X-ray Crystal Structure of an IκBβ·NF-κB p65 Homodimer Complex
2003; Elsevier BV; Volume: 278; Issue: 25 Linguagem: Inglês
10.1074/jbc.m301022200
ISSN1083-351X
AutoresShiva Malek, D. Huang, Tom Huxford, Sankar Ghosh, Gourisankar Ghosh,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoWe report the crystal structure of a murine IκBβ·NF-κB p65 homodimer complex. Crystallographic models were determined for two triclinic crystalline systems and refined against data at 2.5 and 2.1 Å. The overall complex structure is similar to that of the IκBα·NF-κB p50/p65 heterodimer complex. One NF-κB p65 subunit nuclear localization signal clearly contacts IκBβ, whereas a homologous segment from the second subunit of the homodimer is mostly solvent-exposed. The unique 47-amino acid insertion between ankyrin repeats three and four of IκBβ is mostly disordered in the structure. Primary sequence analysis and differences in the mode of binding at the IκBβ sixth ankyrin repeat and NF-κB p65 homodimer suggest a model for nuclear IκBβ·NF-κB·DNA ternary complex formation. These unique structural features of IκBβ may contribute to its ability to mediate persistent NF-κB activation. We report the crystal structure of a murine IκBβ·NF-κB p65 homodimer complex. Crystallographic models were determined for two triclinic crystalline systems and refined against data at 2.5 and 2.1 Å. The overall complex structure is similar to that of the IκBα·NF-κB p50/p65 heterodimer complex. One NF-κB p65 subunit nuclear localization signal clearly contacts IκBβ, whereas a homologous segment from the second subunit of the homodimer is mostly solvent-exposed. The unique 47-amino acid insertion between ankyrin repeats three and four of IκBβ is mostly disordered in the structure. Primary sequence analysis and differences in the mode of binding at the IκBβ sixth ankyrin repeat and NF-κB p65 homodimer suggest a model for nuclear IκBβ·NF-κB·DNA ternary complex formation. These unique structural features of IκBβ may contribute to its ability to mediate persistent NF-κB activation. NF-κB is a family of inducible transcription factors central to the regulation of diverse biological activities such as immune and inflammatory responses, development, and apoptosis (1Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5646) Google Scholar, 2Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4657) Google Scholar, 3Gilmore T.D. Oncogene. 1999; 18: 6842-6844Crossref PubMed Scopus (360) Google Scholar). Five polypeptide subunits, p50, p52, p65 (RelA), c-Rel, and RelB, constitute the mammalian NF-κB family. Although expressed in most cells, the majority of NF-κB homo- and heterodimers with transcriptional activation potential are tightly regulated through stable association with the IκB inhibitor proteins IκBα and IκBβ (4Verma I.M. Stevenson J.K. Schwarz E.M. Van Antwerp D. Miyamoto S. Genes Dev. 1995; 9: 2723-2735Crossref PubMed Scopus (1667) Google Scholar). Various inducing signals, such as cytokines, growth factors, and bacterial and viral products, lead to phosphorylation and eventual proteasome-mediated degradation of NF-κB-associated IκB proteins (5Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4155) Google Scholar). IκB proteolysis coincides with the release of free NF-κB, which binds specifically to DNA enhancer elements in the nucleus and modulates levels of target gene transcription. IκBα and IκBβ bind and inhibit the same NF-κB homo- and heterodimers and have been shown to be capable of functionally replacing one another (6Cheng J.D. Ryseck R.P. Attar R.M. Dambach D. Bravo R. J. Exp. Med. 1998; 188: 1055-1062Crossref PubMed Scopus (88) Google Scholar). Although they function together in regulating the activity of most NF-κB dimers, IκBα and IκBβ do so by differing mechanisms. Due to its rapid degradation and nearly immediate resynthesis upon induction of NF-κB, IκBα participates in fast, transient NF-κB activation (7Sun S.-C. Ganchi P.A. Ballard D.W. Greene W.C. Science. 1993; 259: 1912-1915Crossref PubMed Scopus (973) Google Scholar, 8Arenzana-Seisdedos F. Thompson J. Rodriguez M.S. Bachelerie F. Thomas D. Hay R.T. Mol. Cell. Biol. 1995; 15: 2689-2696Crossref PubMed Google Scholar, 9Hoffmann A. Levchenko A. Scott M.L. Baltimore D. Science. 2002; 298: 1241-1245Crossref PubMed Scopus (1524) Google Scholar). IκBβ, on the other hand, responds to a subset of NF-κB inducers and is degraded with slower kinetics (10Thompson J.E. Phillips R.J. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Abstract Full Text PDF PubMed Scopus (699) Google Scholar, 11Tran K. Merika M. Thanos D. Mol. Cell. Biol. 1997; 17: 5386-5399Crossref PubMed Google Scholar). The reason behind the slower IκBβ degradation is unknown, although it may result, in part, from differences in subcellular localization exhibited by IκBβ·NF-κB and IκBα·NF-κB complexes. Whereas IκBα·NF-κB complexes shuttle in and out of the nucleus, IκBβ·NF-κB complexes remain constitutively cytoplasmic in resting cells (12Huang T.T. Miyamoto S. Mol. Cell. Biol. 2001; 21: 4737-4747Crossref PubMed Scopus (91) Google Scholar, 13Malek S. Chen Y. Huxford T. Ghosh G. J. Biol. Chem. 2001; 276: 45225-45235Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 14Tam W.F. Sen R.J. J. Biol. Chem. 2001; 276: 7701-7704Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 15Ghosh S. Karin M. Cell. 2002; 109: S81-S96Abstract Full Text Full Text PDF PubMed Scopus (3329) Google Scholar). Finally, under some activating conditions, hypophosphorylated IκBβ has been shown to bind nuclear, DNA-bound NF-κB complexes, resulting in a stable nuclear ternary IκBβ·NF-κB·DNA complex (16Suyang H. Phillips R. Douglas I. Ghosh S. Mol. Cell. Biol. 1996; 16: 5444-5449Crossref PubMed Google Scholar, 17DeLuca C. Petropoulos L. Zmeureanu D. Hiscott J. J. Biol. Chem. 1999; 274: 13010-13016Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Together, these characteristics unique to IκBβ serve to mediate the persistent activation of NF-κB observed in many disease states (18Phillips R.J. Ghosh S. Mol. Cell. Biol. 1997; 17: 4390-4396Crossref PubMed Google Scholar, 19Newton T.R. Patel N.M. Bhat-Nakshatri P. Stauss C.R. Goulet Jr., R.J. Nakshatri H. J. Biol. Chem. 1999; 274: 18827-18835Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). To investigate the differences in NF-κB regulation exhibited by related IκB inhibitor proteins, we have determined the x-ray crystal structure of IκBβ bound to the NF-κB p65 homodimer from two different crystal forms at 2.5 and 2.1 Å. The complex crystal structure shows many similarities with that of the previously determined IκBα·NF-κB p50/p65 heterodimer complex (20Huxford T. Huang D.B. Malek S. Ghosh G. Cell. 1998; 95: 759-770Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar, 21Jacobs M.D. Harrison S.C. Cell. 1998; 95: 749-758Abstract Full Text Full Text PDF PubMed Scopus (712) Google Scholar). For example, IκBβ contacts the dimerization domains and one nuclear localization signal (NLS) 1The abbreviations used are: NLS, nuclear localization signal; ARD, ankyrin repeat domain; PEST, proline, glutamic acid, serine, and threonine-rich region; NES, nuclear exit signal. of the dimeric NF-κB transcription factor in a manner similar to IκBα. Our present structure shows that the C-terminal NLS-containing segment of the second p65 subunit displays weak electron density representative of a loose or non-uniformly bound region. Furthermore, a large insertion within the ankyrin repeat domain of IκBβ does not adopt a folded structure. The structure further reveals that the last ankyrin repeat and the PEST sequence of IκBβ bind NF-κB differently as compared with the homologous segments of IκBα. Taken together, these observations suggest a molecular mechanism for persistent NF-κB activation and DNA binding of nuclear IκBβ·NF-κB complexes. Crystallization and X-ray Diffraction Data Collection—Protein expression and crystallization have been reported previously (22Huxford T. Malek S. Ghosh G. J. Biol. Chem. 2000; 275: 32800-32806Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Substitution of potential phosphorylation sites within the IκBβ PEST region to phosphomimetic amino acids fails to increase IκBβ·NF-κB binding affinity. Nevertheless, we used an IκBβ construct with five C-terminal serine residues replaced by glutamates. Recombinant murine IκBβ (amino acids 50–331) and murine NF-κB p65 homodimer (residues 191–325) were expressed in Escherichia coli, purified separately, and finally purified as a stable complex. Both crystal forms were prepared by the hanging drop vapor diffusion method in 8–10% PEG 8000 and sodium citrate, pH 5.6 (crystal form I) or pH 5.8 (crystal form II). Crystals were soaked in cryoprotectant buffer solution containing 20% glycerol and flash cooled under liquid nitrogen. X-ray diffraction data for crystal form I were collected using a MAR345 image plate mounted on a Rigaku FR5 rotating anode x-ray generator equipped with Charles Supper focusing mirrors. Crystal form II data were collected at the Advanced Light Source beamline 5.0.2 at Berkeley National Laboratory using an ADSC Quantum4 charge-coupled device detector. All data were processed and scaled using the programs DENZO and SCALE-PACK from the HKL suite (23Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38777) Google Scholar). Structure Solution and Refinement—Initial solution of the complex was obtained by molecular replacement using AMoRe (24Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5038) Google Scholar). A modified IκBα·NF-κB p50/p65 heterodimer complex structure with the dimerization domains of the p50/p65 heterodimer replaced by a p65 homodimer and the NLS polypeptides removed together with the ankyrin repeat domain (ARD) of IκBα was used as a search model. A rotational search followed by Patterson correlation refinement gave a clear solution with a peak of 8.4 σ. No translation search was necessary because the IκBβ·NF-κB complex crystallized in the P1 space group. The orientations and positions of individual domains were refined by rigid body refinement in crystallography NMR software (25Brünger A.T. Adams P.D. Clore G.M. Gros P. Grosse-Kuntsleve R.W. Jiang J.-S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 10: 25-26Google Scholar). Clear electron density for the NLS polypeptide of p65 subunit A appeared after the first round of crystallography NMR software refinement and phase calculation. Electron density for the subunit B NLS appeared gradually over several cycles of model building and refinement. Validity of the new electron density was confirmed by omit map calculation. Amino acid replacement and model adjustment were carried out based on 2FO-FC maps using the programs O (26Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13036) Google Scholar) and TOM (27Cambillau C. Horjales E. J. Mol. Graph. 1987; 5: 174-177Crossref Scopus (161) Google Scholar). Atomic models for the structure were refined by simulated annealing using crystallography NMR software with a maximum likelihood target function and a flat bulk solvent correction. Amino acids in the insert area of IκBβ and the residues in the NLS regions of the p65 homodimer were added gradually during refinement. Throughout the course of refinement, NCS restraints were applied for the dimerization domains of the p65 homodimer. Refinement of temperature (B) factors resulted in a final working R-factor of 20.0% and free R-factor of 27.9% for all data between 30.0 and 2.5 Å resolution. Ninety-nine percent of the amino acid residues lie in the most favored (87%) and additional allowed (12%) regions of the Ramachandran plot. The crystal form II structure was solved by molecular replacement using the refined atomic coordinates from the structure of crystal form I. Details and statistics of data collection and refinement are shown in Table I. Schematic representations of the complex crystallographic model were prepared in MOL-SCRIPT (28Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), RASTER3D (29Merritt E.A. Bacon D.J. Methods Enzymol. 1997; 277: 505-524Crossref PubMed Scopus (3880) Google Scholar), GRASP (30Nicholls A. Bharadwaj R. Honig B. Biophys. J. 1993; 64: A166Google Scholar), and O. Atomic coordinates for the IκBβ·NF-κB p65 homodimer complex crystal structure have been deposited in the Protein Data Bank and have been assigned the codes 1K3Z (crystal form I) and 1OY3 (crystal form II).Table ISummary of crystallographic analysisData collectionCrystal form ICrystal form IIX-ray sourceHomeALS 5.0.2Wavelength (Å)1.54181.1Resolution (Å)2.52.06Space groupP1P1Unit cell parametersa (Å)46.4048.62b (Å)48.9254.79c (Å)59.4759.57α (°)95.17102.56β (°)91.8096.07γ (°)105.4591.90Reflections (total/unique)110,726/16,126159,589/31,263I/σ7.9 (3.0)aParentheses indicate data for highest resolution shell.11.1 (2.0)Completeness (%)93 (72)90 (75)RsymbRsym = Σ|Iobs — Iavg|/ΣIavg. (%)7.9 (25.3)6.4 (40.9)RefinementResolution range (Å)30.0-2.530.0-2.06Reflections15,38231,809No. of atoms3,8093,875StereochemistryBond length (Å)0.0070.006Bond angle (°)1.341.30R-factorRworkcRwork = Σ|Fobs — Fcalc|/ΣFobs. (%)20.022.9RfreedRfree was calculated with 5% of data. (%)27.926.1a Parentheses indicate data for highest resolution shell.b Rsym = Σ|Iobs — Iavg|/ΣIavg.c Rwork = Σ|Fobs — Fcalc|/ΣFobs.d Rfree was calculated with 5% of data. Open table in a new tab IκBβ·NF-κB p65 Homodimer Complex Crystallization and Structure Solution—Because of difficulties in obtaining single IκBβ·NF-κB complex crystals suitable for x-ray diffraction analysis, a combinatorial approach to complex formation and crystallization was used (22Huxford T. Malek S. Ghosh G. J. Biol. Chem. 2000; 275: 32800-32806Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). All of the NF-κB amino acids necessary for dimer formation, DNA binding, nuclear localization, and IκB binding are contained within the conserved 300-amino acid Rel homology region. The Rel homology region consists of N-terminal and C-terminal (dimerization) domains followed by a flexible region containing a type I nuclear localization signal (NLS) known as the NLS polypeptide. IκBβ contains a centrally located ARD consisting of six ankyrin repeats. The ARD is flanked on the N terminus by the signal response region and on the C terminus by a PEST sequence. Inserted between ankyrin repeats three and four of the IκBβ ARD is a nonconserved sequence of 47 amino acids referred to throughout this paper as the IκBβ insert (Fig. 1). Preliminary in vitro characterization of IκBβ·NF-κB complex formation revealed that the NF-κB p65 subunit N-terminal domain does not contribute significantly to complex binding stability (13Malek S. Chen Y. Huxford T. Ghosh G. J. Biol. Chem. 2001; 276: 45225-45235Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Furthermore, the signal response region and extreme C terminus of IκBβ (amino acids 332–359) were not necessary for stable complex formation. Crystals suitable for x-ray diffraction studies were obtained that contain the IκBβ ARD and PEST and the C-terminal domain and NLS polypeptide of NF-κB subunit p65. Crystals formed in two related triclinic space groups. The first (crystal form I) was solved at 2.5 Å resolution by molecular replacement using an engineered IκB·NF-κB complex as a search model (see "Experimental Procedures"). The second structure (crystal form II) was solved by molecular replacement and refined to 2.1 Å resolution. The two structure models are virtually identical, and atomic coordinates from crystal form I only were used to generate figures. Overall Structure of the IκBβ·NF-κB p65 Homodimer Complex—The global architecture of the IκBβ·NF-κB p65 homodimer complex is similar to that of the IκBα·NF-κB p50/p65 heterodimer complex except for the absence of the p65 N-terminal domain, which is not essential for IκBβ binding (Fig. 2, A and B). IκBβ exhibits the familiar ARD fold (31Gorina S. Pavletich N.P. Science. 1996; 274: 1001-1005Crossref PubMed Scopus (403) Google Scholar, 32Batchelor 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, 33Brotherton D.H. Dhanaraj V. Wick S. Brizuela L. Domaille P.J. Volyanik E. Xu X. Parisini E. Smith B.O. Archer S.J. Serrano M. Brenner S.L. Blundell T.L. Laue E.D. Nature. 1998; 395: 244-250Crossref PubMed Scopus (187) Google Scholar, 34Russo A.A. Tong L. Lee J.O. Jeffrey P.D. Pavletich N.P. Nature. 1998; 395: 237-243Crossref PubMed Scopus (423) Google Scholar, 35Venkataramani R. Swaminathan K. Marmorstein R. Nat. Struct. Biol. 1998; 5: 74-81Crossref PubMed Scopus (78) Google Scholar, 36Yang Y. Nanduri S. Sen S. Qin J. Structure. 1998; 6: 619-626Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 37Michel F. Soler-Lopez M. Petosa C. Cramer P. Siebenlist U. Müller C.W. EMBO J. 2001; 20: 6180-6190Crossref PubMed Scopus (61) Google Scholar, 38Mosavi L.K. Minor Jr., D.L. Peng Z.Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16029-16034Crossref PubMed Scopus (287) Google Scholar). The first two ankyrin repeats of IκBβ contact a segment encompassing the NLS of one p65 subunit, whereas the last three repeats rest against the relatively flat surface formed by the p65 dimerization domains, making only sporadic contacts (Fig. 2, C and D). Only five IκBβ residues C-terminal to the ARD display clear electron density, and none of them contacts p65. The symmetrical p65 subunits contact IκBβ differently. To differentiate between the two p65 subunits, we refer to them as subunit A and subunit B. Subunit B occupies a position analogous to the p50 subunit of the IκBα·NF-κB p50/p65 heterodimer complex. Structural Features of IκBβ—The core ankyrin repeat structure of all ARD-containing proteins is conserved (39Groves M.R. Barford D. Curr. Opin. Struct. Biol. 1999; 9: 383-389Crossref PubMed Scopus (282) Google Scholar, 40Sedgwick S.G. Smerdon S.J. Trends Biochem. Sci. 1999; 24: 311-316Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar). Each repeat contains two anti-parallel α helices followed at nearly a right angle by a loop of variable length. Each repeat begins and ends in a short β hairpin turn. Consecutive helix pairs form a contiguous stack that is stabilized through intra- and inter-repeat hydrophobic interactions. Conserved small and bulky amino acid positions give rise to the curvature of the ankyrin repeat domain. IκB proteins constitute a subfamily within the ankyrin repeat superfamily by virtue of their higher sequence homology and structural similarities both within and outside of the ARD (41Bork P. Proteins. 1993; 17: 363-374Crossref PubMed Scopus (451) Google Scholar). Chief among these differences are inserts within some repeats. The most striking example is the large 47-residue insert after repeat three of IκBβ (Fig. 1). The IκBα protein, by comparison, contains only six additional amino acids inserted at this position. Of the 47 residues inserted with the IκBβ ARD, electron density is clear for only 11 in one of the crystal forms and 12 in the other. This suggests that most of the insert is inherently disordered. In addition to the differences in insert length, sequence variations and structural differences between IκBβ and IκBα with functional consequences occur at the C-terminal portion of the sixth ankyrin repeat and the PEST sequence. Interactions between IκBβ and the p65 Homodimer—Formation of the complex between IκBβ and the NF-κB p65 homodimer buries ∼4000 Å2 solvent-accessible surface area in discontinuous patches (Fig. 2, C and D). The bent shape and apparent structural rigidity of the ARD limits the potential contacts between IκBβ and the relatively planar surface created upon dimerization of NF-κB p65 subunits, giving rise to the disconnected nature of the protein-protein interface. The majority of specific interactions are made between ankyrin repeats one and two of IκBβ and the NLS-containing C-terminal portion of p65 subunit A (NLS polypeptide; p65 amino acids 291–319). These interactions are similar to those observed between IκBα and NF-κB (42Baeuerle P.A. Cell. 1998; 95: 729-731Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar, 43Cramer P. Müller C.W. Structure. 1999; 7: R1-R6Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Furthermore, the structure indicates that a similar NLS polypeptide segment from p65 subunit B is also poised to contact IκBβ. In addition to the NLS contacts, IκBβ uses the inner helices from its final three ankyrin repeats to contact the platform created upon NF-κB p65 subunit dimerization. Both of these components of the IκBβ·NF-κB protein-protein interface are discussed in turn in the sections that follow. Contacts between the NLS Polypeptides of p65 and IκBβ— The NLS polypeptide of p65 subunit A encompasses residues 291–319 and makes extensive contacts with the first two ankyrin repeats of IκBβ, burying more than half the total accessible surface area of the entire IκBβ·NF-κB p65 homodimer complex. The NLS polypeptide of p65 subunit A forms two helices with an approximately orthogonal relative orientation (Fig. 3, A and B). The last four amino acids of the first helix, Lys301, Arg302, Lys303, and Arg304, constitute the functional NLS. Three of these, Lys301, Arg302, and Arg304, contact seven amino acids from the first ankyrin repeat of IκBβ. These are Asp59, Ile67, Asn91, His68, Gln69, Glu56, and Asp57. Except for the substitution of Gln69 to Glu, these amino acids are identical in IκBα. Therefore, it is not surprising that the p65 NLS contacts made by IκBα and IκBβ are highly homologous between the two complexes. The similarity in contacts suggests that these IκB proteins have evolved to interact specifically with the amino acid sequences neighboring the NF-κB p65 subunit NLS. Indeed, we recently showed the NLS polypeptide region of the NF-κB p65 subunit to be the chief specificitydetermining motif in the interaction of NF-κB dimers and IκBα (44Huxford T. Mishler D. Phelps C.B. Huang D.B. Sengchanthalangsy L.L. Reeves R. Hughes C.A. Komives E.A. Ghosh G. J. Mol. Biol. 2002; 324: 587-597Crossref PubMed Scopus (28) Google Scholar). In all, 17 residues of the p65 subunit A NLS polypeptide are involved in contacting IκBβ, in contrast to 14 in the IκBα complex. In addition to salt bridges and polar contacts, the NLS polypeptide makes extensive van der Waals contacts, mainly with the hydrophobic residues on top of the IκBβ first ankyrin repeat (Fig. 3, C and D). In the IκBα·NF-κB complex, the extended conformation of the NLS polypeptide C terminus is stabilized primarily by stacking interactions between Phe77 of IκBα and Phe318 of p65. The IκBβ amino acid homologous to IκBα Phe77 is Ala61. As a result of this substitution, in the IκBβ·p65 homodimer complex the p65 subunit NLS polypeptide C terminus forms a hairpin structure that is stabilized by stacking interactions between Phe73 and Phe76 of IκBβ and Phe318 of p65. Consequently, this region of the IκBβ·NF-κB p65 homodimer complex buries more than 300 Å2 greater solvent-accessible surface area as compared with the analogous portion of the IκBα·NF-κB p50/p65 complex. This suggests that the p65 NLS polypeptide may play a more critical role in contributing to the stability of IκBβ·NF-κB complexes than in the corresponding IκBα complexes. Finally, the increase in observed interactions between IκBβ and p65 in this region of the complex may compensate for the observed lack of interactions between the p65 homodimer and the sixth ankyrin repeat of IκBβ (see below), thus maintaining overall similar stabilities of IκBα·NF-κB and IκBβ·NF-κB complexes (10Thompson J.E. Phillips R.J. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Abstract Full Text PDF PubMed Scopus (699) Google Scholar, 13Malek S. Chen Y. Huxford T. Ghosh G. J. Biol. Chem. 2001; 276: 45225-45235Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). We observe that the electron density corresponding to the NLS polypeptide of the p65 subunit B is present, although considerably weaker than the rest of the molecule in the first of the two crystalline systems. Although continuous backbone density is observed for roughly 15 residues, the side chain densities are unclear, therefore preventing us from unambiguously modeling the NLS polypeptide (Fig. 3E). This difficulty is compounded by the lack of connectivity of backbone density to the core of the dimerization domain. Clear electron density is further lacking in the second crystalline system. It is likely, therefore, that this ambiguous positioning of the p65 subunit B NLS polypeptide and its associated poor electron density are due to extremely weak interactions within this region of the complex. Contacts between the p65 Dimerization Domain and IκBβ— Amino acid residues from the last three ankyrin repeats of IκBβ that contact the dimerization platform of p65 are depicted schematically in Fig. 1. In principle, homologous residues from these three repeats of IκBβ should be able to contact the identical amino positions as in the IκBα complex. Indeed, most of the contacts between ankyrin repeats four and five of IκBβ and the p65 homodimer are conserved from the IκBα complex. However, the inner helix of IκBβ ankyrin repeat six is positioned at a greater distance from the p65 subunit B relative to the IκBα·NF-κB p50/p65 heterodimer complex (Fig. 4A). This is illustrated by the different projections of Arg260 in IκBα and the homologous Arg284 residue of IκBβ (Fig. 4B). Structural differences within this region are also noted in the IκBβ PEST sequence. Although few residues from the IκBβ PEST are ordered, they project away from the protein-protein interface. As a result, more than 300 Å2 buried surface area is lost in this portion of the IκBβ·NF-κB complex as compared with the corresponding region of the IκBα complex. The lack of sequence homology between the sixth repeat of IκBα and IκBβ is most likely responsible for the structural differences displayed by the two proteins in this region. We conclude that the PEST of IκBβ plays a clearly different role in NF-κB recognition than the PEST of IκBα, which is involved in contacting the NF-κB p65 subunit N-terminal domain and disrupting DNA binding. Conserved Features of IκB·NF-κB Complexes—The structure presented here reveals that one p65 NLS polypeptide (subunit A) binds IκBβ in a mode similar to the p65 NLS polypeptide in the IκBα·NF-κB p50/p65 complex (20Huxford T. Huang D.B. Malek S. Ghosh G. Cell. 1998; 95: 759-770Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar, 21Jacobs M.D. Harrison S.C. Cell. 1998; 95: 749-758Abstract Full Text Full Text PDF PubMed Scopus (712) Google Scholar). Three of four basic NLS amino acids directly contact IκBβ, and therefore this NLS appears to be completely masked. Amino acids flanking the NLS also mediate multiple contacts with IκB molecules in both complexes. These extensive interactions are responsible for high affinity binding of the complex (13Malek S. Chen Y. Huxford T. Ghosh G. J. Biol. Chem. 2001; 276: 45225-45235Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). It is possible that in all physiological IκB·NF-κB complexes, one NLS is always completely masked in a manner similar to that observed in these two IκB·NF-κB complexes. Subcellular Distribution of IκB·NF-κB Complexes—With the exception of nuclear Bcl-3·NF-κB complexes, inhibition of NF-κBbyIκB has long been attributed to cytoplasmic retention of IκB·NF-κB complexes. Under this model, IκB was thought to inhibit NF-κB function by masking the nuclear localization signals of NF-κB subunits. However, crystallographic analyses of the IκBα·NF-κB p50/p65 heterodimer complex revealed that although IκBα binds the p65 NLS, it fails to directly contact the NLS of p50 (20Huxford T. Huang D.B. Malek S. Ghosh G. Cell. 1998; 95: 759-770Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar, 21Jacobs M.D. Harrison S.C. Cell. 1998; 95: 749-758Abstract Full Text Full Text PDF PubMed Scopus (712) Google Scholar). Subsequent in vivo immunofluorescence studies showed that IκBα·NF-κB complexes shuttle between the cytoplasm and nucleus in resting cells (15Ghosh S. Karin M. Cell. 2002; 109: S81-S96Abstract Full Text Full Text PDF PubMed Scopus (3329) Google Scholar, 45Johnson C. Van Antwerp D. Hope T.J. EMBO J. 1999; 18: 6682-6693Crossref PubMed Google Scholar, 46Huang T.T. Kudo N. Yoshida M. Miyamoto S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1014-1019Crossref PubMed Scopus (329) Google Scholar, 47Tam W.F. Lee L.H. Davis L. Sen R. Mol. Cell. Biol. 2000; 20: 2269-2284Crossref PubMed Scopus (134) Google Scholar). Nuclear import of the IκBα·NF-κB p50/p65 heterodimer complex is imparted by the free p50 NLS, whereas the one free p65 subunit NLS is sufficient to allow the IκBα·NF-κB p65 homodimer complex to shuttle (13Malek S. Chen Y. Huxford T. Ghosh G. J. Biol. Chem. 2001; 276: 45225-45235Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Active export of the complex from the nucleus relies on nuclear export signals located in the N-terminal signal response region of IκBα and the transactivation domain of the p65 subunit (45Johnson C. Van Antwerp D. Hope T.J. EMBO J. 1999; 18: 6682-6693Crossref PubMed Google Scholar, 46Huang T.T. Kudo N. Yoshida M. Miyamoto S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1014-1019Crossref PubMed Scopus (329) Google Scholar, 48Arenzana-Seisdedos F. Turpin P. Rodriguez M. Thomas D. Hay R.T. Virelizier J.L. Dargemont C. J. Cell Sci. 1997; 110: 369-378Crossref PubMed Google Scholar, 49Harhaj E.W. Sun S.C. Mol. Cell. Biol. 1999; 19: 7088-7095Crossref PubMed Scopus (86) Google Scholar). In contrast to IκBα, IκBβ·NF-κB complexes remain exclusively within the cytoplasm of resting cells (12Huang T.T. Miyamoto S. Mol. Cell. Biol. 2001; 21: 4737-4747Crossref PubMed Scopus (91) Google Scholar, 13Malek S. Chen Y. Huxford T. Ghosh G. J. Biol. Chem. 2001; 276: 45225-45235Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 14Tam W.F. Sen R.J. J. Biol. Chem. 2001; 276: 7701-7704Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 15Ghosh S. Karin M. Cell. 2002; 109: S81-S96Abstract Full Text Full Text PDF PubMed Scopus (3329) Google Scholar). The present structure reveals that the NLS of p65 subunit B is not tightly bound. This mode of binding is not likely to prevent the complex from entering the nucleus. We therefore conclude that our structure is representative of a conformation adopted by a nuclear IκBβ·p65 homodimer complex. We have shown in the accompanying paper that, in conjunction with the small Ras-like GTPase κB-Ras, the non-homologous insert between ankyrin repeats three and four of IκBβ plays an in vivo role in masking this second NF-κB subunit NLS (55Chen Y. Wu J. Ghosh G. J. Biol. Chem. 2003; 278: 23101-23106Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). As a result, nuclear localization of the IκBβ·NF-κB complex is blocked. A Structural Model of the IκBβ·NF-κB·DNA Complex—Several studies have shown that nuclear IκBβ·NF-κB complexes can bind DNA (16Suyang H. Phillips R. Douglas I. Ghosh S. Mol. Cell. Biol. 1996; 16: 5444-5449Crossref PubMed Google Scholar, 17DeLuca C. Petropoulos L. Zmeureanu D. Hiscott J. J. Biol. Chem. 1999; 274: 13010-13016Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 18Phillips R.J. Ghosh S. Mol. Cell. Biol. 1997; 17: 4390-4396Crossref PubMed Google Scholar, 19Newton T.R. Patel N.M. Bhat-Nakshatri P. Stauss C.R. Goulet Jr., R.J. Nakshatri H. J. Biol. Chem. 1999; 274: 18827-18835Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). It has also been shown in vitro that E. coli-expressed, hypophosphorylated IκBβ can complex simultaneously with p65 and DNA to form a ternary complex (11Tran K. Merika M. Thanos D. Mol. Cell. Biol. 1997; 17: 5386-5399Crossref PubMed Google Scholar). The IκBβ·NF-κB complex structure suggests that the N-terminal domains of the p65 homodimer, which contain critical DNA recognition elements, should be free to bind to DNA even upon complex formation of full-length p65 with the wild type IκBβ (Fig. 5). The sixth ankyrin repeat of IκBβ, which lies on the same plane as the bottom of the p65 dimerization domains, does not sterically hinder DNA-contacting amino acid residues from the p65 dimerization domains. In fact, positively charged arginine residues located in the final β turn and the sixth ankyrin repeat of IκBβ could potentially augment affinity of the ternary complex. The PEST sequence of IκBβ, which contains several acidic residues, may exert unfavorable electrostatic repulsion to the DNA phosphodiester backbone. However, because the PEST sequence exhibits some flexibility, it could likely move away from the DNA-protein interface. Additional factors bound to neighboring sites on the target DNA could also serve to neutralize the negative charge of the IκBβ PEST and therefore enhance stability of the ternary complex in a promoter-dependent manner. Furthermore, it is also possible that the observed differential phosphorylation patterns of the IκBβ PEST sequence might play a fundamental role in regulating formation of IκBβ·p65·DNA ternary complexes. Our structure suggests that nuclear IκBβ·p65 complexes may bind to specific NF-κB promoters and modulate gene expression in a manner similar to Bcl-3·p50 or Bcl-3·p52 complexes (50Franzoso G. Bours V. Park S. Tomita-Yamaguchi M. Kelly K. Siebenlist U. Nature. 1992; 359: 339-342Crossref PubMed Scopus (270) Google Scholar, 51Bours V. Franzoso G. Azarenko V. Park S. Kanno T. Brown K. Siebenlist U. Cell. 1993; 72: 729-739Abstract Full Text PDF PubMed Scopus (434) Google Scholar). The x-ray crystal structure of Bcl-3 has been reported recently (37Michel F. Soler-Lopez M. Petosa C. Cramer P. Siebenlist U. Müller C.W. EMBO J. 2001; 20: 6180-6190Crossref PubMed Scopus (61) Google Scholar). The authors propose a ternary complex between DNA bound p50 and Bcl-3 that closely resembles our model. As in the case of Bcl-3, which functions as either a repressor or an activator of transcription depending upon its concentration and phosphorylation states, nuclear IκBβ·NF-κB complexes may also exhibit similarly complex DNA binding properties (52Bundy D.L. McKeithan T.W. J. Biol. Chem. 1997; 272: 33132-33139Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). We wish to acknowledge G. McDermott and staff at the Advanced Light Source beamline 5.0.2 of Berkeley National Laboratory, C. Phelps for aid in synchrotron x-ray data collection, and B. Nolen and members of the G. Ghosh laboratory for critical reading of the manuscript. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences Division of the United States Department of Energy under Contract No. DE-AC03-76SF00098 at Lawrence Berkeley National Laboratory.
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