y0 (minimum polarization) and rises to
y1 (maximum polarization), the following
equation was used,
|
(Eq. 2)
|
where b represents Kd as a
function of x = [CREB], and c represents
the nonspecific constant as a linear additive function
(cx).
 |
RESULTS |
Fig. 1 depicts two magnified views
of a portion of the CREB bZIP-consensus CRE complex as reported by
Schumacher et al. (20). Key residues in the basic and
leucine zipper domains are highlighted. The continuous
-helical
chains are symmetrically apposed at the C-terminal leucine zipper and
are fastened by an electrostatic interaction between Arg314
and Glu319 as well as hydrogen bonds between
Tyr307 and Glu312. The helices pass across the
DNA through the major groove, where several residues from the basic
region make protein-DNA contacts. The most extraordinary feature of the
complex is the hydrated Mg2+ ion, which is juxtaposed
between the two
-helices and appears to stabilize the dimer fork on
the CRE sequence.

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 1.
Views of the CREB bZIP-DNA complex depicting
key protein-protein and protein-divalent cation interactions.
a, view of the CREB bZIP and the side chains of the residues
selected for study (Glu319-Arg314,
Tyr307-Glu312, and Lys304), along
with their contact distances (Å). The hydrated magnesium ion
(gray) complexed with six water molecules occupies the dimer
cavity. b, magnification of the dimer cavity with a view of
the hexahydrated magnesium ion contacted by the Lys304 side
chains, centered above the CRE illustrated at the base of the dimer
fork as a Cory-Pauling-Kolton space-filling model.
|
|
A lysine residue (Lys304) in the basic region of each
subunit provides a hydrogen bond to one of the coordination waters of
the Mg2+ ion (Fig. 1b). Thus, we hypothesized
that Lys304 was essential for Mg2+ ion binding.
To test this idea, we mutated Lys304 to Ala and performed
thermal stability and fluorescence polarization assays on the mutated
and wild-type CREB proteins in the presence or absence of
Mg2+ ions. Alanine was chosen for this substitution because
it is predicted to maintain the helical structure of CREB bZIP.
Moreover, the related factor CAAT/enhancer-binding protein
contains
an Ala in the corresponding position. The thermal stability of each CREB protein was determined by measuring the intrinsic tyrosine fluorescence (contributed by Tyr307 and Tyr336)
as a function of temperature. These studies indicated that the conformational stability of the wild-type protein was unaffected by the
presence or absence of Mg2+ ions,
as indicated by the unchanged
Tm values (Fig. 2a and Table
I; Tm = 45.0 ± 0.2 °C with Mg2+, Tm = 43.2 ± 0.4 °C without Mg2+).
Similarly, the K304A mutation did not have an adverse effect on the
conformational stability of the protein (Fig. 2a and Table I; Tm = 47.0 ± 0.3 °C with
Mg2+, Tm = 46.7 ± 0.3 °C
without Mg2+), indicating that divalent ions are not
necessary for maintaining structure in the absence of DNA. In contrast,
fluorescence polarization measurements indicated that the absence of
Mg2+ ions markedly decreased the binding affinity of
wild-type CREB for the CRE (Fig. 2b and Table
II; Kd = 1.8 ± 0.2 nM with Mg2+, Kd > 20 nM without Mg2+), as reported previously (20).
Moreover, no binding of CREB bZIP K304A to the CRE was detected even in
the presence of Mg2+ ions (Fig. 2b). Thus, the
enhancement of CREB binding to the consensus CRE mediated by
Mg2+ depends upon the integrity of Lys304.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 2.
Involvement of Mg2+ and
Lys304 for binding to the consensus CRE. a,
representative plot of the thermal denaturation of wild-type CREB
(closed circles, with Mg2+,
Tm = 45.0 °C; open
triangles, without Mg2+, Tm = 43.0 °C) and the K304A mutant (open squares;
Tm = 46.0 °C). F represents
the emission intensity at 300 nm (y axis) plotted against
temperature (Celsius; x axis). Fmin
and Fmax represent the observed
minimum and maximum tyrosine flourescence values. Each sample was
remelted after the protein was cooled and refolded, and each condition
was repeated at least twice. b, representative DNA binding
isotherms of the wild-type (open circles, with
Mg2+, Kd = 1.8 nM;
open triangles, without Mg2+,
Kd > 20 nM) and K304A proteins
(open squares; Kd not
determined) bound to the consensus CRE. Measurements, reported as
millipolarization units (mP; y axis), were
normalized and plotted against CREB bZIP concentration (nM;
X axis). The binding curves were repeated at least three
times for each condition.
|
|
View this table:
[in this window]
[in a new window]
|
Table II
The effects of Mg2+ (10 mM) on the affinity of CREB
bZIP for various CREs
Values represent Kd ± S.E. For not determined (ND)
values, no specific binding was detected up to the concentrations
indicated in the figures.
|
|
With the exception of the tyrosine aminotransferase (TAT) CRE, the
binding properties of CREB for nonconsensus CRE sequences have been
poorly explored. We recently reported that CREB binds the nonconsensus
PEPCK CRE with high affinity (24). This finding is not
altogether surprising, because this sequence differs from the
somatostatin CRE by only a single nucleotide (TTACGTCA; the
single change in boldface type). However, these studies were performed
under standard conditions, in the presence of Mg2+ ions. To
test whether CREB binding to nonconsensus CREs similarly required
Mg2+, we reanalyzed binding to the PEPCK element in the
presence and absence of divalent cation. High affinity binding was
detected in both cases (Fig. 3,
a and b, and Table II; Kd = 1.2 ± 0.4 nM with Mg2+,
Kd = 1.4 ± 0.2 nM without
Mg2+). Similar findings were obtained in binding assays
performed using full-length CREB (data not shown). Consistent with
these observations, the K304A mutant bound equally to the PEPCK CRE in
the presence or absence of Mg2+ ions (Fig. 3c
and Table II; Kd = 2.0 ± 0.4 nM
with Mg2+, Kd = 2.0 ± 0.2 nM without Mg2+). These studies indicated that
the mechanisms of CREB binding to the PEPCK and consensus CREs must be
fundamentally different.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 3.
CREB binding to the high affinity,
nonconsensus PEPCK CRE sequence does not require Mg2+.
Representative fluorescence polarization plots are shown of the
wild-type CREB bZIP with Mg2+ (Kd = 1.2 nM) (a) and without Mg2+
(Kd = 1.4 nM) (b) and the
K304A mutant (with or without Mg2+, Kd = 2.0 nM) (c) bound to the PEPCK CRE. Measurements
were plotted as in Fig. 2. The binding curves were repeated at least
three times for each condition.
|
|
Additional binding studies were performed using the c-Fos and TAT CREs.
The rat c-Fos promoter contains a CRE element that differs from
the somatostatin CRE by two nucleotides, in the seventh and eighth
positions (TGACGTAG). The binding affinity of CREB for the
c-Fos CRE had not been determined using equilibrium measurements in
solution. The TAT promoter contains a CRE that differs from the
consensus sequence by three nucleotides (CTGCGTCA) and has
been characterized as a prototypical low affinity binding site (25).
The binding affinity of CREB for the TAT CRE was determined to be 11 nM by Richards et al. (15). We analyzed the TAT
and c-Fos CREs in the presence and absence of Mg2+ ions and
found that in both instances CREB bZIP binding did not require
Mg2+ (Figs. 4 and
5; Kd = ~5
nM for the c-Fos CRE and 10 nM for the TAT CRE
in the presence or absence of Mg2+ ions). This
Mg2+ independence of binding to the c-Fos and TAT elements
was supported by the ability of CREB bZIP K304A to bind to both
sequences irrespective of Mg2+ (Figs. 4c and
5c).

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 4.
CREB binding to the moderate affinity,
nonconsensus rat c-Fos CRE does not require Mg2+.
Representative fluorescence polarization plots are shown of the
wild-type CREB bZIP with Mg2+ (Kd = 5.0 nM) (a) or without Mg2+
Kd = 4.5 nM (b) and the K304A
mutant (with or without Mg2+, Kd = 5.5 nM) (c) bound to the c-Fos CRE. Measurements
were plotted as in Fig. 2. The binding curves were repeated at least
three times for each condition.
|
|

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 5.
CREB binding to the low affinity,
nonconsensus TAT CRE does not require Mg2+.
Representative fluorescence polarization plots are shown of the
wild-type CREB bZIP with Mg2+ (Kd = 10.0 nM (a) or without Mg2+
(Kd = 10.0 nM) (b) and the
K304A mutant (with or without Mg2+, Kd = 6.3 nM) (c) bound to the TAT CRE. Measurements
were plotted as in Fig. 2. The binding curves were repeated at least
three times for each condition.
|
|
Two key determinants of dimerization unique to CREB family members are
interactions between Glu319-Arg314 and
Tyr307-Glu312. To investigate the
contributions of these interactions to protein stability and DNA
binding, we utilized CREB derivatives generated by mutating
Tyr307 to Phe or Arg and Glu319 to Ala or Lys.
The Y307F substitution was designed to test the importance of the
Tyr307-Glu312 hydrogen bond (Fig.
1a). The Y307R substitution was designed to replace the
hydrogen bond with an electrostatic interaction, which was predicted to
increase the stability of a potential dimer structure. As compared with
wild-type CREB (Fig. 2a), the Y307F mutant shows a dramatic
reduction in thermal stability in 50 mM NaCl (Fig.
6a and Table
III; Tm = 24.3 ± 0.6 °C), while the Y307R mutation had a stabilizing effect under
this ionic condition (Fig. 6b and Table III;
Tm = 55.3 ± 0.3 °C). However, the Y307R
mutant was irreversibly denatured at physiological ionic strength (150 mM NaCl), indicating that this concentration of NaCl can
interfere with the formation of the
Arg307-Glu312 salt bridge (data not shown).
Fig. 7b shows that the Y307F
mutant also exhibited a temperature-dependent decrease in
binding to the CRE, as determined by fluorescence polarization assays.
Compared with wild-type CREB (Fig. 7a and Table
IV; Kd = 2.0 ± 0.1 nM at 25 °C, Kd = 15 ± 1.1 nM at 37 °C), DNA binding of the Y307F protein was
greatly diminished at 25 °C and was abolished at 37 °C (Fig.
7b and Table IV). Thus, the interaction between Tyr307 and Glu312 is crucial for dimerization
and subsequent DNA binding.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 6.
Contribution of the
Tyr307-Glu312 hydrogen bonds to CREB
stability. Representative thermal melting plots of Y307F
(Tm = 25.0 °C) (a) and Y307R
(Tm = 55.0 °C) (b) are shown. Tyrosine
fluorescence was measured and plotted as in Fig. 2a. Each
sample was remelted after the protein was cooled and refolded, and each
condition was repeated at least twice.
|
|

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 7.
Tyr307-Glu312 is
required for DNA binding. Representative fluorescence polarization
plots are shown of wild-type CREB bZIP at 25 °C (open
circles; Kd = 2.0 nM) and
37 °C (open squares; Kd = 15.0 nM) (a) and Y307F at 25 °C
(open circles; Kd > 25 nM) and at 37 °C (open squares;
Kd not determined (n.d.)) (b)
bound to the consensus CRE. Measurements were plotted as in Fig. 2. The
binding curves were repeated a minimum of three times for each
condition.
|
|
The importance of the Glu319-Arg314
interaction to dimer stability and specificity was tested by measuring
the thermal stability of proteins containing mutations of
Glu319 to Ala or Lys. As shown in Fig.
8, both mutations reduced CREB protein
stability compared with the wild type CREB bZIP control (Fig.
2a). The E319A substitution reduced dimer stability in an ionic strength-independent manner (Fig. 8a and Table III;
Tm = 29.5 ± 0.8 °C; and data not shown),
and the E319K mutant appeared completely unfolded at 4 °C,
regardless of the ionic strength of the buffer (Fig. 8b and
data not shown).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 8.
Contribution of the
Glu319-Arg314 electrostatic interaction to
CREB stability. Representative thermal melting plots are shown of
E319A (Tm = 29.5 °C) (a) and E319K
(Tm not determined (n.d.))
(b). Tyrosine fluorescence was measured and plotted as in
Fig. 2. Each sample was remelted after the protein was cooled and
refolded, and each condition was repeated at least twice.
|
|
Despite the lowered thermal stability, both derivatives were able to
bind to the consensus CRE, in the presence of Mg2+ ions,
although with greatly reduced affinity at 25 °C (Fig.
9a and Table IV, E319A
Kd = 65.0 nM ± 1.1; Fig. 9b,
E319K Kd > 250 nM). Consistent with
their Tm values, binding of both Glu319
substitutions to the CRE was completely abolished at 37 °C (Fig. 9,
a and b, and Table IV).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 9.
Glu319 mutants decrease DNA
binding. Representative fluorescence polarization plots are shown
of E319A (open circles, 25 °C,
Kd = 65 nM; open
squares, 37 °C, Kd not determined
(n.d.)) (a) and E319K (open
circles, 25 °C, Kd > 250 nM; open squares = 37 °C, Kd not
determined (n.d.)) (b) bound to the consensus
CRE. Measurements were plotted as in Fig. 2. The binding curves were
repeated at least three times for each condition.
|
|
 |
DISCUSSION |
In this study, we examined the contributions of key residues of
the CREB bZIP to structural stability and DNA binding. For these
determinations, we utilized thermal melting and fluorescence polarization assays. The melting assays measured the transitions of the
mutant and wild-type CREB bZIP proteins from the folded to the unfolded
states during thermal denaturation by tracking the emission spectra
from tyrosine residues. In the context of the folded protein, the
tyrosine fluorescence is quenched, presumably by neighboring residues
(26). Upon thermal unfolding of the protein, this quenching is removed,
and the tyrosine fluorescence increases. Indirect measurements of bZIP
dimer association (using circular dichroism in the absence of DNA)
indicate that dimer formation occurs at micromolar concentrations of
peptide (27, 28). However, there has been some disagreement as to
whether CREB exists as a dimer or monomer in solution in the absence of DNA. Previously, our laboratory used fluorescence resonance energy transfer to determine that the Kd for CREB
dimerization in the absence of DNA was 0.6 nM (29), but
equilibrium sedimentation studies suggested that CREB exists primarily
in a monomeric form (30). The melting curves in the current study were
carried out using relatively high concentrations of CREB protein (5 µM), and we have interpreted these results assuming that,
under our experimental conditions, CREB exists in the dimeric form.
Consistent with this interpretation, mutations that were expected to
disrupt dimerization caused observable changes in Tm
and reductions in DNA binding, while the substitution in the basic
region (K304A) did not alter the Tm value from that
obtained for wild-type CREB bZIP. Thus, the thermal melting analyses
described herein can be interpreted reasonably as an indirect measure
of dimer stability. The finding that the thermally unstable E319K
mutant binds to the CRE with low affinity (Kd = 250 nM) suggests that if this form is monomeric, then a single
bZIP helix interacts rather poorly with DNA. Alternately, it may be
that the CRE can recruit and stabilize the very low concentration of
the dimeric form of this mutant, or that it overcomes the interhelical
repulsion between Lys319 and Lys314' at high
protein concentrations.
Role of Mg2+ Ions on CREB bZIP Stability and DNA
Binding--
We utilized fluorescence polarization assays to obtain
equilibrium measurements of protein-DNA binding in solution. Precise quantitation of these interactions might not be achieved by using the
more standard gel mobility shift assays, because the proteins can
dissociate from the DNA within the gel matrix, and the ionic environment is difficult to control during electrophoresis.
We first tested whether Lys304, which interacts with the
hexahydrated Mg2+ ion, was necessary for CREB dimerization
and DNA binding. The importance of Lys304 in CREB function
was tested previously by Dwarki et al. (31). Their study
examined the properties of a CREB K304E mutant in an effort to
determine whether the predicted electrostatic repulsion between the Glu
and the negatively charged phosphate backbone of the CRE would be
deleterious for CREB function. As predicted, this mutant failed to bind
the CRE in vitro or to activate transcription in
vivo. The present study took advantage of the crystal structure of
the CREB bZIP·CRE complex to examine the role of Lys304
specifically in Mg2+ ion-mediated DNA sequence recognition.
We mutated the Lys304 to Ala, the residue that occupies
this position in the related transcription factor CAAT/enhancer-binding
protein
. The thermal stability of the CREB bZIP K304A mutant was
the same as that of the wild-type CREB protein, irrespective of
Mg2+ (summarized in Table I). Therefore, we suspect that
the stability of the mutant dimer is not impaired. However, binding of
wild-type CREB bZIP to the consensus CRE was highly dependent upon
Mg2+ (summarized in Table II). In the absence of
Mg2+, CREB bound poorly to the consensus CRE, and no
binding was detected with K304A, regardless of whether Mg2+
was included in the buffer. It is possible for Lys304 to
interact with the phosphate backbone of the DNA in the absence of the
hexahydrated Mg2+ ion (determined by manipulation of the
complex in RASMOL), which would be consistent with the ability of the
wild-type CREB protein to bind to the CRE at low affinity in the
absence of Mg2+. Elimination of the positive charges
contributed by the Lys304 side chain would diminish its
interaction with DNA, regardless of the presence of Mg2+.
Alternately, the presence of a Mg2+ ion might physically
obstruct the binding of the K304A mutant to the consensus CRE, if the
Ala substitution effectively removed a
Mg2+-dependent spacing requirement from the
dimer fork. Nonetheless, our data indicate that both a Mg2+
ion and Lys304 are required for CREB to bind with high
affinity to the consensus CRE but do not contribute to CREB stability.
Role of Mg2+ Ions for CREB bZIP Binding
Specificity--
The next set of experiments tested whether the
Mg2+ ion requirement for CREB binding was unique to the
consensus CRE (Table II). The PEPCK, TAT, and c-Fos genes are
all regulated by CREB via nonconsensus CREs. The PEPCK CRE differs from
the consensus CRE at the
3 base position (
4,
3,
2,
1/1, 2, 3, 4 corresponds to TGAC/GTCA), in which there is a pyrimidine for purine
substitution (TTACGTCA), the TAT CRE contains substitutions
at the
4,
3, and
2 base positions with a pyrimidine for purine
substitution in the
3 base position (CTGCGTCA), and the
rat c-Fos CRE contains substitutions in the 3 and 4 base positions
(TGACGTAG), with a purine for pyrimidine substitution at the
3 base position. The binding affinities of CREB for the PEPCK and TAT
elements were determined previously to be 2.0 and 11.0 nM,
respectively (see Refs. 24 and 15, respectively). The binding
affinity of CREB for the c-Fos element was determined in the present
study to be 4.5-5.0 nM, approximately 3-fold lower than
that for the consensus CRE. CREB bZIP binding to the lower affinity TAT
CRE, the moderate affinity c-Fos CRE, and the high affinity PEPCK CRE were all independent of Mg2+ (Table II). Similarly, the
K304A mutant was capable of recognizing each of the nonconsensus CREs
but not the consensus CRE. These studies indicate that deviation from
the consensus CRE sequence by even one nucleotide can result in
significant changes in CREB binding under specific ionic conditions.
The CREB bZIP·CRE crystal structure (20) reveals that there are
specific 2-fold symmetry-related contacts made by each CREB monomer to
nucleotides of base pairs
2/2 and
4/4. Specifically, the
C-
of residue Ala297 makes van der Waals contacts
with
2/2 thymine, and
4/4 thymine is engaged in van der Waals
contacts with the C-
of Ala296 and the C-
of
Ser300. Loss of these van der Waals contacts could explain
the lower affinity of CREB for the TAT CRE. The cytosine of base pair
3/3 makes a hydrogen bond to the side chain of Asn293. A
cytosine to adenine substitution at this base pair position, such as
with the PEPCK CRE, could, with slight rearrangement, permit the same
interaction between Asn293 and the N-6 of adenine.
The moderate affinity of CREB bZIP for the c-Fos CRE might result from
the poorer hydrogen bond that would form between N-293 and O-6 of
guanine
4/4, which would require side chain rearrangement. Such a
rearrangement could also explain the altered Mg2+
requirement. The fact that the CREB K304A mutant also distinguished consensus from nonconsensus CREs indicates that the Mg2+
effect does not occur through nonspecific changes in CRE structure. Rather, the palindromic nature of the consensus CRE must provide an
interface that is dependent upon Mg2+ for CREB recognition.
Role of Glu319-Arg314 and
Tyr307-Glu312 on CREB bZIP Stability and DNA
Binding--
We next tested the prediction that the
Glu319-Arg314 and the
Tyr307-Glu312 interactions were critical for
CREB dimerization by examining their affect on CREB bZIP stability
(summarized in Table III). The interactions of these residues are
unique to members of the CREB family. We found that the E319K protein
was less stable than the E319A mutant and that both had significantly
lower Tm values than wild-type CREB bZIP. However,
even the E319K mutant was capable of low affinity binding to the CRE,
suggesting that it might be able to interact with DNA as a monomer or
as a DNA-stabilized dimer. Interestingly, naturally occurring E319K
mutants of CREB have been detected in Raji B lymphocytes and C6 glioma
cells (32, 33). It is possible that other members of the CREB gene
family mediate responses to cAMP in these cells.
The importance of the interhelical hydrogen bond formed between
Tyr307 and Glu312 was tested by mutating
Tyr307 to Phe and Arg. The Phe substitution decreased
protein stability, and the Arg substitution increased protein stability
(although only at the lower salt concentration), as predicted (Table
III). Furthermore, the unstable CREB Y307F mutant had a reduced ability to bind to the CRE (Table IV). These data support a dual role for the
Tyr307-Glu312 interaction, one that serves to
promote dimerization specificity and a second that stabilizes the dimer
on the DNA.
Conclusions--
Our studies support the principal predictions of
the CREB bZIP·CRE crystal structure and confirm the functional roles
of the key residues that define the unique features of transcription factors in the CREB gene family. The importance of Mg2+
ions in the association of CREB with the consensus CRE suggests that
this divalent cation may have a regulatory role for discrimination at
the promoter level. How the difference in Mg2+ sensitivity
of consensus and nonconsensus CREs might affect the regulation of
specific genes remains to be determined, however. In neurons,
depolarization induces calcium fluxes that cause a 3-10-fold increase
in the concentration of free Mg2+ (34, 35). It is possible
that these Mg2+ changes could regulate the binding of CREB
to some cellular genes, but not others, depending on the presence of
particular CRE sequences in the relevant transcriptional control
regions. This complexity and the potential increase in regulatory
control have not been appreciated previously.
 |
ACKNOWLEDGEMENTS |
We thank John Notis for assistance with the
fluorescence polarization measurements, plasmid construction, and
graphics and Dr. James Lundblad and members of the Goodman
laboratory for helpful advice and comments.
 |
FOOTNOTES |
*
This work was supported by grants from the National
Institutes of Health.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.
¶
To whom correspondence should be addressed: Vollum Inst.,
Oregon Health Sciences University, 3181 S. W. Sam Jackson Park
Rd., Portland, OR 97201-3098. Tel.: 503-494-5078; Fax: 503-494-4353; E-mail: goodmanr@ohsu.edu.
Published, JBC Papers in Press, December 24, 2000, DOI 10.1074/jbc.M010263200
 |
ABBREVIATIONS |
The abbreviations used are:
CREB, cAMP response
element-binding protein;
bZIP, basic leucine zipper;
CRE, cAMP response
element;
CREM, CRE modulator;
ATF-1, activating transcription factor-1;
TAT, tyrosine aminotransferase;
PEPCK, phosphoenolpyruvate
carboxykinase.
 |
REFERENCES |
1.
|
Shaywitz, A. J.,
and Greenberg, M. E.
(1999)
Annu. Rev. Biochem.
68,
821-861[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Sheng, M.,
and Greenberg, M. E.
(1990)
Neuron
4,
571-582[Medline]
[Order article via Infotrieve]
|
3.
|
Ginty, D. D.,
Bonni, A.,
and Greenberg, M. E.
(1994)
Cell
77,
713-725[Medline]
[Order article via Infotrieve]
|
4.
|
Enslen, H.,
Sun, P. Q.,
Brickey, D.,
Soderling, S. H.,
Klamo, E.,
and Soderling, T. R.
(1994)
J. Biol. Chem.
269,
15520-15527[Abstract/Free Full Text]
|
5.
|
Montminy, M. R.,
Sevarino, K. A.,
Wagner, J. A.,
Mandel, G.,
and Goodman, R. H.
(1986)
Proc. Natl. Acad. Sci. U. S. A.
83,
6682-6686[Abstract]
|
6.
|
De Cesare, D.,
and Sassone-Corsi, P.
(2000)
Nucleic Acids Res.
64,
343-369
|
7.
|
Ellenberger, T.
(1994)
Curr. Opin. Struct. Biol.
4,
12-21
|
8.
|
Kerppola, T.,
and Curran, T.
(1995)
Nature
373,
199-200[CrossRef][Medline]
[Order article via Infotrieve]
|
9.
|
Hai, T. W.,
Liu, F.,
Coukos, W. J.,
and Green, M. R.
(1989)
Genes Dev.
3,
2083-2090[Abstract]
|
10.
|
Laoide, B. M.,
Foulkes, N. S.,
Schlotter, F.,
and Sassone-Corsi, P.
(1993)
EMBO J.
12,
1179-1191[Abstract]
|
11.
|
Loriaux, M. M.,
Brennan, R. G.,
and Goodman, R. H.
(1994)
J. Biol. Chem.
269,
28839-28843[Abstract/Free Full Text]
|
12.
|
Yun, Y. D.,
Dumoulin, M.,
and Habener, J. F.
(1990)
Mol. Endocrinol.
4,
931-939[Abstract]
|
13.
|
Benbrook, D. M.,
and Jones, N. C.
(1990)
Oncogene
5,
295-302[Medline]
[Order article via Infotrieve]
|
14.
|
Sellers, J. W.,
Vincent, A. C.,
and Struhl, K.
(1990)
Mol. Cell. Biol.
10,
5077-5086[Medline]
[Order article via Infotrieve]
|
15.
|
Richards, J. P.,
Bachinger, H. P.,
Goodman, R. H.,
and Brennan, R. G.
(1996)
J. Biol. Chem.
271,
13716-13723[Abstract/Free Full Text]
|
16.
|
Landschultz, W. H.,
Johnson, P. F.,
and McKnight, S. L.
(1988)
Science
240,
1759-1764[Medline]
[Order article via Infotrieve]
|
17.
|
Ellenberger, T. E.,
Brandl, C. J.,
Struhl, K.,
and Harrison, S. C.
(1992)
Cell
71,
1223-1237[Medline]
[Order article via Infotrieve]
|
18.
|
Glover, J. N. M.,
and Harrison, S. C.
(1995)
Nature
373,
257-261[CrossRef][Medline]
[Order article via Infotrieve]
|
19.
|
Keller, W.,
Konig, P.,
and Richmond, T. J.
(1995)
J. Mol. Biol.
254,
657-667[CrossRef][Medline]
[Order article via Infotrieve]
|
20.
|
Schumacher, M. A.,
Goodman, R. H.,
and Brennan, R. G.
(2000)
J. Biol. Chem.
275,
35242-35247[Abstract/Free Full Text]
|
21.
|
Mansoor, S. E.,
Mchaourab, H. S.,
and Farrens, D. L.
(1999)
Biochemistry
38,
16383-16393[CrossRef][Medline]
[Order article via Infotrieve]
|
22.
|
Eftink, M. R.
(1994)
Biophys. J.
66,
482-501[Abstract]
|
23.
|
Pace, N. C.,
and Scholtz, M. J.
(1997)
in
Protein Structure: A Practical Approach
(Creighton, T. E., ed)
, pp. 299-321, Oxford University Press, New York
|
24.
|
Fass, D. M.,
Craig, J. C.,
Impey, S.,
and Goodman, R. H.
(2001)
J. Biol. Chem.
276,
2992-2997[Abstract/Free Full Text]
|
25.
|
Nichols, M.,
Weih, F.,
Schmid, W.,
DeVack, C.,
Kowenz-Leutz, E.,
Luckow, B.,
Boshart, M.,
and Schutz, G.
(1992)
EMBO J.
11,
3337-3346[Abstract]
|
26.
|
Ross, J. B. A.,
Laws, W. R.,
Rousslang, K. W.,
and Wyssbrod, H. R.
(1999)
in
Topics in Fluorescence Spectroscopy
(Lakowicz, J. R., ed), Vol. 3
, pp. 1-63, Plenum Press, New York
|
27.
|
Weiss, M. A.,
Ellenberger, T. E.,
Wobbe, C. R.,
Lee, J. P.,
Harrison, S. C.,
and Struhl, K.
(1990)
Nature
347,
575-578[CrossRef][Medline]
[Order article via Infotrieve]
|
28.
|
Santiago-Rivera, Z. I.,
Williams, J. I.,
Gorenstein, D. G.,
and Andrisani, O. M.
(1993)
Protein Sci.
2,
1461-1471[Abstract/Free Full Text]
|
29.
|
Lundblad, J. R.,
Kwok, R. P. S.,
Laurance, M. E.,
Huang, M. S.,
Richards, J. P.,
Brennan, R. G.,
and Goodman, R. H.
(1998)
J. Biol. Chem.
273,
19251-19259[Abstract/Free Full Text]
|
30.
|
Wu, X.,
Spiro, C.,
Owen, W. G.,
and McMurray, C. T.
(1998)
J. Biol. Chem.
273,
20820-20827[Abstract/Free Full Text]
|
31.
|
Dwarki, V. J.,
Montminy, M.,
and Verma, I. M.
(1990)
EMBO J.
9,
225-232[Abstract]
|
32.
|
Moreno, C. S.,
Beresford, G. W.,
Louis-Plence, P.,
Morris, A. C.,
and Boss, J. M.
(1999)
Immunity
10,
143-150[Medline]
[Order article via Infotrieve]
|
33.
|
Short, M. L.,
Manohar, C. F.,
Furtado, M. R.,
Ghadge, G. D.,
Wolinsky, S. M.,
Thimmapaya, B.,
and Jungmann, R. A.
(1991)
Nucleic Acids Res.
19,
4290[Medline]
[Order article via Infotrieve]
|
34.
|
Kato, H.,
Gotoh, H.,
Kajikawa, M.,
and Suto, K.
(1998)
Brain Res.
779,
339-333
|
35.
|
Brocard, J. B.,
Rajdev, S.,
and Reynolds, I. J.
(1993)
Neuron
11,
751-757[Medline]
[Order article via Infotrieve]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.