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• W2163515708 abstract "We have determined the binding energies of complexes formed between IκBα and the wild type and mutational variants of three different Rel/NF-κB dimers, namely, the p50/p65 heterodimer and homodimers of p50 and p65. We show that although a common mode of interaction exists between the Rel/NF-κB dimers and IκBα, IκBα binds the NF-κB p50/p65 heterodimer with 60- and 27-fold higher affinity than the p50 and p65 homodimers, respectively. Each of the three flexibly linked segments of the rel homology region of Rel/NF-κB proteins (the nuclear localization sequence, the dimerization domain, and the amino-terminal DNA binding domain) is directly engaged in forming the protein/protein interface with the ankyrin repeats and the carboxyl-terminal acidic tail/PEST sequence of IκBα. In the cell, IκBα functions to retain NF-κB in the cytoplasm and inhibit its DNA binding activity. These properties are a result of the direct involvement of the nuclear localization sequences and of the DNA binding region of NF-κB in complex with IκBα. A model of the interactions in the complex is proposed based on our observations and the crystal structures of Rel/NF-κB dimers and the ankyrin domains of related proteins. We have determined the binding energies of complexes formed between IκBα and the wild type and mutational variants of three different Rel/NF-κB dimers, namely, the p50/p65 heterodimer and homodimers of p50 and p65. We show that although a common mode of interaction exists between the Rel/NF-κB dimers and IκBα, IκBα binds the NF-κB p50/p65 heterodimer with 60- and 27-fold higher affinity than the p50 and p65 homodimers, respectively. Each of the three flexibly linked segments of the rel homology region of Rel/NF-κB proteins (the nuclear localization sequence, the dimerization domain, and the amino-terminal DNA binding domain) is directly engaged in forming the protein/protein interface with the ankyrin repeats and the carboxyl-terminal acidic tail/PEST sequence of IκBα. In the cell, IκBα functions to retain NF-κB in the cytoplasm and inhibit its DNA binding activity. These properties are a result of the direct involvement of the nuclear localization sequences and of the DNA binding region of NF-κB in complex with IκBα. A model of the interactions in the complex is proposed based on our observations and the crystal structures of Rel/NF-κB dimers and the ankyrin domains of related proteins. rel homology region nuclear localization sequence signal response domain glutathione S-transferase. The Rel/NF-κB family of dimeric transcription factors is ubiquitous in all human cell types. NF-κB regulates the expression of a variety of genes essential for cellular immune responses, inflammation, and growth and development (1Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4602) Google Scholar, 2Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2016) Google Scholar, 3Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5591) Google Scholar). Transcriptionally active NF-κB dimers form through the combinatorial assembly of the five monomeric polypeptides, p50, p65, p52, c-Rel, and RelB. Each monomer shares an approximately 300-amino acid region known as the rel homology region (RHR).1Within the RHR of the NF-κB polypeptides are all of the amino acid residues required for subunit dimerization, specific DNA binding, and nuclear localization (2Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2016) Google Scholar, 3Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5591) Google Scholar). Crystallographic analyses of the p50RHR (4Ghosh G. van Duyne G. Ghosh S. Sigler P.B. Nature. 1995; 373: 303-310Crossref PubMed Scopus (511) Google Scholar, 5Muller C.W. Rey F.A. Sodeoka M. Verdine G.L. Harrison S.C. Nature. 1995; 373: 311-317Crossref PubMed Scopus (475) Google Scholar) and the dimerization domain of p65 (p65ddNLS) (6Huang D.-B. Huxford T. Chen Y.-Q. Ghosh G. Structure. 1997; 5: 1427-1436Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) revealed that the 13 carboxyl-terminal amino acids containing the nuclear localization sequence (NLS) are not part of the dimerization domain of p50 and p65. The carboxyl-terminal NLS segments of p50 and p65 are not visible in the electron density maps and are presumably unstructured in solution. Subsequent crystal structures of the RHR dimers of p52 (7Cramer P. Larson C.J. Verdine G.L. Muller C.W. EMBO J. 1997; 16: 7078-7090Crossref PubMed Google Scholar), p65 (8Chen Y.Q. Ghosh S. Ghosh G. Nat. Struct. Biol. 1998; 5: 67-73Crossref PubMed Scopus (201) Google Scholar), and p50/p65 (9Chen F.E. Huang D.B. Chen Y.Q. Ghosh G. Nature. 1998; 391: 410-413Crossref PubMed Scopus (339) Google Scholar) used smaller RHR versions that exclude the carboxyl-terminal 13 amino acids. These structures display folds similar to p50RHR. Together, these structures indicate that the RHR of the NF-κB transcription factors is composed of three mutually independent modules. 1) An amino-terminal domain, composed of roughly 200 amino acids, assumes an immunoglobulin-like tertiary fold and is primarily responsible for conferring DNA binding specificity. 2) The central dimerization domain, approximately 100 amino acids in length, also exhibits an immunoglobulin fold. 3) The NLS, composed of 13 residues at the carboxyl terminus, appears to be flexible in solution (Fig. 1 A, top panel). The nuclear translocation and DNA binding activities of the NF-κB proteins are inhibited through association with a member of the IκB family of transcription factor inhibitors (3Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5591) Google Scholar). A host of extracellular stimuli trigger various signal transduction cascades, which converge at the phosphorylation of IκBα or IκBβ in complex with a dimer of NF-κB (2Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2016) Google Scholar, 10Traenckner E.B. Pahl H.L. Henkel T. Schmidt K.N. Wilk S. Baeuerle P.A. EMBO J. 1995; 14: 2876-2883Crossref PubMed Scopus (934) Google Scholar, 11Brown K. Franzoso G. Baldi L. Carlson L. Mills L. Lin Y.C. Gerstberger S. Siebenlist U. Mol. Cell. Biol. 1997; 17: 3021-3027Crossref PubMed Google Scholar, 12Verma I.M. Stevenson J.K. Schwarz E.M. Van Antwerp D. Miyamoto S. Genes Dev. 1995; 9: 2723-2735Crossref PubMed Scopus (1665) Google Scholar). The phosphorylated IκB proteins become targets for ubiquitination and subsequent proteosome-mediated degradation (13Chen Z. Hagler J. Palombella V.J. Melandri F. Scherer D. Ballard D. Maniatis T. Genes Dev. 1995; 9: 1586-1597Crossref PubMed Scopus (1172) Google Scholar). Free NF-κB dimers can then readily translocate the nuclear envelope and bind to their specific DNA target sites. An analogous mechanism for activation of transcription exists in Drosophila melanogaster development. The morphogen regulatory transcription factor Dorsal exists in an inactive cytoplasmic complex with its inhibitor, Cactus. These two proteins are structurally and functionally related to the NF-κB and IκB proteins, respectively (14Geisler R. Bergmann A. Hiromi Y. Nusslein-Volhard C. Cell. 1992; 71: 613-621Abstract Full Text PDF PubMed Scopus (196) Google Scholar, 15Kidd S. Cell. 1992; 71: 623-635Abstract Full Text PDF PubMed Scopus (184) Google Scholar, 16Steward R. Science. 1987; 238: 692-694Crossref PubMed Scopus (344) Google Scholar). Members of the IκB family of proteins contain six to seven homologous copies of an approximately 33-amino acid sequence known as the ankyrin repeat (3Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5591) Google Scholar). The three-dimensional structures of four different ankyrin repeat containing proteins (17Gorina S. Pavletich N.P. Science. 1996; 274: 1001-1005Crossref PubMed Scopus (396) Google Scholar, 18Luh F.Y. Archer S.J. Domaille P.J. Smith B.O. Owen D. Brotherton D.H. Raine A.R. Xu X. Brizuela L. Brenner S.L. Laue E.D. Nature. 1997; 389: 999-1002Crossref PubMed Scopus (89) Google Scholar, 19Venkataramani R. Swaminathan K. Marmorstein R. Nat. Struct. Biol. 1998; 5: 74-81Crossref PubMed Scopus (78) Google Scholar, 20Batchelor A.H. Piper D.E. de la Brousse F.C. McKnight S.L. Wolberger C. Science. 1998; 279: 1037-1041Crossref PubMed Scopus (266) Google Scholar) show that ankyrin repeats assume a unique structural scaffold in which a “finger-like” β-hairpin is projected from the core helix-turn-helix element. The ankyrin repeat domain of IκBα, consisting of six imperfect repeats, is preceded by a 70-amino acid amino-terminal segment and followed by a 42-amino acid carboxyl-terminal region (21Jaffray E. Wood K.M. Hay R.T. Mol. Cell. Biol. 1995; 15: 2166-2172Crossref PubMed Google Scholar) (Fig. 1 A, bottom panel). The amino-terminal segment, referred to as the signal response domain (SRD), receives signals through the phosphorylation of serines at positions 32 and 36. The SRD is not known to play any role in NF-κB binding (11Brown K. Franzoso G. Baldi L. Carlson L. Mills L. Lin Y.C. Gerstberger S. Siebenlist U. Mol. Cell. Biol. 1997; 17: 3021-3027Crossref PubMed Google Scholar, 22Whiteside S.T. Ernst M.K. LeBail O. Laurent-Winter C. Rice N. Israel A. Mol. Cell. Biol. 1995; 15: 5339-5345Crossref PubMed Google Scholar, 23Brockman J.A. Scherer D.C. McKinsey T.A. Hall S.M. Qi X. Lee W.Y. Ballard D.W. Mol. Cell. Biol. 1995; 15: 2809-2818Crossref PubMed Google Scholar). The carboxyl-terminal segment is rich in proline, glutamic acid/aspartic acid, serine, and threonine residues (the PEST sequence). This PEST sequence is a common feature implicated in high turnover rate among short-lived proteins (24Rogers S. Wells R. Rechsteiner M. Science. 1986; 234: 364-368Crossref PubMed Scopus (1963) Google Scholar). Partial or complete deletion of the acidic PEST region reduces the ability of IκBα to inhibit DNA binding of certain NF-κB dimers (25Ernst M.K. Dunn L.L. Rice N.R. Mol. Cell. Biol. 1995; 15: 872-882Crossref PubMed Google Scholar, 26Leveillard T. Verma I.M. Gene Expr. 1993; 3: 135-150PubMed Google Scholar, 27Naumann M. Wulczyn F.G. Scheidereit C. EMBO J. 1993; 12: 213-222Crossref PubMed Scopus (124) Google Scholar). Other IκB family proteins also contain a similar domain structure, with the notable exceptions of p105 and p110, which contain NF-κB p50 and p52 sequences, respectively, at their amino termini. It is generally believed that the IκB proteins retain NF-κB in the cytoplasm by precluding the NLS of the NF-κB RHR from being recognized by the nuclear transport machinery (1Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4602) Google Scholar, 2Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2016) Google Scholar). In addition, studies have shown that one IκBα molecule interacts with one NF-κB dimer (25Ernst M.K. Dunn L.L. Rice N.R. Mol. Cell. Biol. 1995; 15: 872-882Crossref PubMed Google Scholar). However, it is not known whether the IκB proteins directly contact the NLS or mask it by steric hindrance (28Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2935) Google Scholar, 29Beg A.A. Ruben S.M. Scheinman R.I. Haskill S. Rosen C.A. Baldwin Jr., A.S. Genes Dev. 1992; 6: 1899-1913Crossref PubMed Scopus (614) Google Scholar). Furthermore, it has not been determined whether one or both of the NF-κB NLSs are involved in this process. One other intriguing property of IκBα is that in the post-induction stage, newly synthesized IκBα can enter the nucleus, where it is capable of dissociating transcriptionally competent NF-κB/DNA complexes (30Tran K. Merika M. Thanos D. Mol. Cell. Biol. 1997; 17: 5386-5399Crossref PubMed Google Scholar,31Arenzana-Seisdedos F. Thompson J. Rodriguez M.S. Bachelerie F. Thomas D. Hay R.T. Mol. Cell. Biol. 1995; 15: 2689-2696Crossref PubMed Google Scholar). It remains to be seen whether this inhibition of DNA binding activity of IκBα occurs through interaction with the DNA binding residues of NF-κB. There exists no clear evidence as to why the NF-κB p50/p65 heterodimer is preferentially recognized by IκBα over the p50, p65, or c-Rel homodimers. Furthermore, the role of the p50 subunit of the p50/p65 heterodimer in its interaction with IκBα is not known. Finally, it remains unclear what role, if any, the amino-terminal DNA binding domain of the NF-κB RHR plays in the NF-κB/IκB complex. Using fluorescence polarization competition experiments, we have determined the equilibrium dissociation constants of the complexes between NF-κB (wild type and mutant p50 and p65 homodimers and the p50/p65 heterodimer) and IκBα. We show that IκBα recognizes the NF-κB dimers with variable affinities. In binding to the heterodimer, IκBα directly contacts the NLSs of both subunits in addition to making contacts with the dimerization domains and the DNA binding loop L1 of the p65 subunit. We suggest that the mode of interaction between each of the NF-κB dimers and IκBα is similar and that the relative amounts of various NF-κB dimers and their affinities for IκBα are the determinants of their cytoplasmic retention. The cloning, expression, and purification of the NF-κB subunits and amino-terminal deletion mutants has been described previously (4Ghosh G. van Duyne G. Ghosh S. Sigler P.B. Nature. 1995; 373: 303-310Crossref PubMed Scopus (511) Google Scholar, 6Huang D.-B. Huxford T. Chen Y.-Q. Ghosh G. Structure. 1997; 5: 1427-1436Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 8Chen Y.Q. Ghosh S. Ghosh G. Nat. Struct. Biol. 1998; 5: 67-73Crossref PubMed Scopus (201) Google Scholar, 9Chen F.E. Huang D.B. Chen Y.Q. Ghosh G. Nature. 1998; 391: 410-413Crossref PubMed Scopus (339) Google Scholar). The purification of p50dd/p65ddNLS, the p50RHR/p65RHR heterodimer, and its derivatives was accomplished by unfolding and refolding of purified components (9Chen F.E. Huang D.B. Chen Y.Q. Ghosh G. Nature. 1998; 391: 410-413Crossref PubMed Scopus (339) Google Scholar). In a second method, p50 and p65 have been coexpressed in Escherichia coli and purified as heterodimers. The full-length IκBα (residues 1–317) and IκBαΔSRD (residues 67–317) proteins were expressed as glutathioneS-transferase (GST) fusion proteins in a pGEX vector (Novagen). They were both purified in a similar manner. The clone was transformed into BL21[DE3] cells and induced with 0.1 mmisopropylthio-β-d-galactoside overnight at room temperature. After cell lysis by sonication, the crude lysate was loaded onto a reduced glutathione-Sepharose column, followed by a Q Sepharose column and finally a Superdex75 gel filtration column (Amersham Pharmacia Biotech). The peak fractions were collected and stored at −80 °C. Untagged IκBαΔSRD (residues 67–317) and IκBαΔSRDΔPEST (residues 67–277) were cloned into a pET3a vector (Novagen) and purified as the GST-fusion proteins with the exception that the glutathione-Sepharose step was ignored. A 39-bp fluorescein labeled DNA was purchased from Yale University oligonucleotide synthesis facility. The DNA had the following sequence: 5′-fluorescein-GATCGCTGGGGACTTTCCAGGGAGGCGTGGCCTGAGTCC-3′. The HIV-κB target site is shown in boldface. The complementary strand was synthesized on a Cyclone Plus DNA synthesizer. After deblocking, the oligonucleotides were purified over a Q-Sepharose column. Peak fractions were pooled and concentrated. Equimolar concentrations of the sense and antisense strands were mixed and annealed. The BIAcore biosensor system allows the monitoring of macromolecular reactions in real time (32Jonsson U. Fagerstam L. Lofas S. Stenberg E. Karlsson R. Frostell A. Markey F. Schindler F. Ann. Biol. Clin. 1993; 51: 19-26PubMed Google Scholar). The BIAcore system, sensor chips CM5 (certified), the amine coupling kit, and the anti-GST binding kit were obtained from BIAcore, Inc. The buffer used for all experiments was 10 mm HEPES (pH7.4), 150 mm NaCl, 3.4 mm EDTA, and 0.005% (v/v) Surfactant P20. To immobilize IκBα, an anti-GST antibody was first immobilized on the chip surface via amine coupling using the kit provided by the sensor chip manufacturer. Next, GST-IκBα was injected across the surface, and approximately 350 response units were captured onto the surface by the antibody per binding experiment. Two GST-IκBα constructs were tested with similar results: GST-IκBαΔSRD and GST-IκBα. Various rel/NF-κB constructs were then injected across the chip surface, and binding was monitored. At the end of each run, the GST-IκBα and NF-κB were removed from the surface with 10 μl of 10 mm glycine at pH 2.2, leaving active antibody on the surface. Each surface was used multiple times. To detect nonspecific binding to the GST and the immobilized antibody, control runs were performed with only antibody on the chip surface and with GST immobilized via the antibody on the surface. No significant nonspecific binding was observed. All experiments were performed at 25 °C and at a flow rate of 40 μl/min. All sensorgrams reported have been blank subtracted for any bulk refractive index effects. All fluorescence polarization measurements were made using the Beacon 2000 Fluorescence Polarization System (PanVera Corp.). We used fluorescence polarization to determine the DNA binding affinities of several NF-κB dimers (33LeTilly V. Royer C.A. Biochemistry. 1993; 32: 7753-7758Crossref PubMed Scopus (149) Google Scholar). Serial dilutions of NF-κB were added to constant amounts of labeled DNA. The fluorescence polarization values were recorded once the system had reached equilibrium (approximately 50 min). All measurements were taken at 37 °C in 10 mm Tris (pH 7.5) and 50 mmNaCl. The fractional occupancy was calculated as described in Equation 1. K D was calculated as the concentration of NF-κB at 0.5 fractional occupancy, as follows,Fractional occupancy=P−PD/PND−PD(Eq. 1) where P is polarization in millipolarization units,P D is polarization of free DNA, and P ND is polarization of DNA saturated with NF-κB. In the presence of IκBα and DNA, NF-κB links two competing equilibrium processes (see Equation 2). If the dissociation constant of one process is known, the dissociation constant of the second process can be derived from a competition assay. This assay probes the shift in equilibrium of one reaction when in the presence of a competing inhibitor. Having characterized the affinity of NF-κB for a particular κB-DNA target site, we endeavored to determine its affinity for IκBα binding by a fluorescence polarization competition assay. To perform this competition assay, varying concentrations of IκBα were mixed with constant amounts of NF-κB and labeled DNA. The system was allowed to reach equilibrium (approximately 1 h). We observed an increase in polarization with increased IκBα concentration. This corresponds to the generation of free DNA in solution upon the addition of IκBα to the preformed NF-κB/DNA complexes. Control experiments were performed to check for any nonspecific DNA binding by IκBα. IκBα does not bind DNA even at extremely high concentrations (50 μm). The competition assay binding curves were analyzed for IC50values the concentration of IκBα at 0.5 fractional occupancy. TheK I value (the dissociation constant for the NF-κB/IκBα interaction) was derived using the following values according to Equation 5: the DNA binding affinity of NF-κB (K D), the IC50 value, [NF-κB]total, and [DNA]total, as follows,NF­κB+DNA ⇄ KD NF­κB/DNA+ IκB KI⇅KD=[NF­κB][DNA][NF­κB/DNA]PD−PP−PND=[NF­κB/DNA][DNA] NF­κB/IκB KI=[NF­κB][IκB][NF­κB/IκB](Eq. 2) where [NF-κB]total = [NF-κB] + [NF-κB/IκB] + [NF-κB/DNA], [DNA]total= [DNA] + [NF-κB/DNA], and [IκB]total = [IκB] + [NF-κB/IκB]. Solving for [NF-κB]total in terms of [NF-κB], [IκB]total, [DNA], KD, and KI yields the following expression,[NF­κB]total=[NF­κB]{(KI+[NF­κB])(KD+[NF­κB])+[IκB]total(KD+[NF­κB]+[DNA]total(KI+[NF­κB])}(KI+[NF­κB])(KD+[NF­κB])(Eq. 3) At the midpoint of the titration,[NF­κB]=KD and PD−PP−PND=1(Eq. 4) if [DNA]total and [NF-κB]total are constant and [IκB] is varied. Then, at the IC50 of the competition binding curve, the following holds true. KI=−2 KD2−2 KDIC50−[DNA]total KD+2 KD[NF­κB]total2 KD+[DNA]total−2[NF­κB]total(Eq. 5) For each NF-κB dimer, the K I values were calculated as an average of three individual experiments. Various ratios of [NF-κB]/[DNA] were tested, with similar results. There was less than a 20% error between individual experiments. All runs were performed in 10 mm Tris (pH 7.5) and 50 mmNaCl at 37 °C. 10% native polyacrylamide gels were prepared in 0.25× Tris borate EDTA. The gels were filtered and degassed. Individual or complexed proteins were prepared in 10 mm Tris (pH 7.5), 4% glycerol, 2 mm β-mercaptoethanol, and 50 mm NaCl. Reactions were allowed to reach equilibrium at room temperature for 1 h. Native gel loading buffer (50 mm Tris, pH 7.5, 0.1% bromphenol blue, 10% glycerol, and 1.25 mmβ-mercaptoethanol) was then added to each sample. The gels were run in 0.25× Tris borate EDTA for 1.5 h at 3 mA. To evaluate the contributions of the three segments within the RHR of NF-κB in complex formation with IκBα, a series of structure-based deletion mutants of the homodimers and of the heterodimer were prepared. Care was taken to ensure that the integrity of the three-dimensional fold of these mutants would remain intact. The flexible activation domain of p65, which follows the NLS, has been shown not to participate in the interaction with IκBα (3Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5591) Google Scholar) and was therefore not included in this study. A list of the constructs pertinent to our studies is shown in Fig. 1, A and B. All proteins have been expressed in E. coli and purified to near homogeneity, as shown in Fig. 1 C. We first determined the affinity of the wild type heterodimer (p50RHR/p65RHR) for the HIV-κB DNA using 0.1 nm labeled DNA and increasing amounts of the heterodimer. From the saturation binding curve, the equilibrium dissociation constant (K D) of the NF-κB p50/p65-κB DNA complex was observed as 4.7 nm (Fig. 2 A). We then performed a competition assay using the wild type p50RHR/p65RHR and IκBαΔSRD (IκBα with the amino-terminal signal response domain removed). These experiments show that IκBα binds the wild type heterodimer with an equilibrium dissociation constant (K I) of 3.0 nm (Fig. 2 A). In an effort to determine the contribution of the SRD of IκBα, we repeated the fluorescence polarization competition experiment using the full-length IκBα and IκBαΔSRD as glutathione S-transferase fusion proteins (GST-IκBα and GST-IκBαΔSRD). The inhibition curves resulting from the full-length GST-IκBα, GST-IκBαΔSRD, and IκBαΔSRD are nearly identical (Fig. 2 B). Further controls confirmed that GST does not interact nonspecifically with the NF-κB or the labeled DNA (data not shown). These results confirm that the SRD of IκBα does not contribute to binding of the NF-κB p50/p65 heterodimer. Therefore, we chose to use IκBαΔSRD in our assays for measuring the NF-κB/IκBα binding constants. We next investigated the role of the carboxyl-terminal acidic tail/PEST sequence of IκBα in the inhibition of DNA binding by the p50/p65 heterodimer. We performed the fluorescence polarization competition experiment previously described using a mutant IκBα with both the SRD and PEST sequence removed (IκBαΔSRDΔPEST). Even at a 150-fold excess of IκBαΔSRDΔPEST, only a slight decrease in the polarization was observed (Fig. 2 B). This result identifies the acidic tail/PEST sequence as the necessary element that confers DNA-inhibitory binding activity on IκBα. It is well understood that the nuclear localization of NF-κB dimers is mediated by the nuclear localization signals located at the carboxyl termini of the RHR. However, the exact mode of NLS masking by IκBα remains unclear. To delineate the role of NLSs of the NF-κB heterodimer, we have made three heterodimer variants in which the NLS is individually and doubly removed (p50RHR/p65RHRs, p50RHRs/p65RHR, and p50RHRs/p65RHRs,where s refers to the shortened RHR, with its NLS removed). As seen in Fig. 3 A, the mutant heterodimer with deleted p65 NLS (p50RHR/p65RHRs) has over 5-fold lower affinity for IκBα compared with that of the wild type heterodimer (K I ∼ 16.9 nm p50RHR/p65RHRsversus 3.0 nm p50RHR/p65RHR). On the other hand, p50RHRs/p65RHR binds IκBα with an affinity of 3.1 nm. When both the NLSs are removed, the heterodimer shows a 9-fold (K I ∼ 28.0 nm) defect compared with the wild type heterodimer (Table I). Clearly, the p65NLS is essential for IκBα binding. The role of the p50 NLS is not apparent from the p50RHRs/p65RHR binding profile, which looks similar to the wild type heterodimer. However, if the NLS of p50 was not involved in any contact with IκBα, we would expect the p50RHR/p65RHRs and the p50RHRs/p65RHRs binding profiles to be identical. The 1.6-fold defect in p50RHRs/p65RHRs binding affinity when compared with p50RHR/p65RHRs indicates that the p50NLS does in fact contribute to IκBα binding. Therefore, both the NLSs of p50 and p65 appear to be involved in direct interactions with IκBα. These results establish, for the first time, a specific role for the p50 subunit of the heterodimer in IκBα binding specificity. The p50 subunit is not, it seems, simply an inert participant in the complex formation by nature of its ability to dimerize with the p65 subunit.Table IKI and ΔG values calculated from the fluorescence polarization competition assaysDimerK IΔGnmkcal/molp50RHR/p65RHR3.0 ± 0.7−11.6p50RHRs/p65RHR3.1 ± 0.7−11.6p50RHR/p65RHRs16.9 ± 1.3−10.6p50RHRs/p65RHRs28.0 ± 2.3−10.3p50RHR/p65RHR(ΔL1)172.7 ± 25.9−9.2p65RHR/p65RHR82.1 ± 14.0−9.7p65RHRs/p65RHRs143.3 ± 11.2−9.3p50RHR/p50RHR181.3 ± 16.4−9.2p50RHRs/p50RHRs1518.3 ± 110.4−7.92Each K I value reported is an average of three individual experiments. The K I values were calculated using the equations discussed under “Experimental Procedures.” The K D values (the DNA binding constants) of the various homo- and heterodimers were determined independently. Open table in a new tab Each K I value reported is an average of three individual experiments. The K I values were calculated using the equations discussed under “Experimental Procedures.” The K D values (the DNA binding constants) of the various homo- and heterodimers were determined independently. In support of our results obtained from the fluorescence polarization competition assay, we have performed biomolecular interaction assays on wild type and shortened NF-κB proteins using BIAcore technology. The sensorgram shown in Fig. 3 B provides two important points: first, the nature of the interaction between the wild type heterodimer and IκBα is complex, and both the association and dissociation are multiphasic; and second, the p50RHRs/p65RHRs heterodimer is defective for IκBα binding when compared with the wild type heterodimer. The role of the p50NLS was further probed by using two truncated heterodimeric p50/p65 constructs, in which the amino-terminal domain of the p50 subunit was deleted (p50ddNLS/p65RHR and p50dd/p65RHR). As seen in Fig. 3 C, when the NLS of the p50 subunit is deleted (p50dd/p65RHR), the heterodimer becomes a poorer substrate for IκBα compared with the one with the NLS. Again, this result underscores the importance of the p50 NLS in IκBα binding. The involvement of the acidic tail of IκBα in the inhibition of DNA binding activity of NF-κB led researchers to speculate that the amino-terminal DNA binding domain of NF-κB is involved in contacting IκBα (30Tran K. Merika M. Thanos D. Mol. Cell. Biol. 1997; 17: 5386-5399Crossref PubMed Google Scholar, 34Diehl J.A. McKinsey T.A. Hannink M. Mol. Cell. Biol. 1993; 13: 1769-1778Crossref PubMed Google Scholar, 35Kerr L.D. Inoue J. Davis N. Link E. Baeuerle P.A. Bose Jr., H.R. Verma I.M. Genes Dev. 1991; 5: 1464-1476Crossref PubMed Scopus (118) Google Scholar, 36Kumar S. Gelinas C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8962-8966Crossref PubMed Scopus (27) Google Scholar). Kumar and Gelinas (36Kumar S. Gelinas C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8962-8966Crossref PubMed Scopus (27) Google Scholar) found that a small peptide homologous to the v-Rel amino-terminal DNA binding domain competed with the full-length v-Rel for IκBα binding. In order to investigate whether the amino-terminal DNA binding domain of p65 may be involved in interacting with IκBα, we constructed an NF-κB p50/p65 heterodimer in which a segment of 15 amino acids (residues 30–45 of p65) has been replaced by three glycine residues at loop L1 of p65. Four of the five residues that mediate direct DNA base-specific contacts, Arg33, Arg35, Tyr36, and Glu39 are located within this 15-amino acid segment. As expected, the mutant heterodimer binds to HIV-κB DNA with reduced affinity (237.8 nm). However, this affinity is high enough to perform the fluorescence polarization competition assay with IκBα. We were able to determine that IκBα binds to this mutant heterodimer with a KI of 172.7 nm (Table I). This indicates a 57-fold reduction in binding affinity compared with the wild type heterodimer, showing that the amino-terminal domain of the p65 subunit is involved in contacting IκBα. In vivo transfection followed by immunoprecipitation and qualitative DNA binding inhibition using a gel retardation assay showed that p65 homodimer can be retained in the cytoplasm by IκBα with high efficiency (25Ernst M.K. Dunn L.L. Rice N.R. Mol. Cell. Biol. 1995; 15: 872-882Crossref PubMed Google Scholar). Therefore, one of the objectives of this study was to determine whether the p65 homodimer has an" @default.
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• W2163515708 title "IκBα Functions through Direct Contacts with the Nuclear Localization Signals and the DNA Binding Sequences of NF-κB" @default.
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• W2163515708 doi "https://doi.org/10.1074/jbc.273.39.25427" @default.
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