Mechanism of κB DNA binding by Rel/NF-κB dimers
Christopher B. Phelps, Lei Lei Sengchanthalangsy, Shiva Malek & Gourisankar Ghosh*
Department of Chemistry and Biochemistry, University of California-San Diego
MC 0359
9500 Gilman Drive
La Jolla CA 92093
Running Title: NF-κB/DNA Binding
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Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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Summary:
The DNA binding of three different NF-κB dimers, the p50 and p65 homodimers and the
p50/p65 heterodimer, has been examined using a combination of gel mobility shift and
fluorescence anisotropy assays. The NF-κB p50/p65 heterodimer is shown here to bind
the κB DNA target site of the immunoglobulin κ enhancer (Ig-κB) with an affinity of
approximately 10 nM. The p50 and p65 homodimers bind to the same site with roughly
5 and 15-fold lower affinity, respectively. The nature of the binding isotherms indicates
a cooperative mode of binding for all three dimers to the DNA targets. We have further
characterized the role of pH, salt, and temperature on the formation of the p50/p65
heterodimer/Ig-κB complex. The heterodimer binds to the Ig-κB DNA target in a pH
dependent manner, with the highest affinity between pH 7.0 and 7.5. A strong salt
dependent interaction between Ig-κB and the p50/p65 heterodimer is observed, with
optimum binding occurring at monovalent salt concentrations below 75 mM, with
binding becoming virtually non-specific at a salt concentration of 200 mM. Binding of
the heterodimer to DNA was unchanged across a temperature range between 4 to 42 °C.
The sensitivity to ionic environment and insensitivity to temperature indicate that NF-κB
p50/p65 heterodimers form complexes with specific DNA in an entropically driven
manner.
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Introduction:
The Rel/NF-κB transcription factors constitute one of the most important families
of regulatory transcription factors. Members of the Rel/NF-κB family are essential for
diverse biological functions such as the regulation of innate and adaptive immunity,
development, and apoptosis in a wide array of eukaryotes from Drosophila to man (1-4).
Like most transcription factors, dimers of NF-κB proteins modulate transcription by
directly binding to enhancer sequences located in the regulatory regions of numerous
genes. These DNA sequences are collectively known as κB DNA sequences. In
mammals, the Rel/NF↑κB dimers arise from five polypeptides, p50, p52, p65, cRel and
RelB. The most abundant of these dimers are the p50/p65 heterodimer and the p50
homodimer. The existences of some, but not all, of the other possible dimers have been
shown to exist in cells.
The NF↑κB family can be divided into two subgroups based on the presence or
absence of an activation domain. p50 and p52 do not contain a distinct activation domain
and belong to class I. The other three members constitute the class II sub family. It is
generally believed that the homodimers of p50 and p52 and the p50/p52 heterodimer
function as transcriptional repressors. The remaining combinations of dimeric NF↑κB
proteins, containing at least one monomer of p65, cRel, or RelB, act as activators.
Rel/NF-κB proteins share a region that shows over 45% sequence similarity
across the entire family. This region, known as the rel homology region (RHR), is
responsible for DNA binding and subunit dimerization. High-resolution x-ray crystal
structures of RHRs are known for four homodimers, p50, p52, p65 and cRel in their
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DNA-bound conformations(5-8). These structures show that, as expected, Rel/NF-κB
proteins also share similar structures. Most of the RHR is folded into two
immunoglobulin-like domains connected by a 10 amino acid linker; the N-terminal
domain confers sequence specificity in DNA binding and the C-terminal domain is
involved in dimerization as well as DNA backbone recognition. These structures show
that, unlike most other transcription factors, NF-κB dimers do not use any secondary
structure for contacting DNA. All the DNA contacting residues emanate from loops
connecting secondary structures. Crystal structures of these complexes suggest that in
their free form the N-terminal domains should be flexible with respect to the
dimerization domain.
Recently, the NMR structures of a 16 bp duplex DNA containing the κB target
from the HIV-LTR, which is identical to the κB site in the immunoglobulin light chain κ
gene (Ig-κB), and a mutant form of the target site that abolishes DNA binding have been
solved (9,10). These show that the phosophodiester bonds of the sugar-phosphate
backbone of the native duplex preferentially adopt a distinct conformation in the 5’ and
3’ regions of the κB site. The mutant site is incapable of adopting the native DNA’s
conformation, suggesting that κB-DNA sequence also plays a role in NF-κB/DNA
complex formation. The combined flexibility of the NF-κB dimers and their target DNA
allows NF-κB to adopt multiple conformations in a promoter specific manner.
Among NF-κB’s most well characterized DNA
targets are the κB DNA sites of the immunoglobulin light chain κ gene and HIV-LTR
(Ig-κB) and the interferon β gene (IFN-κB). A crystal structure of the NF-κB p50/p65
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heterodimer bound to the Ig-κB DNA target has been completed (11). In order to
understand the mechanism of DNA binding by NF-κB, thermodynamic parameters need
to be determined for various NF-κB dimers and κB DNA target sites. In this study we
have analyzed binding of Ig-κB and IFN-κB DNA targets with three different NF↑κB
dimers: p50 homodimer, p65 homodimer, and p50/p65 heterodimer, using both a gel
mobility assay and a solution based fluorescence anisotropy assay. The binding of NF-
κB p50/p65 heterodimer to Ig-κB DNA has been further tested for its dependence on pH,
salt, and temperature.
Materials and Methods:
Materials. 5′ Fluorescein labeled oligonucleotides were purchased from the Keck
Oligonucleotide Synthesis Facility at Yale University. Unlabeled oligonucleotides were
synthesized using a Milligen/Biosearch Cyclone Plus DNA Synthesizer. Electrophoresis
and fluorescence polarization chemicals were purchased from Fisher Scientific, except
for MOPS and CAPSO buffers, which were purchased from Sigma. T4-polynucleotide
kinase was purchased from New England Biolabs. [γ↑32P]-ATP and poly(dI-dC) carrier
DNA were purchased from Amersham Pharmacia Biotech. The Nucleotide Removal Kit
was purchased from Qiagen. All proteins were purified according to the following
references:(5,6,8,12).
Site-Directed Mutagenisis. Monomeric p50 and p65 mutants were generated
through a two-step PCR strategy using internal primers. The N- and C-terminal primers
for both mutants were the same as those used for the wild type proteins (12). For the p50
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Y267D/L269D mutant the internal primers used were:
N-terminal: 5’-GGGGAGGAGATTGATCTAGATTGTGACAAGGTTC-3’
C-terminal: 5’-GAACCTTGTCACAATCTAGATCAATCTCCTCCCC-3’.
For the p65 F213D/L215D mutant the internal primers used were:
N-terminal: 5’-GGGGATGAGATCGATCTAGATTGCGACAAGGTG-3
C-terminal: 5-CACCTTGTCGCAATCTAGATCGATCTCATCCCC-3.
Electrophoretic Mobility Shift Assay (EMSA). The oligonucleotide used for the
EMSAs was 5′-TCTGAGGGACTTTCC TGATC-3′, which contains the heterodimer
target site Ig-κB (underlined). This oligonucleotide was annealed to its complimentary
strand and end radiolabeled with 32P using T4-polynucleotide kinase and [γ↑32P]-ATP.
The labeled DNA was then purified using a Nucleotide Removal Kit. Binding reactions
were performed using constant DNA concentration (100 pM for the p50/p65 heterodimer
or 1 nM for the p50 and p65 homodimers) in 20 µL of binding buffer [20 mM Tris (pH
8.0), 50 mM NaCl, 1 mM MgCl2, 1 mM DTT, 1 µg poly(dI-dC) DNA, 0.25 mg/mL
bovine serum albumin, and 5% glycerol (v/v)] at 20°C for 30 minutes. The reaction
mixes were then loaded onto a 6% 0.25X TBE polyacrylamide gel and run for 2 hrs at
120 V. The gels were then dried and exposed to a phosphor image storage plate for a
Molecular Dynamics Storm 860 scanner, which was used to visualize the gels. Gels were
quantified using ImageQuant version 1.2 from Molecular Dynamics.
Fluorescence Anisotropy Assay (FAA). Two 5′ fluorescein labeled
oligonucleotides were used for these assays. A 39-mer, containing the Ig-κB target site
from the HIV-LTR (underlined) 5′-GATCGCTGGGGACTTTCCAGGGAGGCGTG
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GCCTGAGTCC-3′ and a 17-mer containing the IFN-κB site (underlined) 5′-
AGTGGGAAATTCC TCGG-3′. Both were annealed to their complimentary strands prior to use.
p50, p65, or the p50/p65 heterodimer were then serially diluted into 0.6 mL binding
reactions. After the dilutions, each tube was blanked and the labeled oligonucleotides
were added at constant concentration (100 pM, 1 nM, or 10 nM for p50/p65, p50, and
p65 respectively), and the reactions were incubated at 20°C for 45 minutes to 1 hour. For
the monomeric p50 (Y267D/L269D) reactions were set-up using a hairpin
oligonucleotide with the sequence 5’-AAAGTCCCCACCCCCTGGGGACTTT-3’
containing the p50 Ig-κB half-site from the HIV-LTR (underlined) added to the titrated
protein at 1 nM. The anisotropy value of each reaction tube was then measured using a
Beacon 2000 Fluorescence Polarization Analyzer (Panvera, WI). Buffers used in the
assays were as follows: Temperature dependence – 20 mM Tris (pH 8.0), 50 mM NaCl;
Salt Dependence – 20 mM Tris (pH 8.0), and 0, 25, 50, 75, 100, 150, and 200 mM NaCl
or KCl; pH dependence – 20 mM buffer (pH 6.0, 6.2, and 6.5 MES, pH 6.8 and 7.0
MOPS, pH 7.5, 8.0, and 8.5 Tris, and pH 9.0 CAPSO). All salt and pH experiments were
carried out at 37°C, temperature dependence assays were carried out at 4, 8, 16, 22, 30,
37, and 42°C.
Data Analysis. First, the fraction of DNA bound in each reaction was determined.
For EMSA the fraction bound was determined by integrating the area under the peaks for
each band and dividing the area of the bound DNA band by the total area of the bound
and free DNA bands. For the FAAs fraction bound was calculated by subtracting the
experimentally determined polarization value for free DNA from the observed
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polarization value for each data point, then dividing each by the polarization value for
NF-κB saturated DNA. The apparent dissociation constant (Kapp) was determined
graphically as the point where fraction bound equals 0.5. Data from all homodimer
experiments were globally fit to a cooperative binding model using the following
equation:
([NF-κB]/Kmonomer) + ([NF-κB]2/aK2monomer)
Fraction DNA Bound (FB) = __________________________________
1 + (2[NF-κB]/Kmonomer) + ([NF-κB]2/aK2monomer) (1
)
Where Kmonomer is the equilibrium dissociation constant of one monomer interacting
with its DNA half site and a is a cooperativity factor for the binding of the second
monomer. The statistical factor of 2 in the denominator arises due to the two equivalent
monomer-binding sites available prior to the binding of the first monomer.
Equation one was modified to determine the cooperativity of p50/p65 binding as follows:([NF-κB]/Kmonomer(p65)+ [NF-κB]/Kmonomer(p50)) + ([NF-κB]2/aKmonomer(p65)
Kmonomer(p50))
F.B. = _________________________________________________________1 + (2[NF-κB]/Kmonomer(p65) + 2[NF-κB]/Kmonomer(p50)) + ([NF-κB]2/aKmonomer(p65)
Kmonomer(p50)) (2)
Where Kmonomer(p65) is the affinity of the p65 monomer for its DNA half site,
Kmonomer(p50) is the affinity of the p50 monomer for its DNA half site, and a is a
cooperativity factor for the binding of the second monomer.
Kapp values from salt dependence FAAs were then fit to the following models to
determine the number of cations and H2O molecules displaced upon NF-κB binding.
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log(Ka,app) = log(K0) Z*ψ*log[NaCl] (3)
Where K0 is the extrapolated apparent Ka at 1 M NaCl concentration, Z is the number of
cations displaced, ψ is the number of cations thermodynamically bound to each DNA
backbone phosphate previously determined to be 0.88 (13).
log(Ka,app) = log(K0) – A*log[NaCl] + B*0.016*[NaCl] (4)
Where K0 is the same as in equation 3 and A is the total ion (cation and anion)
stoichiometry released. B is the number of H2O molecules released upon binding. The
equation is a simplified version of the equation used by Ha, et al. (14) from O’Brien et al.
(15).
Results:
Binding affinities of NF-κB p50 homodimers for κB-DNA targets. We used only
the RHR portions of both p50 and p65 subunits for binding experiments. The RHR of
p50 and p65 homodimers and the p50/p65 heterodimer have been over-expressed and
purified from over-expressing E. coli clones. We have measured the DNA binding of the
p50 homodimer using a gel mobility shift assay. The DNA probe used for this assay was
a 20-mer duplex DNA containing a centrally located 10-bp Ig-κB site. Figure 1 shows
the free and bound DNA for the p50 homodimer, as well as the p65 homodimer and
p50/p65 heterodimer. The data fit best to a cooperative binding model (Equations 1 and
2) describing two subunits assembling sequentially on the DNA. Figure 2 shows the data
for NF-κB p50 homodimer binding to Ig-κB DNA fit to the cooperative model.
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The affinity of NF-κB p50 homodimer for Ig-κB DNA was further examined
using fluorescence anisotropy assays. The binding conditions were similar to those for
gel mobility shift assays. This solution-based assay circumvents the problems of
artifactual dissociation of a protein/DNA complex as it migrates through a gel matrix.
Figure 3 shows titrations of Ig-κB DNA with the three different NF-κB dimers. The
total fluorescence intensity did not change during the assay, indicating that anisotropy
signals were not due to changes in fluorescence lifetime or other experimental artifacts.
To determine the time required for each reaction to reach equilibrium anisotropy a kinetic
experiment was performed in which each sample was measured at different times until no
change in anisotropy was observed. Accordingly, sufficient time was allowed before
recording the final anisotropy value. Control experiments showed that the presence or
absence of carrier DNA poly dI-dC (2µg/mL) and glycerol (5%) had no effect in binding.
Additionally, we have verified the activities of each protein sample used for the assays
by measuring anisotropy at various stoichiometric protein/DNA ratios (over a range from
20/1 to 1/20). We observe that approximately 85% of the NF-κB in each preparation is
fully active (data not shown).
Anisotropy profiles for each binding experiment show an initial plateau indicating
unbound DNA, followed by a rise in anisotropy as proteins bind to DNA, and a final
plateau showing saturated binding. As mentioned previously for EMSA experiments, the
binding data for anisotropy experiments fit the cooperative model. The apparent
dissociation constants obtained from these anisotropy experiments are very similar to
those found in EMSA experiments. Next we measured the affinity of the p50 homodimer
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for the IFN-κB site. These results are presented in Table 1. Our results show that the
NF-κB p50 homodimer has similar affinities for both Ig-κB and IFN-κB DNA targets.
To further investigate the cooperative nature of binding it is important to
determine the affinity of a monomer for its κB half-site target. The cooperative model
predicts that the monomers bind sequentially to their DNA half sites, with the second
monomer binding to its half site with much higher affinity due to its interaction with the
pre-bound first subunit. In order to test this hypothesis we created a monomeric mutant
p50 using information from crystallographic models and biochemical studies of the p50
homodimer (16,17). The tyrosine at position 267 and leucine at position 269 are critical
for subunit dimerization of p50. These residues are located away from the protein/DNA
interface and are not involved in DNA contacts. We have created and purified the
Tyr267Asp/Leu269Asp double mutant to homogeniety. Size exclusion chromatography
clearly shows that the mutant p50 is monomeric even at a high protein concentration (5
mg/mL, Figure 4A). Binding experiments have been performed with a DNA probe that
bears only a single half site (Figure 4B). This eliminates any possible binding of two
molecules of mutant p50 monomer in a non-cooperative manner. The p50 monomer
binds to this target with an affinity of 210 nM (Kmonomer). Using this value in Equation
1 yields a cooperativity factor of 0.050, suggesting that the second subunit binds to the
DNA with 20-times higher affinity compared to the first monomer, 10.5 nM. The
apparent equilibrium constant (Kapp) for 2 monomers binding to DNA is 2.2x10-15 M2.
However, in the pH, salt, and temperature studies we focus on the overall Kapp, the
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concentration where half of the DNA is bound, which represents the affinity of the entire
NF-κB dimer/DNA complex.
Binding affinities of NF-κB p65 homodimer for κB-DNA targets. We have
performed analogous binding experiments with p65 homodimer for both Ig-κB and IFN-
κB DNA targets (table 1). Binding with Ig-κB DNA has been tested through both EMSA
and polarization experiments at pH 8.0. EMSA experiments show that p65 homodimer
binds the DNA with an affinity of 464 nM and fluorescence anisotropy gives a value of
341 nM. At pH 7.5 the p65 homodimer binds Ig-κB more tightly, with an affinity of
approximately 150 nM. We also observe that the binding affinity of p65 homodimer
IFN-κB DNA is similar to its affinity for Ig-κB DNA (414 nM vs. 341 nM at pH 8.0).
The nature of binding isotherms also suggests a cooperative mode of binding. We,
therefore set out to determine the cooperativity of interactions between p65 and κB
targets. We have created monomeric p65 by mutating Phe231 and Leu233 located at the
subunit interface to aspartic acid. These two residues are located at the equivalent
positions to that of Tyr267 and Leu269, respectively, in p50. We over-expressed,
purified and tested the oligomeric nature by size exclusion chromatography. As expected
this double mutant was monomeric. However, the mutant tends to aggregate, preventing
us from using it in binding experiments. We have over-expressed the monomeric DNA
binding N-terminal domain of p65. X-ray crystal structures show that this fragment
provides most of the sequence-specific binding of target DNA while lacking the
phosphate backbone contacts contributed by the dimerization domain. This fragment
binds a κB half site with an affinity of approximately 1,800 nM at pH 7.5. Considering
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this as the absolutely upper limit, and the affinity of p50 RHR monomer, 210 nM, being
the lower limit, we fit the Kmonomer and a values in equation 1, with Kmonomer
constrained to be less than 1,800 nM, to the p65 RHR data at pH 7.5. This yielded a
Kmonomer of 379 nM and a cooperativity value (a) of 0.16 suggesting that the second
molecule of p65 monomer binds the second half site of DNA with 6 to 7-fold higher
affinity.
Binding affinities of p50/p65 heterodimer for κB-DNA targets. In addition to the
homodimers, we have also extensively studied the NF-κB p50/p65 heterodimer. We
have determined the apparent binding affinities of the heterodimer for the Ig-κB DNA
target using both gel mobility shift and fluorescence anisotropy assays. Similar to the
results observed for the homodimers, we do not see any difference in binding affinities
between these two methods. The Kapp values of the p50/p65 heterodimer for Ig-κB are
approximately 20nM at pH 8.0 in both assays. We observe that the heterodimer binds to
IFN-κB with a relatively lower affinity compared to its Ig-κB targets. The apparent
dissociation constants of Ig-κB and IFN-κB for the heterodimer are 19nM and 27nM,
respectively at pH 8.0. Our results show that the p50/p65 heterodimer has the highest
affinity for Ig-κB DNA, p50 homodimer binds with intermediate affinity, whereas p65
shows the lowest binding affinity.
The nature of binding isotherm clearly indicates that the heterodimer binds κB
targets with highest cooperativity of the three dimers tested here. Using the equilibrium
binding constants of the p50 and p65 monomers to their DNA half sites, we observe that
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the cooperativity of the heterodimer is 0.0017 (the second subunit binds 500 times tighter
than the first) using Equation 2.
Effect of pH on complex formation. To test the pH sensitivity of the interactions
between the heterodimer and Ig-κB DNA we performed binding experiments at pH 7.5
and 8.0 using fluorescence anisotropy assay. These experiments showed approximately
2-fold higher affinity at pH 7.5 than at pH 8.0. To observe if both the homodimers also
exhibit a similar binding trend, the homodimers were subjected to similar experiments.
The homodimers did not show a large difference in affinities as was observed for the
heterodimer. Nevertheless, both these dimers did show slightly higher affinities at pH 7.5
compared to pH 8.0. To further investigate the pH dependence of equilibrium binding
constants of the heterodimer/DNA complex, we tested a wider pH range. The apparent
binding constants were determined for the heterodimer/Ig-κB DNA complex at seven
different pH values ranging from 6.0 to 9.0. At pH 6.0 no change in anisotropy was
observed due to background noise, but a change of intensity was recorded with increases
in protein concentration. Therefore, the binding constant was determined from the plot of
increase of fluorescence intensity vs. protein concentration. As shown in Figure 5,
apparent binding constants vary only roughly 2-fold between pH 6.8 and 8.0, with the
highest affinity is observed at pH 7.0. Below pH 6.8 binding constants increase
significantly as pH decreases. Similarly, Kapp increases as pH increases with a five to
six-fold increases of the binding constant at pH 9.0, the highest pH used in the assay.
Effect of salt on complex formation. The dependency of the apparent K for the
p50/p65 heterodimer/Ig-κB DNA complex on salt concentration was determined at pH
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8.0 and 37°C using the anisotropy method. As shown in Table 2, the Kapp of this
complex is highly dependent on the salt concentration. Kapp remained unchanged
between salt concentrations from 0 to 50mM. Whereas Kapp is approximately 20nM at
50mM NaCl it is reduced by a factor of 3-4 at 100mM NaCl concentration. A two orders
of magnitude reduction in Kapp value is observed at 200mM salt concentration. FAA
experiments replacing NaCl with KCl produced no observable changes in the apparent
equilibrium constants. The salt effect on the heterodimer/Ig-κB DNA complex is shown
in a log-log plot of salt concentration vs. Kapp in Figure 6. The plot fits Equations 3 and
4 relating equilibrium binding constants to ion-water models at NaCl concentrations
where binding is salt-dependent. Log Kapp exhibits a linear dependence on log salt
concentrations from 75mM to 200mM. From the fit to these data points it appears that
between 5-6 ions and approximately 430 water molecules are released upon the
protein/DNA complex formation. The release of large numbers of water molecules is a
hallmark of specific, protein/DNA complex formation (18). Similar strong salt
dependency of apparent equilibrium binding constants (Kapp) on salt was also observed
for the p50 homodimer/IFN-κB DNA complex. Like the heterodimer/Ig-κB DNA
complex the binding constants do not change at salt concentrations between 0 to 50mM.
Above 75mM NaCl concentration p50/IFNβ-κB DNA complex is even more salt
dependent than the heterodimer. The binding constant is decreased over 200 fold at
200mM salt compared to 50mM salt concentration.
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Effect of temperature on complex formation. The dependence of Kapp on
temperature at constant salt concentration (50mM) and pH (7.5) was determined for the
heterodimer/Ig-κB DNA complex. The binding constants were measured at seven
different temperatures ranging from 4 degrees to 42 degrees C. The results are shown in
a plot of ln(Kapp) vs. temperature (Figure 7). We do not observe any temperature
dependence of apparent binding constants.
Discussion:
Over the last five years three dimensional x-ray structures of nine different
complexes of DNA-bound NF-κB dimers have been determined. These structures have
provided a wealth of information regarding how these closely related dimers make
contacts with their DNA targets. In order to understand how NF-κB dimers actually
recognize DNA an energetic profile of NF-κB/DNA interactions is essential. In this
study we have determined relative binding affinities of three NF-κB dimers, p50 and p65
homodimers and p50/p65 heterodimer for two different physiological targets. We have
also investigated the effects of monovalent salt concentration, pH and temperature on
DNA binding by the p50/p65 heterodimer.
Binding affinities. We have used two different methods to measure binding
affinities: gel mobility shift assay and solution-based fluorescence polarization assay.
Binding affinities obtained from both these assays are comparable for each of the three
NF-κB/DNA complexes tested: p50/p65/DNA, p50 homodimer/DNA, and p65
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homodimer/DNA complexes. The nature of the binding isotherms indicates a
cooperative mode of binding. The source of cooperation is likely to be the stepwise
binding of NF-κB monomers to DNA half-sites followed by subunit association through
the dimerization domains of each protein subunit. Indeed our thorough investigation of
binding by p50 to Ig-κB DNA clearly suggests that the dimer recognizes the target in a
highly cooperative manner. Our results also show that the major source of the
cooperativity is indeed the dimerization interactions between the two p50 subunits.
Although we could not perform the similar experiment with p65 due to the aggregation
problem of monomeric p65 RHR, binding affinity of p65 monomer was estimated to fall
between the DNA binding affinity of the N-terminal domain p65 and affinity of
monomeric p50 RHR. A binding affinity for p65 monomer for κB DNA of 379 nM is a
good estimate for two reasons. First, this value fits our data best (lowest standard errors).
Second, this value is roughly 2- fold lower than the p50 Kmonomer, which is expected,
because of extra DNA base contacts made by the p50 monomer. Using these Kmonomer
values for p50 and p65 in a cooperative model for heterodimer binding gives a
cooperativity factor of 0.0017. This suggests that the heterodimer binds the DNA much
more cooperatively than either of the homodimers. Nevertheless, the apparent
equilibrium binding constants provide the true affinity of the NF-κB dimer/DNA
complexes. The apparent dissociation constants obtained from our experiments are
somewhat higher than previous reports (19-23). Although we cannot explain the source
of discrepancies, it is important to note that different binding reaction conditions may
influence the relative affinity values.
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Based on the three dimensional structures of several NF-κB/DNA complexes
several important conclusions can be drawn. These complexes approximately bury 3000
to 3800 Å2 solvent exposed surface area, the dimers make 12 to 14 direct base-specific
hydrogen bonds with their DNA target and 25 to 40 non-specific hydrogen bonding
contacts with the backbone of DNA targets (11). None of these numbers are unusual
when compared with other complexes of dimeric transcription factor/DNA complexes.
Whereas no direct relationship exists between number of contacts between two complex
forming macromolecules and the affinity of such a complex, it is not unusual that NF-κB
binds DNA with nanomolar affinity like most other eukaryotic transcription factors.
Incidentally, NFAT, a NF-κB related transcription factor, is known to bind DNA with
much lower affinity. The amino terminal specificity domain of NFAT is structurally very
similar to the N-terminal domain of NF-κB and recognizes specific bases in almost
identical manner to that of NF-κB (24).
pH effect on binding. DNA binding by the NF-κB heterodimer was determined
as a function of pH. The apparent binding constants of the heterodimer/Ig-κB complex
were measured at eight different pH values ranging from pH 6.0 to 9.0 using appropriate
buffers. As presented in Figure 5, the interaction of protein with DNA is optimal
between pH 6.8 to 7.5. The affinity decreases below and above this pH range. However,
the affinity decreases more dramatically at low pH. It is likely that partial protonation of
certain residues such as Glu39 of p65 and Glu60 and His64 of p50 that are directly
involved in DNA contacts are responsible for this effect. Conversely, deprotonation of
DNA backbone contacting residues, Tyr36 and Cys38 of p65 and the corresponding
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Tyr57 and Cys59 of p50 reduce the affinity of protein for the DNA. Studies on the
dimerization affinity of the p50 homodimer show no pH effects on dimer stability over
the range of pH’s used in these assays (16). Thus, the pH dependence of affinity is due to
alterations of the amino acid residues that contribute directly to the NF-κB/DNA
interface.
Salt effect on binding. NF-κB p50/p65 heterodimer binds Ig-κB DNA in a
highly salt-dependent manner. Although no change in the binding constant is observed
at NaCl concentrations between 0 to 50 mM, an increase of only 100 mM NaCl reduces
the affinity by more than an order of magnitude. At 200 mM NaCl the heterodimer binds
Ig-κB practically non-specifically. Similar strong effects of salt on p50 homodimer
binding to IFN-κB DNA suggests that all NF-κB/DNA complexes are formed in a salt-
dependent manner. Additionally, the formation of p50 dimers in the absence of DNA is
not effected by the salt concentrations used here (16).
From the p50/p65 structure it appears that a significant fraction of the binding
affinity of NF-κB /DNA is likely to come from non-specific salt-bridges between the
DNA phosphate backbone and positively charged protein side chains. There are at least
20 such contacts observed between the heterodimer and Ig-κB DNA complex (11).
Additionally, from NMR and molecular modeling studies of the HIV-LTR Ig-κB DNA,
it appears that the dynamics of the phosphate backbone’s conformation in the 5’ and 3’
regions of the κB sequence play an active role in NF-κB recognition (25). Cooperative
interactions with other transcription factors may provide the higher level of specificity at
physiological salt concentrations, which is approximately 175 mM.
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It is interesting to note that during the original purification of the p50/p65
heterodimer it was observed that the protein bound almost as tightly to non-specific
oligonucleotide columns as to specific ones. NF-κB also eluted from the oligonucleotide
columns at much lower salt concentrations than other DNA-binding proteins (0.2 M and
0.4 M, respectively) (26). Our data predicts this weak binding at the salt concentrations
used and the low protein concentration of this initial purification. At this point it is still
unclear why NF-κB’s DNA binding behavior at low salt concentrations (0-50 mM)
differs from that higher concentrations.
Ha et al. (14) have successfully derived an equation describing the effects of
monovalent salt and water on DNA/protein complex formation (Equation 4), which has
been simplified by O’Brien et al. (15). Using this ion displacement model, we calculate
an A value of 6 ions (also the Z value from Equation 3) and a B value of 426 water
molecules released upon complex formation. The crystal structure of the complex shows
that upon association 3800 Å2 of solvent accessible surface area is buried (11).
Considering 9 Å2 as the surface area of a water molecule, theoretically 422 molecules of
water would be released from this complex.
Temperature effect of binding. The dependence of the apparent binding constants
on temperature at constant salt (50 mM NaCl) and pH (7.5) was determined. As shown
in Figure 7 apparent binding constants essentially remain unchanged at temperatures
ranging from 4°C to 42°C. This suggests that the intrinsic enthalpy change upon complex
formation is negligible. It therefore appears that the binding of Ig-κB DNA by NF-κB
p50/p65 heterodimer is an entropic process driven by the release of counterion and bound
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waters. This is not surprising for two reasons. First, release of a large number of water
molecules clearly favors entropy of binding. Second, crystallographic analysis of various
NF-κB/DNA complexes reveals that several DNA contacting amino acid side chains are
most likely pre-organized through interactions with each other. In fact the structures of
the dimerization domains of the p50 and p65 homodimers show that the DNA backbone
contacting residues contributed by the dimerization domain adopt similar conformations
in the unbound form as those found in their respective homodimer/DNA complexes (17).
These observations suggest that the ordering of amino acid side chains, and the resulting
loss of entropy, is minimal in the forming of NF-κB/DNA complexes.
X-ray crystallographic analyses of various NF-κB/DNA complexes have given a
strong foundation upon which to initiate thermodynamic studies of these complexes. In
this report we have shown qualitatively the relative binding behaviors of three NF-κB
dimers, p50, p65, and p50/p65, with two different DNA targets. We have further
investigated the role of pH, monovalent salt, and temperature on the ability of the
p50/p65 heterodimer to recognize Ig-κB DNA. More detailed analyses are essential to
determine the thermodynamic parameters of binding in more quantitative terms.
Acknowledgments – We would like to acknowledge Partho Ghosh, Simpson Joseph, and
the members of the G. Ghosh lab for critical reading of this manuscript, as well as the C.
Zucker lab for the use of the phosphor-imager and storage screens.
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293(1), 139-50
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Footnotes:
† This study was supported by NIH CA-71871 and fellowships from the Alfred P. Sloan
and Hellman foundations, SM is supported by a predoctoral fellowship from the
American Heart Association, and CP is supported by the UCSD Cellular and Molecular
Genetics Training Grant 2-T32-GM07240-24.
* Corresponding author: fax (858) 534-7042, telephone (858) 822-0469, e-mail
1 The abbreviations used are: bp, base pair; dC, deoxtcytidine; dI, deoxyinosine; DTT,
dithiothreitol; EDTA, Ethylenediaminetetraacetic acid; HIV-LTR, Human
Immunodeficiency Virus – Long Terminal Repeat; MES, 2-(N-
Morpholino)ethanesulfonic acid; MOPS, 3-(N-Morpholino)propanesulfonic acid;
CAPSO, 3-(Cyclohexylamino)-2-hydroxy-1-propanesulfonic acid; TBE, Tris borate
with EDTA.
Figure Legends:
FIG 1. Sample electrophoretic mobility shift assays of p50/p65 heterodimer, p50
homodimer, and p65 homodimers. (left to right) DNA concentration was held constant
in each lane and titrated with decreasing NF-κB concentrations. Arrows indicate the
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location of the NF-κB dimer/DNA complex and free duplex (ds) DNA.
FIG 2. NF-κB dimers bind DNA cooperatively. Semi-logarithmic plot of sample DNA
binding data from an electrophoretic mobility shift assay with p50 homodimer. Data
points are represented as l, and the cooperative fit (equation 1) is represented as a solid
line.
FIG 3. The p50/p65 heterodimer binds DNA tighter than either homodimer. Semi-
logarithmic plot of concentration (nM) of p50/p65, p50, and p65 vs. fraction DNA bound
from fluorescence anisotropy data for representative data sets. p50/p65 (l) binds tightest
followed by p50 (n), then p65 (p).
FIG 4. Dimerization is critical for cooperative DNA binding. A. Size exclusion
chromatography traces of the wild-type and Y267D/L239D p50 RHRs showing that the
mutant is monomeric, even at high protein concentration (5 mg/mL). B. Representative
data sets comparing p50 RHR (n) binding to p50 Y267D/L269D (o) on a semi-
logarithmic plot of concentration vs. fraction DNA bound.
FIG 5. pH dependence profile of p50/p65. The apparent dissociation constant (Kapp,
nM) was measured between pH 6.0 and 9.0, with the lowest Kapp at pH 7.0. Error bars
represent one standard deviation from the average observed value from 3 separate FAAs
at each pH.
FIG 6. DNA binding by p50/p65 is strongly salt dependent. Log of average apparent
association constants (M-1) is plotted vs. log NaCl concentration. Log 0, 25, and 50 mM
NaCl are open symbols, and Log 75, 100, 150, and 200 mM NaCl are solid symbols. The
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error bars represent one standard deviation from the average observed value. The curve
was the fit of the NaCl dependent data points (75mM NaCl and above, solid points) to
determine the number of cations and H2O molecules released upon binding, 6 and 426
respectively.
FIG 7. p50/p65s binding to DNA is temperature independent. The average of the natural
log of Kapp (in M) is plotted vs. temperature (from 4-42°C), with error bars representing
one standard deviation from the average of measured values. The change in temperature
has no observable effect on the binding constant of p50/p65.
Tables:
Table 1. NF-κB binding to Ig-κB and IFN-κB DNA. Apparent equilibrium dissociation
constants (Kapp) from fluorescence anisotropy assay (FAA) experiments for NF-κB
binding to Ig-κB and IFN-κB DNA and from electrophoretic mobility shift experiments
on Ig-κB. Errors for K (app and monomer) values are the standard deviation from the
reported average of a minimum of three independent experiments, except the Kmonomer
value for p65, which was derived from fitting to equation 1 (see text). The cooperativity
factors (a) were also derived from global fits using equation 1 for p50 and p65
homodimer and equation 2 for the p50/p65 heterodimer. As such, the reported errors for
these values are the standard errors of the fits.
Ig-κB (FAA-pH 7.5)
Kapp(nM) Kmonomer (nM) a
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p50 54.3 ±1.1 210 ± 22.6 5.0 x 10-2 ± 3.7 x
10-3
p65 150.7 ± 29.2 379 ± 50.6 1.6 x 10-1 ± 4.2 x
10-2
p50/p65 12.8 ± 2.2 1.7 x 10-3 ± 1.6 x 10-4
Kapp(nM)
Ig-κB (EMSA-pH 8.0) Ig-κB (FAA-pH 8.0) IFN-κB (FAA)
p50 85.3 ± 0.6 84.1 ± 16.2 49.2 ± 11.4 (pH 7.5)
p65 464.1 ± 53.0 341.1 ± 43.9 414.9 ± 24.2 (pH 8.0)
p50/p65 16.8 ± 5.6 19.6 ± 3.8 27.3 ± 4.6 (pH 8.0)
Table 2. NaCl dependence of p50/p65 heterodimer binding to Ig-κB DNA.
Fluorescence anisotropy assays at pH 8.0, 37°C were used to determine the apparent
equilibrium constants at NaCl concentrations between 0 and 200 mM. Errors are the
standard deviation from the average of at least three independent measurements at each
salt concentration.
[NaCl] (mM) Kapp (nM)
0 15.1 ± 8.8
25 17.6 ± 7.7
50 19.6 ± 1.4
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75 38.3 ± 9.9
100 68.9 ± 9.7
150 307.4 ± 4.1
200 1445.0 ± 33.6
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Christopher B. Phelps, Lei Lei Sengchanthalangsy, Shiva Malek and Gourisankar GhoshMechanism of kappaB DNA binding by Rel/NF-kappaB dimers
published online May 23, 2000J. Biol. Chem.
10.1074/jbc.M003784200Access the most updated version of this article at doi:
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