Thermodynamics reveal that helix four in the NLS of NF-kappaB p65 anchors IkappaBalpha, forming a very stable complex

Abstract

IkappaBalpha is an ankyrin repeat protein that inhibits NF-kappaB transcriptional activity by sequestering NF-kappaB outside of the nucleus in resting cells. We have characterized the binding thermodynamics and kinetics of the IkappaBalpha ankyrin repeat domain to NF-kappaB(p50/p65) using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). SPR data showed that the IkappaBalpha and NF-kappaB associate rapidly but dissociate very slowly, leading to an extremely stable complex with a K(D,obs) of approximately 40 pM at 37 degrees C. As reported previously, the amino-terminal DNA-binding domain of p65 contributes little to the overall binding affinity. Conversely, helix four of p65, which forms part of the nuclear localization sequence, was essential for high-affinity binding. This was surprising, given the small size of the binding interface formed by this part of p65. The NF-kappaB(p50/p65) heterodimer and p65 homodimer bound IkappaBalpha with almost indistinguishable thermodynamics, except that the NF-kappaB p65 homodimer was characterized by a more favorable DeltaH(obs) relative to the NF-kappaB(p50/p65) heterodimer. Both interactions were characterized by a large negative heat capacity change (DeltaC(P,obs)), approximately half of which was contributed by the p65 helix four that was necessary for tight binding. This could not be accounted for readily by the small loss of buried non-polar surface area and we hypothesize that the observed effect is due to additional folding of some regions of the complex.

Figure 1

A) Ribbon diagram showing the x-ray crystal structure of the NF-κB/IκBα complex. IκBα is colored blue. The NF-κB is colored to show the p50 subunit in green, the p65 RHR and dimerization domain in red and the p65 NLS and NLS extension in violet. This figure was created using PyMOL v. 0.97 (http://pymol.sourceforge.net/) for the PDB coordinates (PDB accession code: 1nfi) . B) Schematic to show the domain structure of the NF-κB p50 (green) p65 (red) heterodimer.

Figure 2

Summary of SPR data collected for the NF-κB/IκBα interaction. In all experiments the NF-κB was immobilized to a streptavidin chip by a biotin tag on the p65 N terminus and IκBα was flowed over at a flow rate of 50 μl per minute. A) Sensorgrams of 0.23 to 4.0 nM IκBα flowed over NF-κB(p50_248-376/p65₁₉-₃₂₅) at 37 °C. B) Sensorgrams of 0.87 to 6.6 nM of IκBα flowed over NF-κB(p50_248-350/p65 _190-321) at 37 °C. C) Sensorgrams of 0.244 to 250 nM of IκBα flowed over NF-κB(p50_248-350/p65 _190-321) at 25 °C. D) Binding isotherm of 50 μM NF-κB(p50_248-376/p65₁₉₀-₃₂₁) titrated in 5μl injections into 3 μM IκBα. The buffer was 150 mM NaCl, 10 mM MOPS, pH 7.5, 0.5 mM EDTA, 0.5 mM sodium azide and the temperature was 31 °C. Data were analyzed using a model for a single set of identical binding sites after the heats of dilution of NF-κB into buffer were subtracted. K_D,obs was 2.2 nM ± 0.6 nM, and the stoichiometry was 0.9. The large error in K_D was due to the high `c' value for the interaction, where c is defined by Wiseman et al. and it is likely that affinity is significantly underestimated.

Figure 3

Summary of SPR data for the NF-κB(p50 _248-350/p65 _190-304) from which the p65 helix four (305-321) was deleted. A) Sensorgrams of 9.8 to 5000 nM concentrations of IκBα flowed over NF-κB at 25 °C kinetic analysis using a simple 1:1 model yielded a K_D,obs of 460 ± 120 nM, B) equilibrium analysis yielded a K_D,obs = 410 ± 100 nM C) sensorgrams of 9.8 to 5000 nM concentrations of IκBα flowed over NF-κB 15 °C. Kinetic analysis yielded a K_D,obs = 158 ± 12, D) equilibrium analysis yielded a K_D,obs = 210 ± 93 nM. The equilibrium response was plotted against the IκBα concentration and a line was fit to R = K_A × [IκBα].R_max/(K_A.[IκBα]+1) where R is the equilibrium response at a specific IκBα concentration, R_max is the response at saturation of the ligand on the chip and K_A = 1/K_D.

Figure 4

ITC binding isotherms for NF-κB binding to IκBα in 150 mM NaCl, 10 mM MOPS, pH 7.5, 0.5 mM EDTA, 0.5 mM sodium azide at 30°C. Experiments were carried out in triplicate and data were analyzed using a model for a single set of identical binding sites after the heats of dilution of NF-κB into buffer were subtracted. A) NF-κB(p50 _248-350/p65 _190-304), K_D,obs = 333 ± 21 × 10^-9 M; n = 1.1 ± 0.01 B) NF-κB(p50 _248-350/p65 _1-304) K_D,obs = 125 ± 8 × 10^-9 M; n = 0.99 ± 0.02 and C) NF-κB(p50 _39-363 /p65 _1-304) K_D,obs = 42 ± 6 × 10^-9 M; n = 0.84 ± 0.01.

Figure 5

ITC binding isotherm for the peptide fragment of NF-κB p65(289-320) binding to IκBα, in 150 mM NaCl, 10 mM MOPS, pH 7.5, 0.5 mM EDTA, 0.5 mM sodium azide at 30°C. Data were analyzed using a model for a single set of identical binding sites after the heats of dilution of NF-κB into buffer was subtracted a K_D,obs of 1.3 ± 0.09 × 10 ^-6 M and a stoichiometry of 1.1 ± 0.05 at 30 °C was determined.

Figure 6

A) Extrapolation of the thermodynamic characteristics of the NF-κB(p50 _248-350/p65_190-304)/IκBα interaction using the Gibbs-Helmholtz relation in the form: ΔG^o_bind (T_o) = ΔH(T_o)-T_o[[ΔH(T)-ΔG^o(T)]/T + ΔCpln(T_o/T], where ΔH is the enthalpy change, ΔG^o_bind is the free energy change upon binding, T_o = 298 K, and T is the absolute temperature. The ΔG_obs of the interaction is most favorable at approximately 20 °C when TΔS_obs = 0 (T_S). Since T_S occurs the midpoint of the experimental range of temperatures investigated the variation in K_D,obs appears small compared to experimental error. B) Temperature dependence of the ΔH_obs for the IκBα / NF-κB (p50 _248-350/p65 _190-321) (blue) and NF-κB-NLS truncation (p50 _248-350/p65 _190-304) (red) interaction. The slope of the line was used to determine a ΔC_P,obs of -1.30 ± 0.03 for the full length NLS and - 0.60± 0.03 for the NLS truncated NF-κB complex with IκBα.

Figure 7

Temperature dependence of the ΔH_obs for the IκBα/NF-κB (p50_248-350/p65_190-321) (blue) and NF-κB p65 homodimer (p65_190-321/p65_190-321) (green) interaction. The trend shows that binding of the p65 homodimer to IκBα has a consistently more favorable ΔH_obs relative to the heterodimer over the experimental temperature range.