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• W2041503007 abstract "Article15 November 2001free access Crystal structure of the ankyrin repeat domain of Bcl-3: a unique member of the IκB protein family Fabrice Michel Fabrice Michel European Molecular Biology Laboratory, Grenoble Outstation BP 181, 38042 Grenoble, Cedex 9, France Search for more papers by this author Montserrat Soler-Lopez Montserrat Soler-Lopez European Molecular Biology Laboratory, Grenoble Outstation BP 181, 38042 Grenoble, Cedex 9, France Search for more papers by this author Carlo Petosa Carlo Petosa European Molecular Biology Laboratory, Grenoble Outstation BP 181, 38042 Grenoble, Cedex 9, France Search for more papers by this author Patrick Cramer Patrick Cramer European Molecular Biology Laboratory, Grenoble Outstation BP 181, 38042 Grenoble, Cedex 9, France Present address: Institute of Biochemistry, Gene Center, University of Munich, Feodor-Lynen-Strasse 25, D-81377 Munich, Germany Search for more papers by this author Ulrich Siebenlist Ulrich Siebenlist Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892 USA Search for more papers by this author Christoph W. Müller Corresponding Author Christoph W. Müller European Molecular Biology Laboratory, Grenoble Outstation BP 181, 38042 Grenoble, Cedex 9, France Search for more papers by this author Fabrice Michel Fabrice Michel European Molecular Biology Laboratory, Grenoble Outstation BP 181, 38042 Grenoble, Cedex 9, France Search for more papers by this author Montserrat Soler-Lopez Montserrat Soler-Lopez European Molecular Biology Laboratory, Grenoble Outstation BP 181, 38042 Grenoble, Cedex 9, France Search for more papers by this author Carlo Petosa Carlo Petosa European Molecular Biology Laboratory, Grenoble Outstation BP 181, 38042 Grenoble, Cedex 9, France Search for more papers by this author Patrick Cramer Patrick Cramer European Molecular Biology Laboratory, Grenoble Outstation BP 181, 38042 Grenoble, Cedex 9, France Present address: Institute of Biochemistry, Gene Center, University of Munich, Feodor-Lynen-Strasse 25, D-81377 Munich, Germany Search for more papers by this author Ulrich Siebenlist Ulrich Siebenlist Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892 USA Search for more papers by this author Christoph W. Müller Corresponding Author Christoph W. Müller European Molecular Biology Laboratory, Grenoble Outstation BP 181, 38042 Grenoble, Cedex 9, France Search for more papers by this author Author Information Fabrice Michel1, Montserrat Soler-Lopez1, Carlo Petosa1, Patrick Cramer1,2, Ulrich Siebenlist3 and Christoph W. Müller 1 1European Molecular Biology Laboratory, Grenoble Outstation BP 181, 38042 Grenoble, Cedex 9, France 2Present address: Institute of Biochemistry, Gene Center, University of Munich, Feodor-Lynen-Strasse 25, D-81377 Munich, Germany 3Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:6180-6190https://doi.org/10.1093/emboj/20.22.6180 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info IκB proteins associate with the transcription factor NF-κB via their ankyrin repeat domain. Bcl-3 is an unusual IκB protein because it is primarily nucleoplasmic and can lead to enhanced NF-κB-dependent transcription, unlike the prototypical IκB protein IκBα, which inhibits NF-κB activity by retaining it in the cytoplasm. Here we report the 1.9 Å crystal structure of the ankyrin repeat domain of human Bcl-3 and compare it with that of IκBα bound to NF-κB. The two structures are highly similar over the central ankyrin repeats but differ in the N-terminal repeat and at the C-terminus, where Bcl-3 contains a seventh repeat in place of the acidic PEST region of IκBα. Differences between the two structures suggest why Bcl-3 differs from IκBα in selectivity towards various NF-κB species, why Bcl-3 but not IκBα can associate with its NF-κB partner bound to DNA, and why two molecules of Bcl-3 but only one of IκBα can bind to its NF-κB partner. Comparison of the two structures thus provides an insight into the functional diversity of IκB proteins. Introduction Bcl-3 is a putative oncoprotein first discovered during studies of B-cell chronic lymphocytic leukemias (Ohno et al., 1990). Bcl-3 belongs to the IκB family of proteins, which modulate the DNA-binding activity and subcellular localization of the transcription factor NF-κB (reviewed in Ghosh et al., 1998). NF-κB regulates the expression of a wide variety of cellular and viral genes, including those involved in immune and stress responses, apoptosis and cellular proliferation. NF-κB is a homo- or heterodimer of proteins belonging to the Rel family, which includes p65 (RelA), RelB, c-Rel, p50 and p52, with the p50–p65 heterodimer constituting the principal NF-κB species. Rel proteins share a 300-residue Rel homology region (RHR) responsible for dimerization, DNA binding, nuclear localization and binding of IκB proteins. Crystal structures of the DNA-bound RHRs of several Rel proteins reveal that the RHR consists of two immunoglobin-like domains followed by a C-terminal basic type I nuclear localization signal (NLS) that is disordered (Ghosh et al., 1995; Müller et al., 1995; Cramer et al., 1997; F.E.Chen et al., 1998; Y.Q.Chen et al., 1998). The N-terminal immunoglobin-like domain (RHR-n) is primarily responsible for DNA binding specificity, whereas subunit dimerization is exclusively mediated by the C-terminal domain (RHR-c). While p65, c-Rel and RelB contain a C-terminal trans-activation domain, p50 and p52 do not and thus behave as transcriptional repressors in their homodimeric forms (Baeuerle and Henkel, 1994). The mammalian members of the IκB family include Bcl-3, the IκB proteins α, β, γ and ϵ, and the Rel protein precursors p100 and p105. These proteins are characterized by N- and C-terminal domains of variable length and sequence and by a conserved central ankyrin (ANK) repeat domain (ARD) that mediates the interaction with NF-κB. The ARD contains six or seven copies of the ANK repeat, a 33-residue sequence motif present in a large number of eukaryotic and prokaryotic proteins (reviewed in Michaely and Bennett, 1992; Bork, 1993). Crystal structures of several such proteins reveal that ANK repeats form an L-shaped structure composed of a β-hairpin and two antiparallel α-helices packing together to yield a left-handed superhelix (reviewed in Sedgwick and Smerdon, 1999). The various IκB proteins target different NF-κB species: IκBα and IκBβ bind preferentially to p50–p65 and p50–c-Rel heterodimers (Thompson et al., 1995), IκBϵ associates only with homo- or heterodimeric complexes containing p65 and c-Rel (Simeonidis et al., 1997; Whiteside et al., 1997), and Bcl-3 interacts specifically with p50 and p52 homodimers (Franzoso et al., 1992; Wulczyn et al., 1992). In contrast, p100 and p105, which are precursors of the Rel proteins p50 and p52, respectively, appear to bind efficiently to all mammalian Rel proteins. The best characterized IκB protein is IκBα, which contains an N-terminal ‘signal-receiving domain’ (SRD) that becomes phosphorylated in response to various extracellular stimuli, an ARD composed of six repeats and a C-terminal PEST domain involved in basal turn over (reviewed in Baeuerle, 1998; Ghosh et al., 1998; Figure 1A). IκBα retains NF-κB in the cytoplasm by masking the latter's NLS. NF-κB-stimulating signals lead to the phosphorylation, polyubiquitylation and proteasome-mediated degradation of IκBα, permitting NF-κB to be translocated to the nucleus via its newly exposed NLS and to bind to target DNA sites. IκBα, which is rapidly resynthesized after degradation, can enter the nucleus and promote the dissociation of NF-κB from DNA. Figure 1.Structure of Bcl-3. (A) Domain organization of Bcl-3 and IκBα. The basic nuclear localization sequence (NLS) of Bcl-3 is indicated in blue, while the leucine-rich NLS and NES of IκBα are in green. The fragment of Bcl-3 that was crystallized spans residues 119–359 and encompasses the entire ARD. (B) Ribbon diagram of the Bcl-3 ARD. The molecule curves towards the α1 helices, which together with the β-hairpins forms the presumed binding surface for p50 and p52 homodimers. This figure and Figures 3B and C, 5 and 6 were made with Bobscript (Esnouf, 1999) and Raster3D (Merritt and Bacon, 1997). Download figure Download PowerPoint The structure of a complex of partially deleted fragments of IκBα and of an NF-κB p50–p65 heterodimer reveals that the ARD of IκBα interacts extensively with the RHR moieties of NF-κB (Huxford et al., 1998; Jacobs and Harrison, 1998). ANK repeats 1 and 2 interact with and induce the p65 NLS, which is unstructured in solution, to adopt a helical conformation. ANK repeats 4–6 contact the p50–p65 dimerization interface located within the two RHR-c domains, while ANK6 and the C-terminal PEST residues interact with the p65 RHR-n domain, which becomes re-oriented with respect to its position when bound to DNA. At present, the only reported structure of an IκB protein is that of IκBα bound to NF-κB. In order to better understand the NF-κB–IκB signaling system, we have undertaken structural studies of Bcl-3. Bcl-3 is unique among the IκB proteins in that it is primarily localized in the nucleus and is not degraded upon activation of NF-κB-stimulating pathways (Franzoso et al., 1993; Nolan et al., 1993; Zhang et al., 1994). Moreover, whereas IκBα and IκBβ belong to a subclass of IκB proteins containing six ANK repeats, Bcl-3 contains seven repeats and lacks an acidic PEST sequence, forming a distinct subclass together with p100, p105 and (probably) IκBϵ. Bcl-3 has been reported to possess various activities, which partly depend on its concentration, phosphorylation state and interaction with other proteins (Wulczyn et al., 1992; Nolan et al., 1993; Bundy and McKeithan, 1997; Dechend et al., 1999). Many of these activities lead to increased transcription from the κB promoter, in contrast to IκBα, which appears to behave exclusively as an inhibitor. Thus, Bcl-3 can cause DNA-bound p50 homodimers to dissociate from the κB site, permitting these inhibiting NF-κB species to be replaced by trans-activating species containing p65, RelB or c-Rel (Franzoso et al., 1992, 1993; Inoue et al., 1993). Bcl-3 can also form a ternary complex with DNA-bound p50 or p52 homodimers and activate transcription directly, an activity that requires both N- and C-terminal domains of Bcl-3 (Bours et al., 1993; Fujita et al., 1993; Pan and McEver, 1995; Hirano et al., 1998). Furthermore, expression of Bcl-3 can cause p50 to be released from cytosolic p105–p50 complexes and to translocate to the nucleus in a trans-activating complex containing Bcl-3 (Watanabe et al., 1997; Heissmeyer et al., 1999). Here we report the crystal structure of the ARD of human Bcl-3 at 1.9 Å resolution. Although the central ANK repeats are highly similar in structure to those of the IκBα ARD, there are several important differences at the N- and C-termini. Comparison of the two structures provides an insight into why Bcl-3 binds specifically to p50 or p52 homodimers whereas IκBα prefers p65–p50 heterodimers, why Bcl-3 but not IκBα can form a complex with its DNA-bound NF-κB partner, and why Bcl-3 can bind its NF-κB partner symmetrically under certain conditions (two Bcl-3 molecules per homodimer) (Bundy and McKeithan, 1997), whereas IκBα can only bind asymmetrically. The structure of the Bcl-3 ARD should also serve as a better model for the other seven-repeat IκB members of its subclass. Results and discussion Structure determination Human Bcl-3 is a 446-residue protein composed of a central ARD flanked by N- and C-terminal domains that are rich in proline and serine residues (Figure 1A). Reasoning that these flanking domains would be poorly ordered and hamper crystallization, we set out to crystallize only the ARD. Accordingly, a 241-residue fragment spanning residues 119–359 was expressed in Escherichia coli and purified to near homogeneity. Although the protein aggregated at the high concentrations normally used during crystallization trials, it remained relatively monodisperse at low concentrations (∼3 mg/ml), yielding small, well-diffracting crystals. Two related monoclinic crystal forms were obtained, each with one molecule per asymmetric unit. The structure was solved by molecular replacement using the ARD of human IκBα and refined at 1.9 Å resolution in both crystal forms (Table I; Materials and methods). The structure is essentially identical in the two forms, apart from a few side chain conformations that reflect differences in crystal packing interactions. All residues of the expressed fragment are well ordered, except for a small number of N- and C-terminal residues (119–124 and 353–359), for which no density is visible in either crystal form. The final model includes residues 125–352 and either 195 (form I) or 198 (form II) water molecules. Table 1. Data collection and refinement statistics Data set Form I Form II Space group P21 P21 Cell parameters a = 31.7 Å a = 31.4 Å b = 51.2 Å b = 50.9 Å c = 64.7 Å c = 59.5 Å β = 102.0° β = 102.0° Beamline ID14-EH1 ID14-EH1 Resolution (Å) 40.0−1.8 40.0−1.9 Completeness (%)a 89.8 (77.8) 98.6 (94.4) Total observations 46 088 49 074 Unique reflections 17 021 14 381 Redundancya 2.71 (1.73) 3.41 (2.68) Rsym (%)a,b 7.2 (69.7) 9.7 (26.0) I/σIa 10.5 (1.65) 15.1 (3.15) Refinement resolution (Å) 20.0−1.86 20.0−1.9 total number of protein atoms 1729 1716 number of water molecules 195 198 R.m.s.d. from ideal geometry bond lengths (Å) 0.007 0.007 bond angles (°) 0.94 1.00 R-factor (%) (reflections)c 19.9 (15 500) 17.7 (13 489) Rfree (%) (reflections)d 22.9 (828) 21.7 (709) a Data for the highest resolution shell are given in parentheses. b Rsym = Σ|I − <I>|/ΣI, where I is the intensity of the individual reflections and <I> is the mean intensity over symmetrically equivalent reflections. c R = Σ||Fobs| − |Fcalc||/Σ|Fobs|, where Fobs and Fcalc are observed and calculated structure factor amplitudes, respectively. d Rfree is the same calculation including only the randomly chosen 5% of reflections not used for refinement. Structure of the Bcl-3 ARD The seven ANK repeats of the Bcl-3 ARD form an elongated structure with approximate dimensions of 75 × 25 × 25 Å (Figure 1B). As in IκBα and other ANK repeat proteins (for review see Sedgwick and Smerdon, 1999), each repeat consists of an initial β-hairpin followed by a helix–turn–helix motif nearly perpendicular to it. The overall shape of the ARD has been compared with that of a ‘cupped hand’ (Jacobs and Harrison, 1998) such that the β-hairpins form the fingers, the α1 ‘inner’ helices form the palm and the α2 ‘outer’ helices form the back of the hand. The ANK repeats stack together with a slight left-handed twist, such that a complete turn of the superhelix would require a total of 32 repeats, with a 245 Å pitch and a 35 Å radius. The seven ANK repeats are fairly uniform in backbone conformation [mean root mean square deviation (r.m.s.d.)Cα = 1.7 Å for all pairwise comparisons], as expected from their degree of sequence similarity (mean pairwise sequence identity = 27%) (Figure 2). The structures of ANK repeats 2, 4 and 6 are the most similar (r.m.s.d.Cα = 0.49−0.78 Å), reflecting their higher sequence identity (30–39%). ANK1 lacks the initial β-hairpin and contains a four-residue insertion after helix α2, as does ANK3, causing the ARD to bend slightly towards the interhelical turns of these repeats. Compared with the other repeats, ANK5 contains an additional residue, Lys266, inserted within the β-hairpin, creating a slightly wider ‘fingertip’ (Figure 2). A similar insertion, Cys215, occurs in the corresponding β-hairpin of IκBα. ANK7 deviates most from the other repeats in sequence (15–27% identity) but adopts a similar overall conformation, except that a three-residue deletion between the two helices results in an unusually short α1 helix (Figure 2). Figure 2.Alignment of the seven ANK repeats in Bcl-3. (A) Stereo view of superimposed Cα atom traces of the ANK repeats. ANK repeats 2, 4 and 6 (black traces) are the most similar, while larger deviations occur in ANK repeats 1, 3, 5 and 7 (green, orange, red and cyan, respectively). (B) Structure-based sequence alignment of the seven ANK motifs. Highly conserved polar, hydrophobic and small residues are highlighted on a red, green and gray background, respectively. The consensus numbering of residue positions shown at the bottom is that of Jacobs and Harrison (1998). Lower case residues at the N- and C-termini are disordered in the crystal structure. The precise limits of the α1 and α2 helices are shown by underlining. Download figure Download PowerPoint A structure-based alignment of the seven ANK motifs (Figure 2B) shows that half of the residue positions are highly conserved. Residues Ala(15) in helix α1 and Leu(27) in helix α2 are invariant across all repeats, and together with five other conserved hydrophobic residues (at positions 12, 14, 16, 24 and 28 on the two helices) are involved in packing interactions within and between repeats, giving rise to a continuous hydrophobic core running the length of the molecule. As noted for IκBα (Huxford et al., 1998; Jacobs and Harrison, 1998), helix α1 contains hydrophobic residues (Ala or Val) smaller than those in helix α2 (mostly Leu), causing the stack of ANK repeats to curve slightly towards the α1 helix. Residue positions 10–13 comprise the signature TPLH motif characteristic of ANK repeats; these form a tight turn initiating helix α1, stabilized by hydrogen bonds involving residues Thr(10) and His(13). A conserved asparagine or aspartic acid residue at position 5 stabilizes the β-hairpin by hydrogen bonding with backbone nitrogen atoms at positions 7 and 9. Conserved glycines at positions 8 and 31 adopt backbone dihedral angles in the αL region of the Ramachandran plot, allowing a sharp turn in chain direction within the β-hairpin and at the end of helix α2, respectively. A small alanine residue is preferred at position 3, where it packs against side chain atoms from the β-hairpin of the preceding repeat, and at position 32, where it is buried against conserved leucine residues at positions 12 and 27 of the same repeat. A conserved hydrophilic residue at position 25 in helix α2 is exposed to the solvent on the convex surface of the ARD. Whereas ANK repeats 2–6 each possess two hydrophobic faces mediating interactions with the preceding and successive repeats, ANK repeats 1 and 7 have a unique solvent-exposed face and thus deviate from the consensus sequence to avoid exposing hydrophobic residues. On the bottom surface of ANK7, two arginines (Arg342 and Arg351) replace the consensus valine or leucine residues at positions 16 and 28, while a third (Arg344) at position 21 replaces the leucine or proline residue usually present in the other repeats. However, a small patch of hydrophobic residues (formed by Met339, Ile347 and Leu338) remains exposed on this surface, which may explain why the protein aggregates at high concentrations. On the top surface of ANK1, alanine and arginine residues are present at positions 23 and 26 on helix α2, which are usually hydrophobic residues in the other repeats, while the four-residue extension at the end of helix α2 contributes three polar residues (Gln151, Gln152 and Arg155) to this surface. Comparison of Bcl-3 and IκBα tertiary structures The ARDs of Bcl-3 and IκBα share 35% sequence identity over the first six ANK repeats (Figure 3A) and, as expected, have very similar overall structures (r.m.s.d.192Cα = 1.5 Å; Figure 3B). This indicates that a large conformational change is not required for IκB proteins to bind their NF-κB partners, as the structures compared are of IκBα in its bound state and Bcl-3 in its unbound state. Minor differences occur in residues joining ANK repeats 3 and 4, where the two proteins contain sequence insertions of differing length. This region contains a short helical segment in IκBα but has no regular secondary structure in Bcl-3 (Figure 3B, red dot). Figure 3.Comparison of the Bcl-3 and IκBα structures. (A) Sequence alignment of Bcl-3 homologs. Secondary structure elements are indicated above the Bcl-3 and below the IκBα sequences. Large insertions in the IκBβ, p100 and p105 sequences are indicated by numbers in parentheses. Residues are highlighted in yellow if identical or nearly identical to those in Bcl-3 [residues considered nearly identical are: (D,E), (R,K), (F,Y), (S,C) and (I,L,V)]. IκBα residues that interact with the p50 or p65 dimerization domains are boxed in red and blue, respectively. Those in contact with the NLS-containing helix of p65 are circled in blue and those in contact with the C-terminal p65 helix (involved in the hydrophobic capping interaction) are circled in red. Circled residues are contacts observed in the structure by Jacobs and Harrison (1998); contacts involving IκBα residues 280 and 281 are observed in that by Huxford et al. (1998). All other contacts are common to both structures. (B) Superimposition of the Bcl-3 and IκBα Cα backbones. Bcl-3 and IκBα are in green and black, respectively. The view is that of Figure 1B rotated by ∼180° about the vertical axis. The IκBα trace was generated by superimposing the two known IκBα structures, as one has a greater number of ordered residues within N-terminal ANK repeats (Jacobs and Harrison, 1998; solid line) while the other has a more complete PEST region (Huxford et al., 1998; broken line). Differences between Bcl-3 and IκBα within residues joining ANK repeats 3 and 4 and within the interhelical turn of ANK6 are marked by a dot and an asterisk, respectively. (C) Differences between Bcl-3 and IκBα within ANK1. Proteins are colored as in (B). The conformational difference of the N-terminal residues is indicated by a double-headed arrow. Residue Glu85 in IκBα is replaced by Gly138 in Bcl-3. Shown in blue are residues at the C-terminus of p65 that form two helices upon binding to IκBα: the first (residues 293–305) contains the basic NLS and the second (residues 305–320) is involved in a hydrophobic ‘capping’ interaction with ANK1. Download figure Download PowerPoint The most significant differences occur at the N- and C-termini. In ANK1, helix α2 in Bcl-3 is two residues longer than in IκBα, causing residues joining ANK1 with ANK2 to follow significantly different courses. More importantly, in IκBα the N-terminal residues 71–76 adopt a β-hairpin conformation similar to that observed in the canonical ANK motif structure, with residues Asp73 and Asp75 interacting with basic residues of the p65 NLS (Figure 3C). In contrast, the N-terminus of Bcl-3 follows a nearly perpendicular course, such that the corresponding Asp126 and Asp128 side chains point in the opposite direction. The absence of the N-terminal hairpin in Bcl-3 cannot be due to the choice of fragment used for crystallization, as the Bcl-3 construct (beginning at residue 119, equivalent to IκBα residue 66) contains three more residues at the N-terminus than the IκBα fragment (residues 69–288) which contains the N-terminal hairpin (Jacobs and Harrison, 1998). Moreover, there is ample room in the crystal for the N-terminal residues of Bcl-3 to form a β-hairpin. Instead, the observed conformation is likely to be due to crystal packing interactions, as Asp125 and Asp128 interact with Arg318 and Arg322 of a neighboring molecule. This suggests that a β-hairpin in ANK1 is relatively unstable or perhaps completely missing in the absence of a basic NLS peptide from a bound NF-κB species. At the C-terminus, ANK repeat 7 of Bcl-3 and the PEST region of IκBα comprise approximately the same number of residues, occupying similar volumes below ANK repeat 6. However, the PEST backbone turns in the opposite direction to that taken by ANK7 residues, such that the C-termini of IκBα and Bcl-3 are on opposite faces of the ARD. Substantial deviations between the two protein backbones (up to a maximum of 5 Å) also occur within the helices and the interhelical turn of ANK repeat 6, where Bcl-3 helices α1 and α2 are slightly longer than their IκBα counterparts (Figure 3B, asterisk). Comparison of Bcl-3 and IκBα molecular surfaces The electrostatic surface potentials of the Bcl-3 and IκBα structures are appreciably different (Figure 4). The ARD of Bcl-3 is considerably less acidic than that of IκBα: the theoretical pI value is 6.6 for Bcl-3 and 5.6 for IκBα (or 4.8 including the PEST sequence). This difference is partly due to the replacement of six glutamic acid residues in the first three ANK repeats of IκBα by uncharged or basic residues in Bcl-3. Consequently, the upper surface of IκBα is more negatively charged than that of Bcl-3 (Figure 4A and D, green dot). The difference in pI is also due to a greater number of basic residues in Bcl-3, which has a remarkable preference for arginine (17 residues) over lysine (only two). Seven arginine residues cluster in the outer helices and interhelical turns of ANK repeats 6 and 7, creating a large basic patch at the bottom of the ARD. In contrast, the corresponding surface of IκBα is highly acidic due to the presence of the PEST sequence (Figure 4, compare A with D and E with H). Figure 4.Comparison of Bcl-3 and IκBα molecular surfaces. The structures of IκBα (A, B, E and F) and Bcl-3 (C, D, G and H) are shown in equivalent orientations. The view of (A)–(D) is orthogonal to that of (E)–(H), which is approximately that of Figure 1B (i.e. with β-hairpins on the left and α2 helices on the right.) (A, D, E and H) Comparison of electrostatic surface potentials. Regions of negative and positive potential are shown in red and blue, respectively. The basic patch at the bottom of Bcl-3 is formed by arginine residues 311, 318, 322, 342, 344, 345 and 351. The corresponding surface of IκBα is formed by the acidic PEST region. (B, C, F and G) Conservation of the NF-κB contact surface. The C-terminal domains of p50 (blue) and p65 (green) are represented as ribbons bound to the surface of IκBα and, to facilitate comparison, to that of Bcl-3. In (B) and (F), regions of the IκBα surface within 4.5 Å of the p50 and p65 RHR-c domains are colored magenta. In (C) and (G), surface-exposed residues, which are identically conserved between Bcl-3 and IκBα, are shown in yellow. The asterisks and triangles indicate regions of the IκBα surface in contact with NF-κB that are composed of residues poorly conserved in Bcl-3. This figure was prepared using GRASP (Nicholls et al., 1991). Download figure Download PowerPoint A large portion of the IκBα surface is involved in recognizing NF-κB (Figure 4B and F). ANK repeats 1 and 2 recognize the NLS-containing C-terminus of p65 (upper part of Figure 4B); the inner helices of ANK repeats 5 and 6 contact the dimerization domain of p65 (lower part of Figure 4B) and the β-hairpins of ANK repeats 4–6 interact with the p50 dimerization domain (Figure 4F). All three regions of the contact surface are relatively well conserved in Bcl-3 (Figure 4, compare B with C, and F with G), and over half of the IκBα residues involved in specific contacts with NF-κB are identical or nearly identical in Bcl-3 (Figure 3A). This suggests that many of the interactions observed in the IκBα–NF-κB complex are preserved in the Bcl-3–(p50)2 and Bcl-3–(p52)2 complexes. Indeed, because of the high degree of sequence and structural similarity between the p50 homodimer and the p50–p65 heterodimer (F.E.Chen et al., 1998), Bcl-3 is likely to bind to the homodimer with a nearly identical relative orientation to that of IκBα bound to the heterodimer. The strip of surface contacting p50 is particularly well conserved (Figure 4F and G), including Tyr181 and Tyr248 in the β-hairpins of ANK repeats 4 and 6 (Bcl-3 residues Tyr232 and Tyr299), which contact a cluster of hydrophobic residues in p50. This makes good sense, as the p50 subunit of the heterodimer recognized by IκBα is common to the homodimer recognized by Bcl-3. Potential differences in recognition of the NF-κB C-terminus and implications for binding symmetry In the IκBα–NF-κB structure, residues C-terminal to the dimerization domain of p65 form two helices. The first, consisting of p65 residues 293–305 and including the basic NLS, interacts with a negatively charged surface on IκBα formed by residues from ANK repeats 1, 2 and, to a lesser extent, 3 (Figures 3A and 4A). The corresponding surface in Bcl-3 is considerably less acidic (Figure 4A and D, green dot), suggesting that Bcl-3 may bind more weakly to a basic NLS. In particular, IκBα residue Glu85, which forms a salt bridge with Arg304 of the p65 NLS, is replaced by a glycine (Gly138) in Bcl-3; the two aspartate residues, which in IκBα interact with NLS residues Lys301 and Arg302, point away from the NLS site in the Bcl-3 crystal structure (although they may adopt a similar orientation in solution or upon the binding of p50 or p52; Figure 3C). The second C-terminal helix of p65 is formed by residues 306–320 and is involved in a hydro phobic ‘capping’ interaction with the top surface of the IκBα ARD, which is relatively poorly conserved in Bcl-3 (Figure 4, compare asterisks in B and C, and F and G). This surface is formed by the unique solvent-exposed face of ANK1, which is considerably less hydrophobic in Bcl-3 than in IκBα. Specifically, three bulky hydrophobi" @default.
• W2041503007 created "2016-06-24" @default.
• W2041503007 creator A5005915670 @default.
• W2041503007 date "2001-11-15" @default.
• W2041503007 modified "2023-10-18" @default.
• W2041503007 title "Crystal structure of the ankyrin repeat domain of Bcl-3: a unique member of the IkappaB protein family" @default.
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