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IκBα is an ankyrin repeat protein that inhibits NF-κB transcriptional activity by sequestering NF-κB outside of the nucleus in resting cells. We have characterized the binding thermodynamics and kinetics of the IκBα ankyrin repeat domain to NF-κB(p50/p65) using surface plasmon (SPR) resonance and isothermal titration calorimetry (ITC). SPR data showed that the IκBα and NF-κB associate rapidly but dissociate very slowly, leading to an extremely stable complex with a KD.obs of approximately 40 pM at 37 °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 the p65. The NF-κB(p50/p65) heterodimer and p65 homodimer bound IκBα with almost indistinguishable thermodynamics except that the NF-κB p65 homodimer was characterized by a more favorable ΔHobs relative to the NF-κB(p50p65) heterodimer. Both interactions were characterized by a large negative heat capacity change (ΔCP,obs), approximately half of which was contributed by the p65 helix four that was necessary for tight binding. This could not be readily accounted for 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.
In resting cells, NF-κB dimers with transcription activation potential are sequestered in the cytoplasm, interacting with a family of inhibitors of kappa B (IκBs) proteins 1. The nuclear factor kappa B (NF-κB) is a family of transcription factors that control cellular signaling, cellular stress responses, cell growth, survival, and apoptosis 2-5. It is known that at lest 58 viral or bacterial products, some 46 stress conditions and chemicals, and at least 32 cytokines and receptor ligands, as well as apoptotic mediators and mitogens, activate the NF-κB signaling system and subsequently the expression of more than 150 target genes 6. IκBα is capable of inhibiting many of the NF-κB family members including the most abundant p50/p65 but also other p65 and cRel-containing homo- and heterodimers (for a review see 3). Following the action of a large number of different stimuli, IκBα is phosphorylated, ubiquinylated, and degraded, freeing the NF-κB nuclear localization signal (NLS) which targets the NF-κB to the nucleus. In resting cells, the NF-κB/IκBα complex appears to be stable 7. A hallmark of the NF-κB/IκBα complex is its high stability in resting cells, with a recent estimate of the IκBα half-life at longer than 48 hrs (Hoffmann et al., unpublished data). This tight regulation is critical, as only a small amount of NF-κB, due to leaky inhibition, is sufficient to give gene expression. This is confirmed by a recent study which showed that upon stimulation, only a small fraction of cytoplasmic NF-κB enters the nucleus and this results in transcriptional activation 8.
The crystal structure of NF-κB(p50/p65) bound to IκBα was determined by two labs simultaneously (Figure 1A) 9,10. The structures reveal an extended protein-protein interface formed between IκBα and NF-κB, and provide a structural basis for the studies presented here. IκBα contains an ankyrin repeat domain, comprising six ankyrin repeat units, and a C terminal PEST region. Each ankyrin repeat consists of about 33 residues and adopts a fold containing a β- hairpin fold followed by two anti-parallel α- helices followed by a short loop. The ankyrin repeat domain of IκBα forms an extensive interface with the NF-κB heterodimer, forming contacts in multiple regions (Figure 1B), burying more than 4000 Å2 of surface area in the interface. The two proteins run antiparallel with ankyrin repeat 1 (AR1) of IκBα being capped by helix four of NF-κB p65 (residues 305-321). AR1 - AR3 contact helix three (residues 289-300) of p65, and AR3 - AR6 and the first part of the IκBα PEST sequence contact the NF-κB p50 and p65 dimerization domains. The PEST sequence also contacts the NF-κB p65 amino-terminal domain. Despite the structural and biochemical information locating the contacting surfaces, identification of the critical determinants of binding affinity has eluded us 11. In this study we address this important issue by taking apart the NF-κB/IκBα complex and carrying out a detailed study of the binding thermodynamics. We have previously investigated the interactions of the NF-κB dimers with IκBα by a fluorescence polarization competition assay 12 and an electrophoretic mobility shift assay (EMSA) 13. However, both techniques have potential limitations and so we have undertaken a more detailed study by direct binding assays using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC).
The binding thermodynamics and kinetics were measured for the interaction between IκBα and full-length NF-κB(p50/p65) heterodimer as well as with fragments of the NF-κB that were missing either the N-terminal domain or helix four from the nuclear localization sequence (NLS) region. The interaction of IκBα with the p65 homodimer was also measured. These experiments helped identify regions of NF-κB that are critical for tight binding affinity and they also revealed the thermodynamics that drive the interaction.
We have previously demonstrated that the free IκBα is highly dynamic in parts and only marginally stable in solution 14. In the work presented here, we also measured the temperature dependence of the binding interaction to investigate the possibility of a folding upon binding mechanism.
This study provides three important characteristics of the NF-κB/IκBα complex. First, the interaction has an extremely low dissociation rate which results in very high affinity and explains the long half-life observed in vivo. Second, the affinity of the interaction critically depends on the p65 NLS and this explains how the NLS is effectively sequestered by IκBα. Third, the thermodynamics help explain how IκBα can be very stable in complex with NF-κB and yet be rapidly degraded.
The binding kinetics and thermodynamics of the IκBα/NF-κB interaction were investigated by surface plasmon resonance (SPR). An N-terminal cysteine was introduced into NF-κB(p65) and the NF-κB(p50248-376/p6519-325 heterodimer was biotinylated and immobilized on a streptavidin chip. Sensorgrams reveal that the IκBα/NF-κB interaction achieved high affinity by a combined fast association rate and a slow dissociation rate at 37 °C (Figure 2A) leading to a KD,obs of 3.9 ± 0.3 × 10-11M (Table 1A). Experiments were carried out at 37 °C to speed up the rate of dissociation of IκBα, which was very slow at 25 °C, relative to the baseline drift of the instrument. Even at 37 °C the dissociation rate was very low, which meant that we did not even begin to cover one half-life of the dissociation event within the 20 min dissociation period. Thus, the dissociation rate we report represents the upper limit of the true kd and the true KD,obs for the complex may be even lower. Disruption of the NF-κB/IκBα complex required a 1 min pulse of 3M urea at 37 °C and 6 M urea at 25 °C for regeneration. It is noteworthy that the NF-κB(p50/p65) heterodimer was only tethered to the chip by the p65 domain, and the non-covalent interactions between the p50 and p65 were not disrupted during regeneration, suggesting that they are very strong. SPR binding data showed that two different IκBα constructs comprising residues 67-287 or 67-317 bound to NF-κB with almost identical kinetics and affinity (KD,obs of 3.9 ± 0.3 and 4.5 ± 1.0 × 10-11 M respectively) (Table 1A) demonstrating that the C-terminal residues of IκBα, which extend the “PEST” sequence, do not have a significant effect on binding. Further experiments were performed using IκBα 67-287 because it was less prone to aggregation than the longer construct.
The amino-terminal deletion construct, p65190-321 (Figure 1B), was generated to investigate the effect of the p65 amino-terminal domain on IκBα binding. The heterodimer was formed using an excess (1000x) of un-tagged p50 and sensorgrams were obtained as for the NF-κB(p50248-376/p6519-325), except that only a 1 min pulse of only 1.5M urea was required for regeneration of the free NF-κB on the chip (Figure 2B). Comparison of the results from NF-κB(p50248-376/p6519-325), which contained the amino-terminal domain of p65 (Table 1A) with results for NF-κB(p50248-350/p65190-321), lacking the amino-terminal domain revealed an approximately 2-fold decrease in ka and an approximate 8-fold increase in kd and a KD,obs of 3.2 ± 1.0 × 10-10 M (Table 1 B). The faster dissociation rate permitted data to be recorded at 25°C (Figure 2C) as well as 37°C (Table 1B) for this complex. As expected the dissociation rate was significantly slower at 25 °C resulting in a KD,obs of 0.91 ± 0.27 × 10-10M.
The C-terminal residues of NF-κB contain the NLS consensus sequence KRKR at residues 301 - 304. This region is flanked by two α-helices, helix three (289-300) and helix four (305-321). Together these motifs are known as the NLS domain (Figure 1B). We investigated the effect of helix four of the NLS on binding. Initial SPR experiments with NF-κB(p50248-350/p65190-304) revealed that the IκBα dissociated orders of magnitude faster; too fast to be quantitated at 37 °C. At 25 °C (Figure 3A) and at 15 °C (Figure 3C), the kd of IκBα was 100 ± 20 and 24 ± 0.95 × 10-3 s-1 respectively, compared to 0.12 ± 0.041 × 10-3 s-1 at 25 °C, for the longer construct (Table 1). Due to the weaker binding affinity, data were also analyzed by equilibrium analysis as shown in Figure 3B and 3D respectively. The results of both the kinetic and equilibrium analyses were in good agreement, confirming the reliability of both modes of analysis. Thus, deletion of helix four of the p65 NLS resulted in an increase of more than 1000 fold in kd. Including the decrease in ka, that was also observed, the affinity of IκBα for this complex was decreased by approximately 10,000 fold by the deletion of helix four of the NLS. Although the NF-κBs that contained helix four bound too tightly to accurately measure the KD,obs by ITC (Figure 2D), the NF-κBs that did not contain helix four bound weakly enough to allow direct comparison between the ITC and SPR data (Figure 4A, Table 2). The results showed good agreement between the two experiments, especially considering the complex nature of the two proteins involved. At 25 and 15 °C the KD,obs estimated by SPR was approximately four and three times weaker respectively than that measured by ITC. The small discrepancy probably results from an effect of the surface change of the carboxymethy dextran chip or of the linker used to immobilize the NF-κB. An electrostatic effect is more likely since the IκBα contains an excess of acidic residues in its C-terminus. The ITC also confirmed the observation that the effect of the N-terminal domain on IκBα binding affinity is small (Table 2, Figure 4B).
Since the NF-κB NLS appeared so important for binding, we generated synthetic peptides spanning the regions of the p65 NLS. Three peptides comprising residues 289-307, 289-314 and 289-320 were synthesized. Only the longest peptide comprising residues 289-320, showed any detectable binding. An expressed version of this peptide that could be purified more readily bound with a KD,obs of 1.3 ± 0.09 × 10 -6 M at 30 °C (Figure 5). The relatively tight binding affinity for such a short segment of NF-κB underscores the importance of the NLS for binding to IκBα. The observation that only peptides containing all of helix four bound IκBα again highlights the importance of this region of the NLS.
Binding of NF-κB to IκBα was investigated over a range of temperatures using ITC. This technique provides a direct measure of the observed enthalpy change of binding (ΔHobs) as well as the binding affinity. When carried out over a range of temperatures (at constant pressure), the observed heat capacity change (ΔCP,obs) and the full thermodynamic profile can be obtained. This was possible for the weaker binding IκBα/NF-κB(p50248-350/p65190-304) complex for which KD,obs and hence ΔGobs and T.ΔSobs as well as the ΔHobs could be accurately determined. The full thermodynamic profile for the interaction is shown in Figure 6A. The compensation of ΔHobs and T.ΔSobs leading to the relatively small change in ΔGobs over the physiological temperature range is apparent from this plot.
For the IκBα/NF-κB(p50248-350/p65190-321) complex containing the full NLS region, binding was too tight to permit determination of KD,obs directly. We were unable to employ a competition binding assay, due to the lack of a suitable competing ligand as used by Freire and co-workers 15,16 and so only the ΔHobs was determined for this interaction (Figure 6B). ΔHobs was linear as a function of temperature up to approximately 33 °C, which is close to the temperature (45 °C) at which IκBα unfolds to a soluble aggregate 14. Above 33 °C the slope curved as also reported by Ladbury and co-workers, for another interacting system 17. As discussed later on, only the linear region of the plot was analyzed yielding a ΔCP,obs = -1.30 ± 0.03 kcal mol-1 K-1 This is a very large negative ΔCP,obs even for such a large interaction interface suggesting additional contributions to the ΔCP,obs that were not anticipated from the structure alone.
Comparison of the temperature dependence of the formation of the two IκBα/NF-κB complexes, one with and one without helix four in the NLS, revealed that deletion of helix four resulted in a ΔCPobs of only -0.60 ± 0.03 compared to -1.30 ± 0.03 when helix four was present. This was a surprisingly large difference in ΔCP,obs for such a small deletion from the C-terminus of NF-κB. Confirming these results, the ΔCP,obs for the NLS peptide (residues 289-320) was measured to be -0.4 ± 0.04 (data not shown).
Binding of the NF-κB(p65/p65) homodimer was measured by SPR to permit comparison with the heterodimer binding experiments. The NF-κB(p65/p65) homodimer was immobilized on the chip by a single biotin linker on one of the p65 subunits. Analysis of the sensorgrams revealed binding characteristics almost indistinguishable from those for the NF-κB(p50/p65) heterodimer (Table 1D). The association rates were similar (2.9 ± 0.3 compared to 1.7 ± 0.32 × 106 M-1 s-1) as were the dissociation rates (1.6 ± 0.14 compared to 0.54 ± 0.10 × 10-3 s-1), leading to less than 2 fold decrease in binding affinity for the p65 homodimer (KD,obs = 0.54 ± 0.08×10-9 M) relative to the hetrodimer (0.32 ± 0.10×10-9 M). This was also confirmed by the ITC data from the weaker binding complexes (Table 3). For example, KD,obs for the analogous hetoerdimer (p5039-363/p651-304) and homodimer (p651-304/p651-304) /IκBα complexes were 42 ± 6.0 and 51 ± 16 respectively. ITC data also showed that IκBα binding to the NF-κB(p65/p65) homodimer is characterized by a large negative ΔCPobs (ΔCP,obs =-1.43 ± 0.05 kcal mol-1 K-1) similar to that observed for the NF-κB(p50/p65) heterodimer complex (Figure 7). Overall the binding of IκBα to the homodimer is characterized by a more favorable (negative) ΔHobs over the entire range of temperatures investigated, suggesting some compensation by a more unfavorable ΔS for the IκBα/homodimer complex.
The NF-κB signaling pathway is a complicated system involving the interactions of a large number of protein/protein and protein/DNA interactions 18. In order to have a complete understanding of the mechanism of regulation in this system, to understand the subtle effects mutations play, and to perhaps build models that predict the system-wide behavior 19, it is essential that the thermodynamics and kinetics of each of the interactions be accurately determined. We have used two direct binding experiments to measure the contributions of the individual domains of p65 to the IκBα/NF-κB interaction. When the entire RHR of p65 was present, the binding affinity was approximately 40 pM, some 25-fold tighter than previously reported 13. Previous measurements of the IκBα/NF-κB binding affinity using a fluorescence polarization competition assay and by an electrophoretic mobility shift assay, reported values of approximately 1 nM 9,13. Both the ITC and the SPR data show that the affinity of NF-κB heterodimers and homodimers containing full-length p65 is much tighter than the previously reported affinities. The agreement between the present data and previously reported data is much better for the weaker binding complexes, suggesting that the EMSA and fluorescence polarization assays were simply underestimating the high affinity of the complexes containing the entire RHR of p65. In resting cells, the IκBα/NF-κB complex is in the cytoplasm awaiting stimulation of the IκB kinase (IKK) that phosphorylates IκBα targeting it for ubiquitinylation and proteasome degradation 3. Attempts to measure the intracellular half-life of the IκBα/NF-κB complex in resting cells report that the complex is exceedingly stable 7. In IKK knock out cells, a half-life of at least 48 hours and perhaps longer has been estimated (A. Hoffmann, unpublished data). Our observations that the affinity of the IκBα/NF-κB complex complex is in the picomolar range, and that the dissociation rate constant is so slow as to be nearly irreversible, are consistent with the long half-life that has been observed in vivo. This is a very important finding because it helps to explain recent results showing that even upon activation, only a small fraction of the NF-κB is actually released from its inhibited state to translocate into the nucleus and turn on transcription 8. The extremely high affinity of the inhibited complex ensures that there is no “leakiness” in this transcriptional inactivation system, and allows for rapid transcriptional activation upon release of inhibition.
We previously employed a structure-based mutagenesis approach to probe the interaction interface of the NF-κB/IκBα complex 11. This investigation showed that when more than 20 residues of IκBα that form contacts with the NF-κB were mutated to alanine only marginal effects on KD were reported. This indicated a lack of a hot spot in the NF-κB/IκBα interface. Mutations that made the most significant effects were that of Tyr 181 to alanine, which makes contact to the p50 dimerization domain, and both Asp 71 and Asp 75 that make contact with Arg 304 located between helix three and helix four in the NF-κB p65 NLS. When both residues are mutated to alanine an approximate 8-fold effect on binding of NF-κB was observed 11. Based on the X-ray crystal structure, these residues seem to be important for anchoring down the NLS. This agrees with our finding that truncation of the NLS by deletion of helix four has a large effect on the KD of the complex, contributing one third (4.5 kcal per mol) of the ΔGobs at 25°C (Table 4). The total surface area buried by this region is only one sixth (818 Å compared to 4902 Å) of the total surface area buried by the whole RHR domain. The large contribution that this small region of p65 makes leads us to predict helix four within the p65 NLS (residues 305-320) will contain as yet unexplored specific contact residues important for the tight binding affinity of the NF-κB/IκBα interaction. This finding is biologically significant because tight binding of the NLS is expected to be important for sequestering the NLS from importin α. The KRKR sequence ending in Arg 304 forms the essential recognition motif for the importin complex 20-22 and atightly bound helix four would most likely prevent specific interaction of the importin with the NLS.
Consistent with previous results, our equilibrium and kinetic binding experiments have revealed that the ΔGobs and ΔCP,obs as well as the ka and kd for the NF-κB(p50p65) and the NF-κB(p65p65) binding IκBα are almost identical 13. Given that there are significant differences in the specific contacting residues between the p50p65 and the p65p65 with IκBα, there are two possible explanations for these results 9. The residues that are non-conserved in p50 relative to p65 might be isoenergetic or enthalpy-entropy compensated in binding IκBα. This possibility is supported by the significantly more favorable ΔHobs, for the NF-κB(p65p65)/IκBα complex, which is compensated by a more unfavorable ΔS contribution relative to the NF-κB(p50p65)/IκBα complex. However, in the absence of a crystal structure of the p65p65/IκBα complex, it is difficult to rationalize this observation in detail. Another possible explanation is that the p65 subunit of the p50p65 contributes the major proportion of the ΔGobs of binding and that the effect of subtle changes in contacting residues between the p50 subunit and the second p65 subunit in the homodimer/IκBα is not significant. This possibility is supported by our finding that the NLS of p65 contributes almost half of the overall affinity of the complex of NF-κB with IκBα.
These biophysical data are important because they allow one to rationalize the biological effects of experiments, such as the p50 and p65 knock out experiments in mice. Mice lacking the p50 subunit of NF-κB show no developmental abnormalities apparently because the p65 homodimer can replace all of the important functions of the normally more abundant p50p65 heterodimer 23. Conversely the p65 knock out is lethal in mice due to TNF-α induced apoptosis 24. In wild type cells, the NF-κB(p50p65) heterodimer is the most abundant form, but this most likely to be due to increased expression levels of the p50 subunit or the increased affinity if the NF-κB p50p65 heterodimer compared to other dimers. Our results suggest that IκBα has evolved to recognize primarily p65 so that it can regulate gene expression by both p65p65 homodimers and by p50p65 heterodimers.
The variation of ΔHobs for IκBα/NF-κB complex was linear over a temperature range of 17 to 33 °C (Figure 6A). At temperatures exceeding 33 °C, some negative curvature of the slope was observed (Supplementary Figure 1). A similar observation was recently been reported by Ladbury and co-workers for the phosphate 5 tetratricopeptide repeat (TPR) domain with Hsp90 17. These researchers were able to show that the nonlinearity was due to an increased proportion of the unfolded form of the protein leading to an additional folding coupled binding component of the ΔHobs. It is also likely that the IκBα is unfolding in our experiments since the unfolding transition for IκBα occurs around 45°C 14. Since the TPR domain reported by Ladbury and co-workers displayed reversible thermal unfolding behavior, the data were analyzed by simultaneously fitting unfolding data and the temperature dependence of ΔHobs measured by ITC. Since IκBα displays irreversible thermal unfolding to an aggregate, we were only able to fit the linear part of the data. Determination of the ΔCP,obs from the linear region of the curve has been shown to be a good approximation up to approximately 15 degrees below the unfolding temperature of the protein, as was the case in our studies 17. By this method, a relatively large ΔCP,obs for IκBα binding (-1.30 ± 0.03 kcal mol-1 K-1) was determined.
Many attempts have been made to relate ΔCP,obs to burial of polar and non polar surface area 25-28 and more recently including the burial of hydroxyl groups 29. However, discrepancies commonly arise when folding or induced fit is coupled to binding as in the case of many protein-DNA interactions 28 or due the presence of large numbers of buried water molecules 30,31, or networks of water molecules near the surface of the interface 32, or linked protonation effects 33,34. While we cannot rule out any of these potential mechanisms leading to such a large ΔCP,obs, a model of folding coupled binding of one or both binding partners could potentially account for the burial of non-polar surface area unanticipated from the structure of the complex. This is supported by our previous finding that parts of IκBα are highly dynamic in solution 14.
A coupled folding and binding mechanism is also consistent with the significant effect of removal of the fourth helix of p65 that caps ankyrin repeat 1 of IκBα. Indeed, removal of helix four in the NLS resulted in a halving of the ΔCP,obs. This was a surprisingly large difference in ΔCP,obs for such a small deletion from the C-terminus of NF-κB. One likely explanation is that helix four `caps' ankyrin repeat 1 of IκBα effectively stabilizing the entire IκBα molecule. If this is the case, the effect of helix four binding may be propagated by strengthening contacts between neighboring repeats of the ankyrin repeat domain of IκBα resulting in a significant folding stabilization. This finding also has potential biological significance. With the tight association we observe for the IκBα/NF-κB complex, it then becomes difficult to imagine how the inhibition can be rapidly released upon IKK activation and proteasome targeting. Now one can imagine that the entire complex is brought to the proteasome, and that degradation occurs beginning with the unstructured N-terminal domain of IκBα. Subsequent degradation of the first ankyrin repeat will then result in the loss of the interaction between helix four of p65 and IκBα, followed by a 1000- fold increase in the kd resulting in rapid dissociation of the complex. The rest of IκBα could then be readily degraded and the NF-κB could then translocate to the nucleus. Thus, helix four may represent the key to unlock the tight complex between NF-κB and IκBα allowing for rapid transcriptional activation upon cellular stimulation.
Direct binding experiments have revealed, for the first time, the almost irreversible binding of IκBα to NF-κB. This helps explain the extremely long in vivo half-life of the complex and the leak-proof nature of the inhibition. The thermodynamics of the NF-κB/IκBα interaction, together with our previous studies of the dynamics of the free IκBα, suggest that the IκBα/NF-κB interaction may involve coupled folding and binding. Deletion of helix four in the NLS of p65, which contacts the first ankyrin repeat of IκBα, dramatically increased the dissociation rate of the complex. This observation suggests a model whereby degradation of IκBα may only need to proceed through the first ankyrin repeat for complete and rapid NF-κB dissociation to occur.
Human IκBα (67-287) was expressed in the Pet 11a vector and purified as previously described 14. Two mutations were introduced into the p65 gene, N-terminal cysteine and a Cys38 to Ser to allow specific biotinylation only at the N-terminus. This gene will be referred to as p65. Murine NF-κB(p50248-376/p6519-325) was co expressed in a modified pET 29b vector and purified as previously described 35. Cells were harvested and sonicated then centrifuged at 12,000 RPM for 45 min and the supernatant loaded onto a tandem fast flow Q and fast flow S column (GE Healthcare) equilibrated in 50 mM NaCl, 25 mM Tris, pH 7.5, 0.5 mM EDTA. After loading the Q column was disconnected and protein fractions eluted from the S with a gradient from 50 to 400 mM NaCl. Fractions were collected and analyzed by SDS PAGE. Bands were visualized by silver staining and fractions with equal intensity of P50 and p65 were collected and pooled. The final step of the purification was size exclusion on an S-200 Superdex column equilibrated in 150 mM NaCl, 10 mM MOPS, pH 7.5, 0.5 mM EDTA, 0.5 mM sodium azide. The purified fractions were biotinylated by incubation with a 1:1 molar ratio of biotin PEO maleimide (Pierce Chemicals), at room temperature for 30 min and purified immediately by size exclusion chromatography on an S200 Superdex 16/60 column. Fractions containing the biotinylated heterodimer were collected and stored at -80 °C in 50 μl portions until used.
All other NF-κB constructs were expressed using a Pet 11a single expression vector and purified using a similar tandem column technique. Again, an N-terminal cysteine was introduced into the p65 as described above. For the NF-κB p65190-321 and NF-κB p50248-350 constructs, E. coli BL21 DE3 cells were grown to an OD of 0.6 and induced at room temperature for 16 hours with 0.1mM IPTG. For the NF-κB p65190-321 the column was equilibrated in 50mM NaCl, 25 mM MES pH 7.0, 10 mM BME, 0.5 mM EDTA. For the NF-κB p50248-350 the column was equilibrated in 50 mM NaCl, 25 mM MES pH 6.2, 10 mM BME, 0.5 mM EDTA and gradient of 50-300 mM NaCl, was run.
For the NF-κB p651-325, NF-κB p651-304 and NF-κB p5039-363 proteins, cells were induced with 0.5 mM IPTG. NF-κB p651-325 the columns were equilibrated in 50 mM NaCl, 25 mM MES, pH 6.5, 10 mM BME, 0.5 mM EDTA, a gradient was run from 50-450 mM NaCl. For NF-κB p651-304 50 mM NaCl, 25 mM Tris, pH 7.0, 10 mM BME, 0.5 mM EDTA and a 50-300 mM gradient was used. For NF-κB p5039-363 the columns were equilibrated in 50 mM NaCl, 25 mM MES, pH 6.2, 10 mM BME, 0.5 mM EDTA. Gradient was run from 50-700 mM NaCl.
Protein concentrations were determined spectrophotometrically from a scan of wavelengths 340-220 nm using the following ε280 values: 24180 for NF-κB p50248-350, 21620 for NF-κB p65190-321, 19060 for NF-κB p65190-304, 36980 for the NF-κB p651-325, 34420 for NF-κB p651-325, and 42100 for NF-κB p5039-363 homodimers and 30580 for NF-κB(p50248-376/p6519-325), 22900 for NF-κB(p50248-350/p65190-321), 21620 for NF-κB(p50248-350/p65190-304), 39540 for NF-κB(p5039-363/p651-325), 38260 for NF-κB(p5039-363/p651-304) heterodimers. For IκBα67-287 an ε280 of 12090 was used. Dimers were formed in vitro by incubating an equimolar amount for two hours at 25 °C and overnight at 4 °C prior to ITC experiments. For SPR experiments, the p65 was biotinylated as already described. A 1000 fold excess of un-biotinylated p50 (for heterodimers) or p65 (for homodimers) was incubated with biotinylated p65 and the equilibrated mixture was immobilized immediately on a streptavidin (SA) SPR chip.
The C-terminal residues 289-320 of NF-κB were expressed in the trp leader vector which contains an octa-histidine tag and thrombin cleavage sequence and drives small peptides into inclusion bodies 36. Inclusion bodies were solubilized 6M guanidine hydrochloride, 50mM Tris, pH 7.4 and the solubilized peptide was captured by Ni-NTA column equilibrated in the same buffer, and a gradient was run to a final concentration of 150 mM NaCl, 50 mM Tris, pH 7.4, 2mM CaCl2. The peptide was cleaved from the column with thrombin on the column for 4 hrs at 25 °C. The final purification step was reverse phase HPLC on a C18 column with a 0-50% acaetonitrile gradient, with 0.1% TFA. The peptide was lyophilized and dissolved in 150mM NaCl, 10mM MOPS, pH 7.5, 0.5mM EDTA and the pH adjusted with 10 M NaOH.
Sensorgrams were recorded on a Biacore 3000 instrument using streptavidin (SA) chips. Biotinylated NF-κB was immobilized on the chip in a high salt buffer (500 mM NaCl, 10mM Tris, pH 7.5, 0.5mM EDTA, 0.5mM sodium azide, 0.005% P20). Sensorgrams were run in the automatic subtraction mode using flow cell 1 (FC 1) as an ummodified reference. Data was collected for FC's 2,3 and 4, which contained varying amounts of NF-κB ligand with the lowest amount immobilized on FC2 and the highest on FC4. Injections were made using the kinject injection mode, alternating highest with lowest concentration samples, with a 5 minute contact time and a1200 second dissociation phase, in all cases except for the weaker interactions where a 3 min contact time and a 3 min dissociation phase was used. The running buffer used for the binding experiments was 150 mM NaCl, 10mM Tris, pH 7.5, 10% (w/v) glycerol, 3mM DTT, 0.5mM sodium azide, 0.2 mM EDTA and 0.005% P20. The glycerol improved the stability of the NF-κB during regeneration. Regeneration was achieved using a one min pulse of a urea solution. The concentration of urea required depended on the NF-κB construct and the experimental temperature and was prepared by diluting a 6M stock into the running buffer. The minimum urea concentration required for complete regeneration under each condition was determined by repeat injections. The data was analyzed using the Bia Evaluation 4.1 software using a simple 1:1 langmuir binding model. Between 3 and 12 sensorgrams were obtained for each construct and condition tested using a range of immobilized NF-κB and IκBα concentrations.
For NF-κB(p50248-376/p6519-325 at 37 °C, 200, 300 and 400 RU of NF-κB were immobilized. 0.23 to 4.0 nM IκBα was injected. A one min pulse of 3M urea was used for regeneration. Lower ligand and analyte concentrations were not employed to avoid the long term noise of the instrument becoming significant relative to the slow dissociation of the IκBα 37.
For NF-κB(p50248-350/p65190-321 sensorgrams were obtained at 25 and 37 °C. NF-κB was immobilized at 50, 75, 100, 150, 200, 250 and 350 RUs. Sensorgrams were recorded using several ranges of IκBα. These were: 0.87 to 9.9 nM IκBα with 200, 250 and 350 RU NF-κB, 0.24 to 20 nM IκBα for 50, 75 and 100 RU of NF-κB and with 0.01 to 5 nM IκBα with 100, 150 and 200 RU of NF-κB at 37 °C. For experiments at 25 °C, concentrations used were 0.87 to 9.9 nM IκBα with 100, 200 and 300 RU NF-κB, 0.24 to 20 and 0.022 to 10 nM IκBα with 50, 75 and 100 RU NF-κB. A one min pulse of 1.5 M urea was used for regeneration at 37 °C and 3 M Urea at 25 °C.
For NF-κB(p50248-350/p6519-304 at 25 °C, 16 to 1000 nM IκBα was used with 100, 200 and 250 RU of NF-κB, and 9.9 to 5000 nM IκBα was used with 50, 75 and 100 RU of NF-κB. At 15 °C, 9.9 to 5000 nM IκBα was used with 50, 75 and 100 RU of NF-κB. No regeneration was required at either temperature with this NF-κB construct because it bound so weakly. Data were analyzed by equilibrium analysis in addition to the kinetic analysis. The equilibrium response was plotted against the IκBα concentration and a line was fit to R = KA × [IκBα].Rmax/(KA.[IκBα]+1) where R is the equilibrium response at a specific IκBα concentration, Rmax is the response at saturation of the ligand on the chip and KA = 1/KD.
For the NF-κB(p50248-350/p6519-304), the effect of the 10% glycerol in the running buffer was assessed by experiments at 25 °C with 9.9 to 5000 nM IκBα and 50, 75 and 100 RU of NF-κB immobilized using a running buffer that was the same as that used for the ITC experiments (150 mM NaCl, 10 mM MOPS, pH 7.5, 0.5 mM EDTA, 0.5 mM sodium azide, 0.005% p20). No significant difference in the binding data was observed using this buffer.
ITC experiments were carried out on a Microcal MCS instrument. IκBα and NF-κB were purified by size exclusion chromatography on an S-75 or S-200 column respectively immediately prior to use. In a typical ITC experiment, 20 15 μl injections of 50 μM NF-κB were made into a 5 μM IκBα solution in the cell. ITC experiment were carried out in a buffer of 150 mM NaCl, 10 mM MOPS, pH 7.5, 0.5 mM EDTA, 0.5 mM sodium azide. Isotherms were analyzed using the Origin software (Microcal) as described elsewhere 38. For the very tight complexes, the KD,obs could not be determined due the high `c' value for the interaction, where c is defined by Wiseman et al. 38.