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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References

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2. Baeuerle, P. A., and Henkel, T. (1994) Annu. Rev. Immunol. 12, 141-179

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4. Siebenlist, U., Franzoso, G., and Brown, K. (1994) Annu Rev Cell Biol 10, 405-

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5. Müller, C. W., Rey, F. A., Sodeoka, M., Verdine, G. L., and Harrison, S. C.

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6. Ghosh, G., van Duyne, G., Ghosh, S., and Sigler, P. B. (1995) Nature 373(6512),

303-310

7. Cramer, P., Larson, C. J., Verdine, G. L., and Muller, C. W. (1997) Embo J

16(23), 7078-90

8. Chen, Y. Q., Ghosh, S., and Ghosh, G. (1998) Nat Struct Biol 5(1), 67-73

9. Tisne, C., Hantz, E., Hartmann, B., and Delepierre, M. (1998) J Mol Biol 279(1),

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10. Tisne, C., Hartmann, B., and Delepierre, M. (1999) Biochemistry 38(13), 3883-

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11. Chen, F. E., Huang, D. B., Chen, Y. Q., and Ghosh, G. (1998) Nature 391(6665),

410-3

12. Chen, F., Kempiak, S., Huang, D., Phelps, C., and Ghosh, G. (1999) Protein

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13. Lohman, T. M., deHaseth, P. L., and Record, M. T. (1980) Biochemistry 19,

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3522-3530

14. Ha, J.-H., Capp, M. W., Hohenwalter, M. D., Baskerville, M., and Record, M. T.,

Jr. (1992) Journal of Molecular Biology 228, 252-264

15. O’Brien, R., DeDecker, B., Fleming, K. G., Sigler, P. B., and Ladbury, J. E.

(1998) Journal of Molecular Biology 279, 117-125

16. Sengchanthalangsy, L. L., Datta, S., Huang, D.-B., Anderson, E., Braswell, E.H.,

Ghosh, G. (1999) J. Mol. Biol. 289, 1029-1040

17. Huang, D.-B., Huxford, T., Chen, Y.-Q., and Ghosh, G. (1997) Structure 5(11),

1427-1436

18. Lundback, T., and Hard, T. (1996) Proceedings of the National Academy of

Sciences, USA 93, 4754-4759

19. Thanos, D., and Maniatis, T. (1992) Cell 71(5), 777-789

20. Urban, M. B., and Baeuerle, P. A. (1990) Genes Dev. 4(11), 1975-1984

21. Matthews, J. R., Nicholson, J., Jaffray, E., Kelly, S. M., Price, N. C., and Hay, R.

T. (1995) Nucleic Acids Res 23(17), 3393-402

22. Fujita, T., Nolan, G. P., Ghosh, S., and Baltimore, D. (1992) Genes Dev 6(5),

775-87

23. Duckett, C. S., Perkins, N. D., Kowalik, T. F., Schmid, R. M., Huang, E. S.,

Baldwin, A. S., Jr., and Nabel, G. J. (1993) Mol Cell Biol 13(3), 1315-22

24. Zhou, P., Sun, L. J., Dötsch, V., Wagner, G., and Verdine, G. L. (1998) Cell

92(5), 687-96

25. Tisne, C., Delepierre, M., and Hartmann, B. (1999) Journal of Molecular Biology

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293(1), 139-50

26. Ghosh, S., Gifford, A. M., Riviere, L. R., Tempst, P., Nolan, G. P., and Baltimore,

D. (1990) Cell 62(5), 1019-1029

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

gghosh@ucsd.edu.

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. 

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