X-ray Crystal Structure of an I (cid:1) B (cid:2) (cid:1) NF- (cid:1) B p65 Homodimer Complex*

We report the crystal structure of a murine I (cid:1) B (cid:2) (cid:1) NF- (cid:1) B p65 homodimer complex. Crystallographic models were determined for two triclinic crystalline sys-tems and refined against data at 2.5 and 2.1 Å. The overall complex structure is similar to that of the I (cid:1) B (cid:3) (cid:1) NF- (cid:1) B p50/p65 heterodimer complex. One NF- (cid:1) B p65 subunit nuclear localization signal clearly contacts I (cid:1) B (cid:2) , 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 I (cid:1) B (cid:2) is mostly disordered in the structure. Primary sequence analysis and differences in the mode of binding at the I (cid:1) B (cid:2) sixth ankyrin repeat and NF- (cid:1) B p65 homodimer suggest a model for nuclear I (cid:1) B (cid:2) (cid:1) NF- (cid:1) B (cid:1) DNA ternary complex formation. These unique structural

NF-B is a family of inducible transcription factors central to the regulation of diverse biological activities such as immune and inflammatory responses, development, and apoptosis (1)(2)(3). Five polypeptide subunits, p50, p52, p65 (RelA), c-Rel, and RelB, constitute the mammalian NF-B family. Although expressed in most cells, the majority of NF-B homo-and heterodimers with transcriptional activation potential are tightly regulated through stable association with the IB inhibitor proteins IB␣ and IB␤ (4). Various inducing signals, such as cytokines, growth factors, and bacterial and viral products, lead to phosphorylation and eventual proteasome-mediated degradation of NF-B-associated IB proteins (5). IB proteolysis coincides with the release of free NF-B, which binds specifically to DNA enhancer elements in the nucleus and modulates levels of target gene transcription.
IB␣ and IB␤ bind and inhibit the same NF-B homo-and heterodimers and have been shown to be capable of functionally replacing one another (6). Although they function together in regulating the activity of most NF-B dimers, IB␣ and IB␤ do so by differing mechanisms. Due to its rapid degradation and nearly immediate resynthesis upon induction of NF-B, IB␣ participates in fast, transient NF-B activation (7)(8)(9). IB␤, on the other hand, responds to a subset of NF-B inducers and is degraded with slower kinetics (10,11). The reason behind the slower IB␤ degradation is unknown, although it may result, in part, from differences in subcellular localization exhibited by IB␤⅐NF-B and IB␣⅐NF-B complexes. Whereas IB␣⅐NF-B complexes shuttle in and out of the nucleus, IB␤⅐NF-B complexes remain constitutively cytoplasmic in resting cells (12)(13)(14)(15). Finally, under some activating conditions, hypophosphorylated IB␤ has been shown to bind nuclear, DNA-bound NF-B complexes, resulting in a stable nuclear ternary IB␤⅐NF-B⅐DNA complex (16,17). Together, these characteristics unique to IB␤ serve to mediate the persistent activation of NF-B observed in many disease states (18,19).
To investigate the differences in NF-B regulation exhibited by related IB inhibitor proteins, we have determined the x-ray crystal structure of IB␤ bound to the NF-B 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 IB␣⅐NF-B p50/p65 heterodimer complex (20,21). For example, IB␤ contacts the dimerization domains and one nuclear localization signal (NLS) 1 of the dimeric NF-B transcription factor in a manner similar to IB␣. 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 IB␤ does not adopt a folded structure. The structure further reveals that the last ankyrin repeat and the PEST sequence of IB␤ bind NF-B differently as compared with the homologous segments of IB␣. Taken together, these observations suggest a molecular mechanism for persistent NF-B activation and DNA binding of nuclear IB␤⅐NF-B complexes.

EXPERIMENTAL PROCEDURES
Crystallization and X-ray Diffraction Data Collection-Protein expression and crystallization have been reported previously (22). Substitution of potential phosphorylation sites within the IB␤ PEST region to phosphomimetic amino acids fails to increase IB␤⅐NF-B binding affinity. Nevertheless, we used an IB␤ construct with five C-terminal serine residues replaced by glutamates. Recombinant murine IB␤ (amino acids 50 -331) and murine NF-B 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 FIG. 1. Sequence alignment of IB␤ and IB␣. The primary sequences of IB␤ and IB␣ are shown. Alignment was generated in ClustalW (53) and rendered in BOXSHADE. Black triangles border IB␤ amino acid residues 50 -331 corresponding to the protein construct used for crystallization in complex with p65. Secondary structural elements present in the structure are depicted schematically above the sequence, with ␣ helices, ␤ strands, and loops denoted by cylinders, arrows, and thin lines, respectively. The IB␤ signal response region includes sites of inducible phosphorylation denoted by asterisks and conserved lysine sites for polyubiquitination (lysines 21 and 22 of IB␣ and lysine 9 of IB␤). The nuclear export sequence in IB␣ is labeled, as are the insert within the third ankyrin repeat of IB␤ and the C-terminal PEST sequences of both IB␣ and IB␤. Serine amino acids within the C-terminal PEST that were mutated to glutamic acid are denoted by asterisks. Amino acids within 3.9 Å of subunit A of the NF-B p65 homodimer are indicated by the presence of a red circle above the single letter primary sequence. Similar orange-colored circles identify IB␤ residues that contact p65 homodimer subunit B. 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 (23). Structure Solution and Refinement-Initial solution of the complex was obtained by molecular replacement using AMoRe (24). A modified IB␣⅐NF-B 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 IB␣ was used as a search model. A rotational search followed by Patterson correlation refinement gave a clear solution with a peak of 8.4 . No translation search was necessary because the IB␤⅐NF-B complex crystallized in the P1 space group. The orientations and positions of individual domains were refined by rigid body refinement in crystallography NMR software (25). 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 2F O -F C maps using the programs O (26) and TOM (27). 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 IB␤ 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 (28), RASTER3D (29), GRASP (30), and O. Atomic coordinates for the IB␤⅐NF-B 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).

IB␤⅐NF-B p65 Homodimer Complex Crystallization and
Structure Solution-Because of difficulties in obtaining single IB␤⅐NF-B complex crystals suitable for x-ray diffraction analysis, a combinatorial approach to complex formation and crystallization was used (22). All of the NF-B amino acids necessary for dimer formation, DNA binding, nuclear localization, and IB binding are contained within the conserved 300amino 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. IB␤ 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 IB␤ ARD is a nonconserved sequence of 47 amino acids referred to throughout this paper as the IB␤ insert (Fig. 1). Preliminary in vitro characterization of IB␤⅐NF-B complex formation revealed that the NF-B p65 subunit N-terminal domain does not contribute significantly to complex binding stability (13). Furthermore, the signal response region and extreme C terminus of IB␤ (amino acids 332-359) were not necessary for stable complex formation. Crystals suitable for x-ray diffraction studies were obtained that contain the IB␤ ARD and PEST and the C-terminal domain and NLS polypeptide of NF-B 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 IB⅐NF-B 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 IB␤⅐NF-B p65 Homodimer Complex-The global architecture of the IB␤⅐NF-B p65 homodimer complex is similar to that of the IB␣⅐NF-B p50/p65 heterodimer complex except for the absence of the p65 Nterminal domain, which is not essential for IB␤ binding (Fig.  2, A and B). IB␤ exhibits the familiar ARD fold (31-38). The first two ankyrin repeats of IB␤ 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 IB␤ residues C-terminal to the ARD display clear electron density, and none of them contacts p65. The symmetrical p65 subunits contact IB␤ 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 IB␣⅐NF-B p50/p65 heterodimer complex.
Structural Features of IB␤-The core ankyrin repeat structure of all ARD-containing proteins is conserved (39,40). Each repeat contains two anti-parallel ␣ helices followed at nearly a The complex is aligned so that the p65 homodimer 2-fold pseudosymmetry axis runs vertically in the plane of the page. Dashed lines indicate disordered regions. IB␤ is shown in cyan, and p65 subunits A and B are shown in red and orange, respectively. The same color scheme is used to represent the three complex components throughout the rest of the figures in this paper. B, the complex is rotated 90°about the vertical axis of p65. C, molecular surface representation of the p65 homodimer. Amino acids from subunit A within 3.9 Å of IB␤ are colored red, and residues from subunit B that contact IB␤ are shown in orange. IB␤ is depicted as a cyan-colored worm. The complex is viewed in an orientation identical to that described in A. D, the molecular surface of IB␤ is depicted, with amino acids that contact p65 homodimer colored cyan. The p65 homodimer subunits A and B are depicted as red and orange backbone worms, respectively. The entire complex is rotated 180°about the vertical axis from the view in C.
right angle by a loop of variable length. Each repeat begins and ends in a short ␤ hairpin turn. Consecutive helix pairs form a contiguous stack that is stabilized through intra-and interrepeat hydrophobic interactions. Conserved small and bulky amino acid positions give rise to the curvature of the ankyrin repeat domain.
IB 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 (41). Chief among these differences are inserts within some repeats. The most striking example is the large 47-residue insert after repeat three of IB␤ (Fig. 1). The IB␣ protein, by comparison, contains only six additional amino acids inserted at this position. Of the 47 residues inserted with the IB␤ 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 IB␤ and IB␣ with functional consequences occur at the Cterminal portion of the sixth ankyrin repeat and the PEST sequence.
Interactions between IB␤ and the p65 Homodimer-Formation of the complex between IB␤ and the NF-B p65 homodimer buries ϳ4000 Å 2 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 IB␤ and the relatively planar surface created upon dimerization of NF-B 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 IB␤ 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 IB␣ and NF-B (42,43). Furthermore, the structure indicates that a similar NLS polypeptide segment from p65 subunit B is also poised to contact IB␤. In addition to the NLS contacts, IB␤ uses the inner helices from its final three ankyrin repeats to contact the platform created upon NF-B p65 subunit dimerization. Both of these components of the IB␤⅐NF-B protein-protein interface are discussed in turn in the sections that follow.
Contacts between the NLS Polypeptides of p65 and IB␤-The NLS polypeptide of p65 subunit A encompasses residues 291-319 and makes extensive contacts with the first two ankyrin repeats of IB␤, burying more than half the total accessible surface area of the entire IB␤⅐NF-B 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, Lys 301 , Arg 302 , Lys 303 , and Arg 304 , constitute the functional NLS. Three of these, Lys 301 , Arg 302 , and Arg 304 , contact seven amino acids from the first ankyrin repeat of IB␤. These are Asp 59 , Ile 67 , Asn 91 , His 68 , Gln 69 , Glu 56 , and Asp 57 . Except for the substitution of Gln 69 to Glu, these amino acids are identical in IB␣. Therefore, it is not surprising that the p65 NLS contacts made by IB␣ and IB␤ are highly homologous between the two complexes. The similarity in contacts suggests that these IB proteins have evolved to interact specifically with the amino acid sequences neighboring the NF-B p65 subunit NLS. Indeed, we recently showed the NLS polypeptide region of the NF-B p65 subunit to be the chief specificitydetermining motif in the interaction of NF-B dimers and IB␣ (44).
In all, 17 residues of the p65 subunit A NLS polypeptide are involved in contacting IB␤, in contrast to 14 in the IB␣ 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 IB␤ first ankyrin repeat (Fig. 3, C and D). In the IB␣⅐NF-B complex, the extended conformation of the NLS polypeptide C terminus is stabilized primarily by stacking interactions between Phe 77 of IB␣ and Phe 318 of p65. The IB␤ amino acid homologous to IB␣ Phe 77 is Ala 61 . As a result of this substitution, in the IB␤⅐p65 homodimer complex the p65 subunit NLS polypeptide C terminus forms a hairpin structure that is stabilized by stacking interactions between Phe 73 and Phe 76 of IB␤ and Phe 318 of p65. Consequently, this region of the IB␤⅐NF-B p65 homodimer complex buries more than 300 Å 2 greater solventaccessible surface area as compared with the analogous portion of the IB␣⅐NF-B p50/p65 complex. This suggests that the p65 NLS polypeptide may play a more critical role in contributing to the stability of IB␤⅐NF-B complexes than in the corresponding IB␣ complexes. Finally, the increase in observed interactions between IB␤ 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 IB␤ (see below), thus maintaining overall similar stabilities of IB␣⅐NF-B and IB␤⅐NF-B complexes (10,13).
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 IB␤-Amino acid residues from the last three ankyrin repeats of IB␤ that contact the dimerization platform of p65 are depicted schematically in Fig. 1. In principle, homologous residues from these three repeats of IB␤ should be able to contact the identical amino positions as in the IB␣ complex. Indeed, most of the contacts between ankyrin repeats four and five of IB␤ and the p65 homodimer are conserved from the IB␣ complex. However, the inner helix of IB␤ ankyrin repeat six is positioned at a greater distance from the p65 subunit B relative to the IB␣⅐NF-B p50/p65 heterodimer complex (Fig. 4A). This is illustrated by the different projections of Arg 260 in IB␣ and the homologous Arg 284 residue of IB␤ (Fig. 4B). Structural differences within this region are also noted in the IB␤ PEST sequence. Although few residues from the IB␤ PEST are ordered, they project away from the protein-protein interface. As a result, more than 300 Å 2 buried surface area is lost in this portion of the IB␤⅐NF-B complex as compared with the corresponding region of the IB␣ complex. The lack of sequence homology between the sixth repeat of IB␣ and IB␤ is most likely responsible for the structural differences displayed by the two proteins in this region. We conclude that the PEST of IB␤ plays a clearly different role in NF-B recognition than the PEST of IB␣, which is involved in contacting the NF-B p65 subunit N-terminal domain and disrupting DNA binding.

Conserved Features of IB⅐NF-B Complexes-
The structure presented here reveals that one p65 NLS polypeptide (subunit A) binds IB␤ in a mode similar to the p65 NLS polypeptide in the IB␣⅐NF-B p50/p65 complex (20,21). Three of four basic NLS amino acids directly contact IB␤, and therefore this NLS appears to be completely masked. Amino acids flanking the NLS also mediate multiple contacts with IB molecules in both complexes. These extensive interactions are responsible for high affinity binding of the complex (13). It is possible that in all physiological IB⅐NF-B complexes, one NLS is always completely masked in a manner similar to that observed in these two IB⅐NF-B complexes.
Subcellular Distribution of IB⅐NF-B Complexes-With the exception of nuclear Bcl-3⅐NF-B complexes, inhibition of NF-B by IB has long been attributed to cytoplasmic retention of IB⅐NF-B complexes. Under this model, IB was thought to inhibit NF-B function by masking the nuclear localization signals of NF-B subunits. However, crystallographic analyses of the IB␣⅐NF-B p50/p65 heterodimer complex revealed that although IB␣ binds the p65 NLS, it fails to directly contact the NLS of p50 (20,21). Subsequent in vivo immunofluorescence studies showed that IB␣⅐NF-B complexes shuttle between the cytoplasm and nucleus in resting cells (15,(45)(46)(47). Nuclear import of the IB␣⅐NF-B p50/p65 heterodimer complex is imparted by the free p50 NLS, whereas the one free p65 subunit FIG. 4. Comparison of the interactions between the sixth ankyrin repeat of IB␤ and IB␣ and the p65 subunit A. A, superposition of the p65 subunit A dimerization domain from the two IB⅐NF-B complexes reveals the overall similarity in position and fold of the complex-associated IB␤ and IB␣. Color scheme is consistent with previous figures, except that the p65 subunit from the IB␣⅐NF-B complex structure is depicted in pink. Ankyrin repeats are numbered. One noticeable difference (enclosed within the dashed black box) is that the sixth ankyrin repeat of IB␤ occupies a position at a greater distance from p65 than does the corresponding segment of IB␣. B, close up stereoview of the boxed area from A. Arg 260 and Trp 258 are labeled as reference points.
NLS is sufficient to allow the IB␣⅐NF-B p65 homodimer complex to shuttle (13). Active export of the complex from the nucleus relies on nuclear export signals located in the N-terminal signal response region of IB␣ and the transactivation domain of the p65 subunit (45,46,48,49). In contrast to IB␣, IB␤⅐NF-B complexes remain exclusively within the cytoplasm of resting cells (12)(13)(14)(15).
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 IB␤⅐p65 homodimer complex. We have shown in the accompanying paper that, in conjunction with the small Ras-like GTPase B-Ras, the non-homologous insert between ankyrin repeats three and four of IB␤ plays an in vivo role in masking this second NF-B subunit NLS (55). As a result, nuclear localization of the IB␤⅐NF-B complex is blocked.
A Structural Model of the IB␤⅐NF-B⅐DNA Complex-Several studies have shown that nuclear IB␤⅐NF-B complexes can bind DNA (16 -19). It has also been shown in vitro that E. coli-expressed, hypophosphorylated IB␤ can complex simultaneously with p65 and DNA to form a ternary complex (11). The IB␤⅐NF-B complex structure suggests that the Nterminal 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 IB␤ (Fig. 5). The sixth ankyrin repeat of IB␤, 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 ␤ turn and the sixth ankyrin repeat of IB␤ could potentially augment affinity of the ternary complex. The PEST sequence of IB␤, 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 IB␤ 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 IB␤ PEST sequence might play a fundamental role in regulating formation of IB␤⅐p65⅐DNA ternary complexes.
Our structure suggests that nuclear IB␤⅐p65 complexes may bind to specific NF-B promoters and modulate gene expression in a manner similar to Bcl-3⅐p50 or Bcl-3⅐p52 complexes (50,51). The x-ray crystal structure of Bcl-3 has been reported recently (37). 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 IB␤⅐NF-B complexes may also exhibit similarly complex DNA binding properties (52).