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Originally published In Press as doi:10.1074/jbc.M600237200 on March 15, 2006

J. Biol. Chem., Vol. 281, Issue 21, 14787-14795, May 26, 2006
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Structural Diversity in p160/CREB-binding Protein Coactivator Complexes*

Lorna Waters{ddagger}12, Baigong Yue§2, Vaclav Veverka{ddagger}, Philip Renshaw{ddagger}, Janice Bramham{ddagger}, Sachiko Matsuda{ddagger}, Thomas Frenkiel, Geoffrey Kelly, Frederick Muskett{ddagger}, Mark Carr, Supported by Wellcome Trust Grants 066047 and 063632{ddagger}3, and David M. Heery§4

From the {ddagger}Department of Biochemistry, Henry Wellcome Building, University of Leicester, Lancaster Road, Leicester LE1 9HN, United Kingdom, §School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom, and Medical Research Council Biomedical NMR Centre, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom

Received for publication, January 10, 2006 , and in revised form, March 14, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Ligand-induced transcription by nuclear receptors involves the recruitment of p160 coactivators such as steroid receptor coactivator 1 (SRC1), in complex with histone acetyltransferases such as CREB-binding protein (CBP) and p300. Here we describe the solution structure of a complex formed by the SRC1 interaction domain (SID) of CBP and the activation domain (AD1) of SRC1, both of which contain four helical regions (C{alpha}1, C{alpha}2, C{alpha}3, and C{alpha}3' in CBP and S{alpha}1, S{alpha}2', S{alpha}2, and S{alpha}3 in SRC1). A tight four-helix bundle is formed between S{alpha}1, C{alpha}1, C{alpha}2, and C{alpha}3 that is capped by S{alpha}3. In contrast to the structure of the AD1 domain of the related p160 protein ACTR in complex with CBP SID, the sequences forming S{alpha}2' and S{alpha}2 in SRC1 AD1 are not involved in the interface between the two domains but rather serve to position S{alpha}3. Thus, although the CBP SID domain adopts a similar fold in complex with different p160 proteins, the topologies of the AD1 domains are strikingly different, a feature that is likely to contribute to functional specificity of these coactivator complexes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The lysine acetyltransferase CBP5 interacts with a large number of nuclear proteins, many of which are transcription factors (1, 2). This is achieved through direct or indirect protein-protein interactions that are mediated by distinct structural domains within the CBP protein such as the KIX, CRD, CH1, CH3, bromodomain and SID domains. The protein-binding domains of CBP display partial specificity, having both distinct and overlapping binding partner profiles, which contributes to the phenomena of synergy and cross-talk between transcription factors (1).

The recruitment of CBP to target gene promoters/enhancers facilitates acetylation of histone N-terminal tails, leading to chromatin remodeling and enhanced gene expression. This has been demonstrated for nuclear receptors, which activate transcription of their target genes in response to ligand binding (3, 4). Ligand-bound receptors undergo a conformational change that stimulates their interaction with cofactors that contain functional LXXLL motifs, such as the p160 coactivators SRC1, TIF2, and ACTR (5, 6). Studies of steroid-regulated gene promoters have revealed that p160s and HAT proteins are among the first cofactors recruited in response to ligand (7, 8). Such temporally ordered recruitment of coactivators to promoters/enhancers is crucial for the sequential chromatin modification and remodeling events preceding transcription (4).

The efficacy of nuclear receptor/cofactor interaction is influenced by a number of determinants, including the precise sequence and number of LXXLL motifs, the sequences flanking core motifs, and other distal sequences (9-11). CBP contains three LXXLL motifs, although they mediate only weak direct interactions with estrogen, androgen, and progesterone receptors (9, 10). Thus, at least in the case of the steroid receptors, efficient recruitment of CBP/p300 and associated factors is achieved indirectly via the p160 proteins (12).

A number of studies have shown that recruitment of CBP/p300 proteins is facilitated by the p160 activation domain AD1, which acts as a potent transcriptional activator in mammalian and yeast cells (3, 12-17). The AD1 domain docks with the SRC1 interaction domain (SID) of CBP, located within the sequence 2058-2130 (12), and this interaction is essential for ligand-dependent transcription mediated by steroid receptors (12, 18). In addition to binding p160s, the SID also facilitates interactions of CBP with activation domains in other nuclear proteins, including the transcription factors Ets1, Ets2, p53, and IRF3, and viral activators such as E1A, KSHV IRF1, and Tax (19-22). Indeed, it has been shown that competition between such proteins for binding to the SID contributes to the negative cross-talk observed between different signaling pathways (22). Similarly, binding of viral proteins to the SID and other CBP/p300 domains not only facilitates viral gene transcription but may also down-regulate expression of host defense genes, through exclusion of host factors from CBP-p300 complexes.

The AD1/SID interaction is also important in MOZ-TIF2-associated leukemogenesis. MOZ-TIF2 is a fusion protein expressed in acute myeloid leukemia blasts containing the inv8(p11;q13) translocation (23). This fusion protein contains the C-terminal sequence of TIF2, including the AD1 domain, facilitating its interaction with CBP/p300, as demonstrated by in vitro interactions and in vivo by using fluorescence resonance energy transfer experiments (24). As a consequence of this interaction, CBP is mislocalized from promyelocytic leukemia bodies, and cellular levels of CBP are depleted, leading to a reduced transcriptional activity of CBP-dependent activators such as nuclear receptors and p53 (24). Consistent with this, AD1 integrity was found to be essential for transformation of hematopoietic progenitors by MOZ-TIF2 in vitro (24, 25) and for induction of acute myeloid leukemias by MOZ-TIF2 in mice (25). Therefore, understanding the structure of the CBP-p160 complexes has relevance to the etiology of acute myeloid leukemia.

Phenotypic differences in knock-out mice indicate that p160s have tissue-specific functions (reviewed in Ref. 26). Similarly, CBP and p300 proteins appear to have distinct roles in vivo, for example in hematopoiesis (27). Thus, the existence of different p160-CBP or p160-p300 complexes in vivo suggests they may have specific albeit partially redundant functions. For example, SRC1 and ACTR interact with thymine DNA glycosylase, whereas TIF2 does not. This is because of the presence of a tyrosine repeat motif (YXXY) in ACTR and SRC1, which is not conserved in the TIF2 sequence (28). Chromatin immunoprecipitation assays investigating cofactor recruitment at the pS2 gene promoter have indicated that the presence of one p160 can exclude recruitment of others (7), although the molecular basis of this is unknown. Another study provided evidence that CBP-p160 complexes are functionally distinct, as it was observed that different p160 combinations can be detected on androgen receptor target gene promoters, possibly through the formation of specific p160 heterodimer pairs (29). Thus, determination of the structures of different complexes will be essential to understand how such selectivity is achieved. In this study, we describe a high resolution solution structure of the complex formed between the SID domain of CBP and the AD1 domain of SRC1. By comparison to the CBP SID/ACTR AD1 structure (30), we show that although the structure of CBP SID is strikingly similar in both complexes, p160 proteins adopt distinct conformations that are likely to be important for their different biological functions.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Protein Expression/Purification—To express the CBP/SRC1 interaction domain complex in Escherichia coli, we obtained a modified pET22b dual expression vector, containing sequences encoding the ACTR AD1 and CBP SID domains, preceded by independent ribosome-binding sites (a gift from Peter Wright, Scripps Institute). To generate the CBP SID/SRC1 AD1 expression vector, the ACTR AD1 sequence was removed by restriction digestion with NcoI and HindIII and replaced with an NcoI/HindIII-digested PCR fragment containing the coding region for residues 920-970 of SRC1 followed by a thrombin cleavage site (underlined) and a polyhistidine tag (KLVPRGSLEHHHHHH). The mouse CBP SID-(2059-2117) and human SRC1 AD1-(920-970) dual expression vector was transformed into E. coli strain B834, which was used to produce either unlabeled, 15N-labeled, or 15N/13C-labeled samples of the CBP SID-SRC1 AD1 complex. For labeled samples, cells were grown in minimal media containing 0.6 g/liter [15N]ammonium sulfate and 2 g/liter [13C]glucose as the sole nitrogen and/or carbon sources. The medium was also supplemented with 50 mg/liter unlabeled methionine, as B834 is a methionine auxotroph. Transformants were grown at 37 °C, and expression of the two proteins forming the complex was induced in mid-log phase by the addition of 1 mM isopropyl-1-thio-beta-D-galactopyranoside. The cells were harvested 3 h post-induction, resuspended in 20 mM sodium phosphate, 100 mM sodium chloride, and 5 mM imidazole buffer (pH 7.0). Lysis was achieved by sonication. Insoluble material was removed by high speed centrifugation, and the cleared lysate was filtered through a 0.2-µm filter prior to chromatography. The histidine-tagged CBP SID-SRC1 AD1 complex was affinity-purified on a HiTrap chelating HP column (Amersham Biosciences) charged with Ni2+ and eluted with a linear gradient of imidazole (5-250 mM). Fractions containing the complex were pooled and subjected to a final purification by gel filtration on a Superdex 75 16/60 pre-packed column (Amersham Biosciences). Typical yields of the >95% pure complex were 10 mg/liter.

Reverse Phase HPLC—Reverse phase HPLC analysis was performed on a C4 column. The individual polypeptides were resolved by applying a two-step linear gradient of acetonitrile (1.6-80%), and the relative amount of each was quantified by absorbance at 215 nm. Eluted peaks were identified by mass spectrometry.

Circular Dichroism Spectroscopy—Far UV CD spectra were obtained from 0.4 mM samples of the CBP SID-SRC1 AD1 complex in a 20 mM sodium phosphate, 100 mM sodium chloride buffer (pH 7.0). Samples were placed in a 0.1-mm path length cell, and spectra were recorded from 180 to 250 nm at a resolution of 1 nm and a scan speed of 20 nm/min, with each spectrum representing the average of 10 accumulations. Prior to analysis, CD spectra were corrected for buffer absorbance, and the raw data were converted to molar CD per residue.

NMR Spectroscopy—NMR spectra were acquired from 0.35-ml samples of 1.5 mM CBP SID-SRC1 AD1 complex in a 20 mM sodium phosphate, 100 mM sodium chloride, 10 µM EDTA, and 0.02% (w/v) sodium azide buffer (pH 7.0), containing either 10% D2O, 90% H2O, or 100% D2O as appropriate. All NMR data were acquired at 25 °C on either an 800-MHz Varian Inova or a 600-MHz Bruker Avance spectrometer. The two-dimensional and three-dimensional spectra recorded to obtain sequence-specific assignments for CBP SID and SRC1 AD1 in complex were as follows: 1H TOCSY with mixing times of 40 and 55 ms (31) and NOESY with an NOE mixing time of 100 ms (32); 15N/1H HSQC; TOCSY-HSQC with a mixing time of 50 ms and NOESY-HSQC with an NOE mixing time of 100 ms (33); 13C/1H HCCH-TOCSY with a mixing time of 20 ms (34), HMQC-NOESY with an NOE mixing time of 100 ms (35); and 15N/13C/1H HNCACB (36) and CBCA(CO)NH (37). Typical acquisition times in F1 and F2 for the three-dimensional experiments were 11-13 ms for 15N, 7.5-9.5 ms for 13C, and 15 ms for 1H, and an acquisition time of 75 ms in F3 (1H). The majority of the three-dimensional spectra were collected over ~88 h, two-dimensional 1H experiments over 8.5-24 h, and 15N/1H HSQC spectra over about 30 min. Typical acquisition times in two-dimensional experiments were either 70 (15N) or 35 ms (1H) in F1 and 250 ms in F2 (1H). The WATERGATE method (38) was used to suppress the water signal when required. The three-dimensional NMR data were processed using NMRPipe (39) with linear prediction used to extend the effective acquisition times by up to 1.5-2-fold in F1 and F2. The spectra were analyzed using the XEASY package (40).

Structure Calculations—A family of converged CBP SID/SRC1 AD1 structures was determined in a two-stage process using the program CYANA (41). Initially, the combined automated NOE assignment and structure determination protocol (CANDID) was used to automatically assign both the intra- and intermolecular NOE cross-peaks identified in three-dimensional 15N- and 13C-edited NOESY spectra. This approach provides a completely unbiased assignment of the NOE peaks, in which all peaks are evaluated as either intra- or intermolecular in origin. Subsequently, several cycles of simulated annealing combined with redundant dihedral angle constraints were used to produce the final converged CBP SID/SRC1 AD1 structures (42). The input for the CANDID stage consisted of essentially complete 15N, 13C, and 1H resonance assignments for the nonexchangeable groups in the CBP SID-SRC1 AD1 complex, two manually picked three-dimensional NOE peak lists corresponding to all NOEs involving amide protons (1179) and all NOEs between aliphatic protons (2371), and one manually picked two-dimensional NOE peak list corresponding to all NOEs involving aromatic side chain protons (127). In addition, the CANDID stage included 80 torsion angle constraints for CBP SID and 64 torsion angle constraints for SRC1 AD1 obtained from analysis of chemical shift data using TALOS (43). Hydrogen bond constraints were also added for regions predicted to contain regular {alpha}-helical secondary structure by both the NOE and chemical shift data, which corresponds to residues Ala2067-Leu2075, Gln2082-Lys2092, and Ala2099-Ala2107 of CBP SID and Glu929-Ser941 of SRC1 AD1. The peak lists were prepared using XEASY, and the intensities of peaks were obtained using the "interactive integration" routine (40). CANDID calculations were carried out using the default parameter settings in CYANA 1.0.6, with the upper limit for NOE-derived distance constraints set at 5.5 Å and chemical shift tolerances set to 0.02 ppm (direct and indirect 1H) and 0.3 ppm (15N and 13C). The final converged CBP SID/SRC1 AD1 structures were produced from 100 random starting coordinates using a standard torsion angle-based simulated annealing protocol combined with 5 cycles of redundant dihedral angle constraints (44, 45). The calculations were based upon 1855 nonredundant NOE-derived upper distance limits (maximum value 6.0 Å), assigned to unique pairs of protons using CANDID, 142 {Phi} and {Psi} torsion angle constraints derived from TALOS, and 104 hydrogen bond constraints for helical residues. Analysis of the family of structures obtained was carried out using the programs CYANA and MOLMOL (41, 46)


Figure 1
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  		FIGURE 1.
Purification and characterization of the CBP SID-SRC1 AD1 complex. A, 15% SDS-polyacrylamide gel showing separation of whole cell lysate from E. coli B834 expressing CBP SID-SRC1 AD1 complex (lane 2). The His6-tagged complex was purified by affinity chromatography. Lanes 3-12 show fractions containing the purified complex after gel filtration. Molecular weight markers are shown in lane 1. B, far UV circular dichroism spectrum acquired for the CBP SID-SRC1 AD1 complex. C, reverse phase HPLC of the purified CBP SID-SRC1 AD1 complex. The areas under the two peaks were used to calculate the ratio of the CBP SID domain to SRC1 AD1 domain in the purified complex. D, 15N/1H HSQC spectrum of the CBP SID-SRC1 AD1 complex. The assignments of the signals from backbone amide groups in both domains are indicated by residue type and number, with the overlapped region between 7.85 and 8.35 ppm in 1H and 118.5 and 121.5 ppm in 15N shown in the expanded region to the right of the complete spectrum. For clarity, residues are numbered according to their position in the domain. Thus, CBP residues Pro2059-Gln2117 are numbered P2-Q60, and SRC1 residues Pro920-Ser970 are numbered P303-S353, with the C-terminal linker to the His tag numbered 354-361. Assignments obtained for a number of side chain NH2 groups are indicated on the spectrum.

 
Sequence-specific Assignments—Sequence-specific backbone resonance assignments (N, NH, C{alpha}, and Cbeta) were obtained for the CBP SID-SRC1 AD1 complex from the identification of intra- and inter-residue connectivities in HNCACB, CBCA(CO)NH, and 15N/1H NOESY-HSQC spectra. Assignments were then extended to the side chain signals using correlations observed primarily in 15N/1H TOCSY-HSQC and 13C/1H HCCH-TOCSY, with additional supporting evidence provided by 15N/1H NOESY-HSQC and 13C/1H HMQC-NOESY spectra where required.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Expression and Purification of the SRC1-CBP Complex—We previously used yeast two-hybrid and in vitro pulldown experiments to define the minimal sequences required for interaction of the CBP SID and SRC1 AD1 domains (12). A modified pET22b dual expression vector (30), containing sequences encoding the SRC1 AD1-(920-970) and CBP SID-(2059-2117) domains, was used to coexpress these polypeptides in E. coli. The addition of a polyhistidine tag at the C terminus of the AD1 domain facilitated purification of the complex, as described under "Experimental Procedures." A highly purified complex containing polypeptides of the expected size was obtained, as determined by SDS-PAGE (Fig. 1A). Far UV CD spectra acquired for the CBP SID-SRC1 AD1 complex indicated that the complex was predominantly helical, having characteristic negative ellipticity peaks at ~209 and 221 nm and a large positive peak at 195 nm (Fig. 1B). Analysis of the spectra using the CDPro software package (47) indicated that the complex contained 54% (±7.3%) helical, 20% (±6.1%) turn, and 25% (±5.7%) random coil secondary structure.

Reverse phase HPLC analysis was performed to determine the stoichiometry of the purified complex. The eluted peaks were identified by mass spectrometry (data not shown). Quantification of the peptide bond absorbance from both peaks confirmed the formation of a 1:1 CBP SID-SRC1 AD1 complex (Fig. 1C).

Sequence-specific Assignments and Structure Calculation for the CBP-SRC1 Complex—Very comprehensive sequence-specific resonance assignments were obtained for the CBP SID-SRC1 AD1 complex despite the relatively poor dispersion observed in spectra, which is illustrated by the HSQC spectrum (Fig. 1D). For example, backbone amide assignments were obtained for all non-proline residues in the complex except as follows: Asn2060, Arg2061, and Gln2117 in CBP SID; Asn927 in SRC1 AD1 (96%); and for all C{alpha} and Cbeta signals apart from the two unlabeled methionines and two residues in CBP SID (Pro2059 and Gln2117) (96%).

The CANDID protocol was effective in determining unique assignments for the NOEs identified in the three-dimensional 15N- and 13C-edited NOESY and the aromatic to aliphatic region of the two-dimensional NOESY. At the end of the final cycle, unique assignments were obtained for 89.8% (1059/1179) of the NOE peaks picked in the 15N/1H NOESY-HSQC spectra, 89.3% (2117/2371) in the 13C/1H HMQC-NOESY spectra, and 92.1% (117/127) in the NOESY spectrum, which produced 1759 nonredundant 1H to 1H upper distance limits. The final family of CBP SID-SRC1 AD1 complex structures was determined using a total of 2005 NMR-derived structural constraints (an average of 18.2 per residue), including 1759 NOE-based upper distance limits (328 intraresidue, 514 sequential (i,i + 1), 643 medium range (i,i ≤ 4), and 274 long range (i,i ≥5) comprising 166 intramolecular NOEs and 108 intermolecular NOEs), 142 backbone torsion angle constraints (71{Phi} and 71{Psi}), and 104 hydrogen bond constraints. Following the final round of CYANA calculations, 37 satisfactorily converged structures were obtained from 100 random starting structures. The converged structures contain no distance or van der Waals violation greater than 0.5 Å and no dihedral angle violations greater than 5°, with an average value for the CYANA target function of 6.17 ± 0.95Å2. The sums of the violations for the upper distance limits, lower distance limits, van der Waals contacts, and torsion angle constraints were 20.7 ± 1.84 Å, 1.5 ± 0.24 Å, 14.8 ± 1.55 Å, and 33.8 ± 6.53°, respectively. Similarly, maximum violations for the converged structures were 0.40 ± 0.05 Å, 0.24 ± 0.08 Å, 0.28 ± 0.04 Å, and 3.44 ± 0.64°, respectively. The NMR constraints and structural statistics for the complex are summarized in Table 1. The family of converged CBP SID-SRC1 AD1 complex structures, together with the NMR constraints, have been deposited in the Protein Data Bank (code 2C52).


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  		TABLE 1
NMR constraints and structural statistics for the CBP SID-SRC1 AD1 complex

 
Structure of the CBP-SRC1 Complex—The solution structure of the CBP SID-SRC1 AD1 complex was determined to high precision, as evident from the superposition of the protein backbone of the family of converged structures (best fit for residues 2063-2113 of CBP SID and 928-963 of SRC1 AD1; see Fig. 2A) and reflected in low root mean standard deviation (r.m.s.d.) values to the mean structure for both the backbone (0.49 ± 0.10 Å) and all heavy atoms (0.95 ± 0.09 Å). The backbone topology of the complex is composed primarily of eight helices linked by turns and loops, including four {alpha}-helices in the CBP SID domain (C{alpha}1, Ser2066-Lys2076; C{alpha}2, Gln2082-Ser2093; C{alpha}3, Pro2095-Thr2106, and C{alpha}3', Tyr2109-Asn2112) and three {alpha}-helices in the SRC1 AD1 domain (S{alpha}1, Glu929-Ser941;S{alpha}2', Glu945-Leu948, and S{alpha}3, Ile957-Gln962). SRC1 AD1 also contains a short 310 helix (S{alpha}2, Glu950-Leu955). The total helical content of the complex is just over 50%, in close agreement with the CD analysis (54% helical structure; see Fig. 1B).

The CBP SID and SRC1 AD1 domains are intimately associated in the complex, with C{alpha}1, C{alpha}2, C{alpha}3, and S{alpha}1 forming a four-helix bundle (Fig. 2B). The overall interface between the two domains is substantial, corresponding to a solvent-inaccessible surface of 1019 Å2 on CBP SID and 1088 Å2 on SRC1 AD1. Helices S{alpha}2' and S{alpha}2 pack together on one side of the complex to stabilize the corner region of the "L"-shaped domain and are not involved in the interface with CBP SID. In contrast, S{alpha}3 serves to cap the four-helix bundle region and forms contacts with C{alpha}2, C{alpha}3, C{alpha}3', and the two serine residues in the PSSP turn of CBP SID. The first two helices of CBP SID (C{alpha}1 and C{alpha}2) lie almost anti-parallel to each other and are separated by a well defined five-residue turn consisting of the sequence SPSSP. The N-terminal portion of C{alpha}2 contains a stretch of five glutamines preceding the more hydrophobic C-terminal half of the helix. C{alpha}3 is inclined away from C{alpha}1 and C{alpha}2 exposing a hydrophobic groove between C{alpha}1 and C{alpha}3, which accommodates S{alpha}1 of the SRC1 AD1 domain. Helices C{alpha}3 and C{alpha}3' are separated by a short region of irregular structure and resemble a single bent helix. The stabilizing interactions between C{alpha}1 and C{alpha}2 and between C{alpha}2 and C{alpha}3 appear to involve no ionic or hydrogen bonds but rely on favorable van der Waals contacts, primarily involving residues Ala2067, Leu2071, and Thr2074 from C{alpha}1; Gln2083, Val2087, Ile2090, and Leu2091 from C{alpha}2; and Leu2097, Phe2101, and Thr2106 from C{alpha}3. Asn2094 also makes van der Waals interactions with both C{alpha}2 and C{alpha}3.

The SRC1 AD1 domain also appears to be primarily stabilized by van der Waals interactions, involving residues Ala931 and Gln935 from S{alpha}1, Lys943 from the unstructured region between S{alpha}1 and S{alpha}2', Thr946 from S{alpha}2', Ala949 that links S{alpha}2' to S{alpha}2, and Gly956 that links S{alpha}2 to S{alpha}3. However, two potential salt bridges are formed between residues Glu945 and Lys959 and between Asp952 and Lys959. The binding of S{alpha}1 in the hydrophobic groove situated between C{alpha}1 and C{alpha}3 is stabilized by van der Waals interactions. The favorable contacts between the N-terminal helices of CBP SID and SRC1 AD1 mainly involve interactions between buried side chains found in the leucine-rich motifs of C{alpha}1 (LXXLLXXL corresponding to Leu2068, Leu2071, Leu2072, Leu2075) and S{alpha}1 (LLXXLXXFL corresponding to Leu932 and Leu936). These motifs resemble the LXXLL and I/LXXI/H/LIXXXIL motifs that mediate the interaction of coactivators and corepressors with nuclear receptors. Additional favorable van der Waals interactions are found between residues Gln935 and Phe939 from S{alpha}1 and the leucine residues of C{alpha}1. Similarly, stabilizing contacts occur between S{alpha}1 and C{alpha}3, involving the leucine-rich motif (Leu933, Leu936, and Leu940) and residues Glu929 and Val937 of S{alpha}1, and residues Gln2096, Leu2097, Ala2100, Phe2101, and Gln2104 of C{alpha}3 in CBP. A potential salt bridge between Lys2076 and Asp944 may also stabilize this region of the complex.


Figure 2
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  		FIGURE 2.
Solution structure of the CBP SID-SRC1 AD1 complex. A, best fit superimposition of the family of 37 converged NMR structures obtained for the CBP SID-SRC1 AD1 complex. The CBP SID domain is shown in blue and SRC1 AD1 in red. B, schematic (ribbon) representation of the backbone topology of the two polypeptides in the complex, using the same orientation as in A, and demonstrating the tight 4-helix bundle conformation. The four helices of CBP SID (C{alpha}1, C{alpha}2, C{alpha}3, and C{alpha}3') and SRC1 AD1 (S{alpha}1, S{alpha}2',S{alpha}2, and S{alpha}3) are indicated, along with the N and C termini of both domains.

 
The fourth helix of SRC1 AD1 (S{alpha}3) fits into a second hydrophobic groove in CBP SID located between the well defined SPSSP turn linking C{alpha}1 and C{alpha}2 and the C terminus of C{alpha}3. This interaction is stabilized by hydrophobic contacts involving Leu955, Ile957, Leu960, Val961, Gln962, and Gly964 of SRC1 AD1 and Ser2079, Ser2080, Gln2084, Arg2105, Lys2108, and Tyr2109 of CBP SID. The five C-terminal residues of SRC1 AD1 appear to loop back toward S{alpha}3 and C{alpha}3', leading to some van der Waals contacts between Leu969 of SRC1 AD1 and Tyr2109 of CBP SID. However, this loop is primarily stabilized by intramolecular van der Waals interactions involving Gln962 and residues within a C-terminal linker sequence originating from the expression vector. In the absence of the linker sequence, contacts between Leu969 and Tyr2109 may not occur, and the C-terminal region of SRC1 AD1, including S{alpha}3, may make additional contacts within the complex, or with other domains/proteins.

Some features of the NMR data obtained for the CBP SID-SRC1 AD1 complex clearly show the presence of a second minor conformational state, which is in exchange with the structure reported here (Fig. 1D). This is clearly indicated by the presence of cross-peaks between signals from backbone amide groups in 15N/1H TOCSY-HSQC spectra, which arise through chemical exchange processes (48). This also results in exchange broadening of a significant number of backbone amide signals. Interestingly, only a few of the TOCSY exchange peaks involve residues in CBP SID, whereas almost all of the residues from Leu936-Val961 in SRC1 AD1 are affected, which corresponds to the six C-terminal residues of S{alpha}1 through to the end of S{alpha}3. This suggests that the SRC1 AD1 domain has a degree of conformational instability, even when bound to CBP SID, which may facilitate the rapid dissociation of the complex. However, we do not exclude the possibility that other protein/protein interactions may stabilize complexes of the full-length proteins.

Comparison of CBP-SRC1 and CBP-ACTR Complexes—The solution structure of CBP SID in complex with the AD1 domain from ACTR has been reported previously (30). Comparison of the two complexes revealed that they have both conserved and distinct structural features (Fig. 3, A and B). The SID adopts similar secondary and tertiary structures, when bound to different AD1 domains (Fig. 3, A and B), as highlighted by the superposition of the SID polypeptide backbone atoms (residues Ile2063, Pro2065-Ala2067, Asp2070-Ser2079, and Gln2085-Lys2108), which yields an r.m.s.d. value of 1.79 Å (Fig. 3C). The most notable difference between the CBP SID polypeptides in the two complexes is the conformation of the sequence composed of five glutamine residues (2082-2086). In the CBP-SRC1 complex this region forms the N terminus of an extended C{alpha}2 helix (Fig. 3, A and C), whereas in the CBP-ACTR complex the three proximal glutamine residues were proposed to form part of a solvent-exposed loop (designated poly(Q); Fig. 3, B and C). This difference may be due to the paucity of NMR signal assignments for residues in this region in the CBP-ACTR complex (30), resulting in fewer NMR constraints on the conformation rather than a true structural difference. Another difference between the CBP SID domains is C{alpha}3, which is 17 residues long (Pro2095-Ala2111) in the CBP/ACTR structure, although in the CBP-SRC1 complex this region forms two shorter helices (C{alpha}3, Pro2095-Thr2106; C{alpha}3', Tyr2109-Asn2112) resembling a kinked helix. The C{alpha}3' helix is inclined away from the rest of the four-helix bundle, which is not observed in the ACTR-AD1 complex.


Figure 3
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  		FIGURE 3.
Comparison of CBP SID/SRC1 AD1 and CBP SID-ACTR AD1 complexes. A and B, equivalent views of the backbone topology of the CBP SID-SRC1 AD1 and CBP SID-ACTR AD1 complexes, respectively, which were obtained by superimposing the CBP SID domain from both complexes (residues Ile2063, Pro2065-Ala2067, Asp2070-Ser2079, and Gln2085-Lys2108). The CBP SID domain is shown in blue; SRC1 AD1 is shown in red and ACTR AD1 in green. C, comparison of the backbone topologies of the two CBP SID domains, which were overlaid on the same residues as in A and B. The CBP SID domains from the SRC1 AD1 and ACTR AD1 complexes are shown in blue and cyan, respectively. D, comparison of the backbone folds of the AD1 domains of SRC1 (red) and ACTR (yellow), which were overlaid on residues Ile2063, Pro2065-Ala2067, Asp2070-Ser2079, Gln2085-Lys2108 of CBP-SID and residues Asp928-Ser941 and Asp1044-Ser1057 of SRC1 and ACTR, respectively.

 
Structural similarity between p160 AD1 domains in the two complexes is confined to the N-terminal helices A{alpha}1 and S{alpha}1, which form four-helix bundles with the CBP SID (Fig. 3, A and B). The remaining sequences in the AD1 domains adopt strikingly distinct topologies as highlighted by superimposing the AD1 polypeptide structures (Fig. 3D). This is also reflected in the significant chemical shift differences for NMR signals from equivalent residues in the two AD1 domains and also for CBP SID residues that make distinct contacts with AD1 in the two complexes, in particular, residues in the C{alpha}3/C{alpha}3' region (data not shown). Although SRC1 AD1 contains three further helical regions (residues S{alpha}2', Glu945-Leu948; S{alpha}2, Glu950-Ser954; and S{alpha}3, Ile957-Gln962), ACTR AD1 contains only two additional helices (A{alpha}2, Leu1064-Leu1071, and A{alpha}3, Ile1073-Gln1079) (Fig. 3D). These distinct topologies are somewhat unexpected given that SRC1 and ACTR share 53% sequence identity and 67% sequence similarity within the AD1 domain. In the CBP SID-ACTR AD1 complex, A{alpha}2 and A{alpha}3 wrap around C{alpha}3 making extensive contacts with this helix, whereas A{alpha}2 occupies a hydrophobic groove between the proposed poly(Q) loop and C{alpha}3 (Fig. 3B). In the CBP SID-SRC1 AD1 complex, only the C-terminal residue (Leu955) of S{alpha}2 (corresponding to Leu1071 in A{alpha}2) makes any contact with the SID domain (Fig. 3A). Instead, S{alpha}3 fills the groove between the PSSP turn and C{alpha}3, effectively capping the four-helix bundle (Fig. 3A). This contrasts with the position of corresponding helix of ACTR (A{alpha}3), which packs against the adjacent hydrophobic face of C{alpha}3 (Fig. 3B).


Figure 4
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  		FIGURE 4.
Critical residues in SRC1 AD1 required for CBP binding. A, sequence alignment of p160 AD1 domains and CBP-binding sequences from viral proteins E1A and TAX. For the viral proteins, low homology sequences are represented in gray, and regions showing homology to helices S{alpha}1/A{alpha}1 and S{alpha}3/A{alpha}3 are in black. SRC1 residues, which were subjected to replacement with alanine and which are required for CBP binding/transcriptional activity of AD1, are highlighted in red. SRC1 residues highlighted in blue had little effect on AD1 transcriptional activity when replaced with alanine (see C, below). The black boxes over the alignments indicate regions of helical secondary structure observed in complexes for SRC1 (S{alpha}1, S{alpha}2',S{alpha}2, and S{alpha}3) and ACTR (A{alpha}1, A{alpha}2, and A{alpha}3). B, reporter (luciferase) assay showing the relative transcriptional activities of GAL4-AD1 (SRC1) or mutants thereof containing alanine replacements for the residues indicated. Reporter activity is presented as fold induction over control (GAL4-DBD) and was normalized to a cotransfected beta-galactosidase reporter. The data presented are averages from triplicates, and the error bars indicate the S.D. DBD, DNA binding domain; WT, wild type. C, ribbon representation of the backbone topology of CBP SID-SRC1 AD1 complex. The side chains of residues involved in two potential inter-molecular salt bridges (Lys2076 of CBP SID and Asp944 of SRC1 AD1; Arg2105 of CBP SID and Asp967 of SRC1 AD1) are highlighted in purple. Note that Arg2105 and Asp952 are spatially distant and cannot form a salt bridge equivalent to that observed between Arg2105 and Asp1086 in the CBP-ACTR complex. The side chains of residues Asp952, Asp958, and Asp960, which are required for AD1 transcriptional activity, are represented in green.

 
The CBP SID-ACTR AD1 complex was reported to contain a salt bridge between residues Asp1068 in A{alpha}2 and Arg2105 in C{alpha}3, coordinating a hydrogen-bonding network involving Asp1060 and Arg2105 and Asp1068 and Tyr2109. This buried charged interaction has been proposed to be important for the specificity of the CBP SID/ACTR AD1 interaction, although it does not contribute directly to the stability of the complex (49). Both of these aspartate residues are conserved in SRC1 AD1 (Asp944 and Asp952); however, they are spatially distant from the C{alpha}3 helix. Asp944 is present in the loop between S{alpha}1 and S{alpha}2', and Asp952 is contained within the S{alpha}2 helix (Fig. 4C). However, it is possible that Asp944 forms a salt bridge with Lys2076 from C{alpha}1. Similarly, an aspartate residue (Asp965) near the C terminus of SRC1 AD1 may form a salt bridge with Arg2105, which is involved in van der Waals contacts between C{alpha}3 and S{alpha}3. As would be expected from the tertiary structures of their respective complexes, the solvent-inaccessible surface areas of CBP SID (1681 Å2) and ACTR AD1 (1839 Å2) domains are greater than those observed for the complex of CBP SID (1019 Å2) and SRC1 AD1 (1088 Å2).

In summary, the backbone structure of the CBP SID and the first helix of AD1 are very similar in both complexes, as reflected in a backbone atom r.m.s.d. value of 2.05 Å (calculated by superimposition of residues Ile2063, Pro2065-Ala2067, Asp2070-Ser2079, and Gln2085-Lys2108 of CBP SID and residues Asp928-Ser941 and Asp1044-Ser1057 of SRC1 AD1 and ACTR-AD1, respectively). The remainder of the p160 AD1 domains adopt very distinct folds, although the C-terminal helices of AD1 appear to be essential to stabilize the complex.

Structural Flexibility of the CBP SID Permits Complex Formation with Multiple Partners—The NMR structure of the CBP SID in isolation (also termed IBiD because of its interaction with IRF3) revealed it to have significant {alpha}-helical content (19), although the conformation of the isolated CBP SID has been proposed to be consistent with that of a molten globule (50). Isolated AD1 polypeptides have little if any intrinsic structure (50)6 but appear to undergo induced folding in complex with CBP SID (30). An important question is whether short conserved structural motifs promote similar modes of binding of different proteins to CBP, or whether different complexes adopt drastically different conformations that influence the overall structure of CBP/p300 proteins, which in turn influences combinatorial complex formation.

This study reveals structural similarity within the {alpha}1 helix of two p160 AD1 domains, which contributes to a four-helix bundle in both the CBP-SRC1 and CBP-ACTR complexes. We have shown previously that the LLXXLXXXL sequence motif within {alpha}1 is conserved in other SID-binding proteins (see Fig. 4A), and that disruption of this sequence in Ets2 and E1A abrogates their binding to the CBP SID (22). Fusion of the SRC1 AD1 sequence to a GAL4 DNA binding domain permits very potent activation of a GAL reporter gene in mammalian cells, through recruitment of endogenous CBP and p300 (12). As shown in Fig. 4B, replacement of the Leu932, Leu933, and Leu936 residues in S{alpha}1 with alanines resulted in loss of AD1 transcriptional activity. This highlights the requirement of the S{alpha}1 for interaction with full-length CBP/p300 in vivo.

The C-terminal helix ({alpha}3) of the SRC1 and ACTR AD1 domains also plays a critical role in formation of the two complexes with CBP SID as discussed above. Interestingly, the sequence of the S{alpha}3 helix of SRC1 AD1 (957IDKLV961) is divergent from that in ACTR and TIF2 (IPELV) (Fig. 4A). The IPELV sequence in ACTR and TIF2 resembles the LPXL motif that has been shown to mediate the interaction of CITED and HIF1{alpha} transcription factors with the CH1 domain of CBP (51). Mutagenesis of the SRC1 AD1 domain in the context of GAL4-AD1 revealed that the D958A or L960A mutations resulted in almost complete loss of reporter activation, whereas K959A retained almost full transactivation function (Fig. 4B). In addition, reporter activation by the V961A mutant was reduced by 70%, suggesting it is important but not essential for CBP/p300 recruitment (Fig. 4B). These results confirm the importance of S{alpha}3 and key residues within that helix for CBP/p300 recruitment.

Replacement of the conserved aspartate residues (D944A and D952A) also resulted in loss of reporter activation by GAL4-AD1 (Fig. 4B). Thus, as suggested for the CBP-ACTR complex (30, 49), salt bridge formation may be important in stabilizing the higher order folding of CBP-p160 complexes. There is evidence to suggest that SRC1 sequences outside of the AD1 domain may influence the interaction with CBP. The signaling molecule 8-bromo-cAMP induces mitogen-activated protein kinase-dependent phosphorylation of SRC1 at Thr1179 and Ser1185, which leads to increased ligand-independent transcriptional activity of the progesterone receptor and enhanced CBP recruitment (52, 53). Similarly, the interaction of ACTR with CBP was abrogated by mutation of residues outside of the AD1 domain, which are known to be phosphorylated in vivo (Thr24, Ser543, Ser857, Ser860, and Ser867) (53). This may reflect binding between additional regions of SRC1 and CBP and/or conformational changes in SRC1 that effect the affinity of the AD1 region for CBP.

We have indicated previously that several CBP SID-binding proteins contain low level sequence homology in the regions of SRC1 AD1 corresponding to S{alpha}1, and to a lesser extent S{alpha}3, that may indicate conserved modes of binding to CBP. An alignment of the sequences of interaction domains of several CBP SID-binding proteins is shown in Fig. 4A. The transactivation domain of human T-cell leukemia Tax protein contains a short sequence (312YTNIPISL319) that is required for binding to CBP SID (21). This sequence may form an amphipathic {alpha}-helix analogous to S{alpha}3 of SRC1 AD1 and play an equivalent role in stabilizing CBP/Tax interactions (Fig. 4A). The adenoviral oncoprotein E1A revealed also sequences essential for CBP binding, which may be functionally and structurally equivalent to SRC1 AD1 S{alpha}1 and S{alpha}3 (22) (Fig. 4A). IRF3 contains a sequence termed the IRF association domain (IAD), which mediates its interactions with CBP SID, and the crystal structure of this complex was recently reported (54). Little structural similarity is evident between the CBP-IRF3 complex and the CBP-p160 structures. Contacts between the IAD and SID are mediated by hydrophobic contacts involving C{alpha}1, C{alpha}2, and C{alpha}3 in CBP SID with two amphipathic {alpha}-helices (H3 and H4) in the IAD containing IXXLI and LXXLVXXXV motifs, respectively. Interestingly, the secondary structure of the SID is similar to that observed in p160 complexes, and we note that the poly(Q) region of CBP is comprised within the C{alpha}2 helix, as in the CBP-SRC1 complex. Thus the SID appears to be able to fold into distinct conformations to accommodate binding to different transcription factors.

In conclusion, the NMR structure reported here reveals both similar and diverse structural features in CBP SID-p160 AD1 complexes, which may reflect functional differences between p160 proteins. Structural versatility of the CBP SID is likely to underpin its ability to assemble different transcription factor-cofactor complexes in a promoter-dependent context.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 2C52) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported in part by Grant 054401/Z/98/B from the Wellcome Trust (to D. M. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by a Ph.D. studentship from the Biotechnology and Biological Sciences Research Council. Back

2 Both authors should be considered as equal first authors. Back

3 To whom correspondence may be addressed: Dept. of Biochemistry, Henry Wellcome Bldg., University of Leicester, Lancaster Rd., Leicester LE1 9HN, UK. Tel.: 44-116-229-7075; Fax: 44-116-229-7018; E-mail: mdc12{at}le.ac.uk. 4 To whom correspondence may be addressed: School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, UK. Tel.: 44-115-951-5087; Fax: 44-115-846-6249; E-mail: david.heery{at}nottingham.ac.uk.

5 The abbreviations used are: CBP, CREB-binding protein; CREB, cAMP-response element-binding protein; SID, SRC1 interaction domain; AD1, activation domain; r.m.s.d., root mean standard deviation; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; HSQC, heteronuclear single quantum coherence; TOCSY, total correlation spectroscopy; HPLC, high pressure liquid chromatography; IRF, interferon regulatory factor; IAD, IRF association domain; SRC1, steroid receptor coactivator 1. Back

6 J. Bramham and D. M. Heery, unpublished results. Back


   ACKNOWLEDGMENTS
 
We are grateful to Peter Wright for the gift of the dual expression plasmid pET22B. We thank Jonas Emsley and Paul McEwan for useful discussions and Sharad Mistry for technical assistance.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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