Nuclear hormone receptors are ligand-activated transcription factors that regulate the expression of genes that are essential for development, reproduction and homeostasis. The hormone response is mediated through recruitment of p160 receptor coactivators and the general transcriptional coactivator CBP/p300, which function synergistically to activate transcription. These coactivators exhibit intrinsic histone acetyltransferase activity, function in the remodelling of chromatin, and facilitate the recruitment of RNA polymerase II and the basal transcription machinery. The activities of the p160 coactivators are dependent on CBP. Both coactivators are essential for proper cell-cycle control, differentiation and apoptosis, and are implicated in cancer and other diseases. To elucidate the molecular basis of assembling the multiprotein activation complex, we undertook a structural and thermodynamic analysis of the interaction domains of CBP and the activator for thyroid hormone and retinoid receptors. Here we show that although the isolated domains are intrinsically disordered, they combine with high affinity to form a cooperatively folded helical heterodimer. Our study uncovers a unique mechanism, called 'synergistic folding', through which p160 coactivators recruit CBP/p300 to allow transmission of the hormonal signal to the transcriptional machinery.

The tandem zinc finger (TZF) domain of the protein TIS11d binds to the class II AU-rich element (ARE) in the 3' untranslated region (3' UTR) of target mRNAs and promotes their deadenylation and degradation. The NMR structure of the TIS11d TZF domain bound to the RNA sequence 5'-UUAUUUAUU-3' comprises a pair of novel CCCH fingers of type CX(8)CX(5)CX(3)H separated by an 18-residue linker. The two TIS11d zinc fingers bind in a symmetrical fashion to adjacent 5'-UAUU-3' subsites on the single-stranded RNA via a combination of electrostatic and hydrogen-bonding interactions, with intercalative stacking between conserved aromatic side chains and the RNA bases. Sequence specificity in RNA recognition is achieved by a network of intermolecular hydrogen bonds, mostly between TIS11d main-chain functional groups and the Watson-Crick edges of the bases. The TIS11d structure provides insights into the RNA-binding functions of this large family of CCCH zinc finger proteins.

The cellular response to low tissue oxygen concentrations is mediated by the hypoxia-inducible transcription factor HIF-1. Under hypoxic conditions, HIF-1 activates transcription of critical adaptive genes by recruitment of the general coactivators CBP͞ p300 through interactions with its ␣-subunit (Hif-1␣). Disruption of the Hif-1␣͞p300 interaction has been linked to attenuation of tumor growth. To delineate the structural basis for this interaction, we have determined the solution structure of the complex between the carboxy-terminal activation domain (CAD) of Hif-1␣ and the zinc-binding TAZ1 (CH1) motif of cyclic-AMP response element binding protein (CREB) binding protein (CBP). Despite the overall similarity of the TAZ1 structure to that of the TAZ2 (part of the CH3) domain of CBP, differences occur in the packing of helices that can account for differences in specificity. The unbound CAD is intrinsically disordered and remains relatively extended upon binding, wrapping almost entirely around the TAZ1 domain in a groove through much of its surface. Three short helices are formed upon binding, stabilized by intermolecular interactions. The Asn-803 side chain, which functions as a hypoxic switch, is located on the second of these helices and is buried in the molecular interface. The third helix of the Hif-1␣ CAD docks in a deep hydrophobic groove in TAZ1, providing extensive intermolecular hydrophobic interactions that contribute to the stability of the complex. The structure of this complex provides new insights into the mechanism through which Hif-1␣ recruits CBP͞p300 in response to hypoxia. H ypoxia-inducible factor 1 (HIF-1) activates genes that are crucial for cell survival under hypoxic conditions. HIF-1 is a basic helix-loop-helix-periodic-aryl hydrocarbon receptorsingle-minded (Per-Arnt-Sim) domain transcription factor composed of two subunits, Hif-1␣ and the aryl hydrocarbon receptor nuclear translocator (1). It induces expression of proteins controlling glucose metabolism, cell proliferation, and vascularization, and thus plays a key role in the pathology of cancer, heart disease, and stroke (2, 3). The oxygen-sensing and transactivating functions of HIF-1 are contained within the Hif-1␣ subunit, which is overexpressed in most human cancers (4). In mice, deletion of Hif-1␣ results in neural and cardiovascular developmental arrest and embryonic death (5), underscoring the vital role of Hif-1␣, and the maintenance of cellular oxygen homeostasis, in the correct functioning of the mammalian cell.Several steps in the signal transduction mechanism by which low cellular oxygen concentration is converted to Hif-1␣-mediated transcription of hypoxic response genes are now known (reviewed in refs. 6 and 7). Under normoxic conditions, Hif-1␣ becomes hydroxylated at a proline in the oxygen-dependent degradation domain, associates with the von Hippel-Lindau tumor suppressor protein, and is rapidly degraded in the cytoplasm via the ubiquitin-proteasome pathway (8-10). Under hypoxic conditions, proline hydroxylation ...

The tumor suppressor activity of p53 is regulated by interactions with the ubiquitin ligase HDM2 and the general transcriptional coactivators CBP and p300. Using NMR spectroscopy and isothermal titration calorimetry, we have dissected the binding interactions between the N-terminal transactivation domain (TAD) of p53, the TAZ1, TAZ2, KIX, and nuclear receptor coactivator binding domains of CBP, and the p53-binding domain of HDM2. The p53 TAD contains amphipathic binding motifs within the AD1 and AD2 regions that mediate interactions with CBP and HDM2. Binding of the p53 TAD to CBP domains is dominated by interactions with AD2, although the affinity is enhanced by additional interactions with AD1. In contrast, binding of p53 TAD to HDM2 is mediated primarily by AD1. The p53 TAD can bind simultaneously to HDM2 (through AD1) and to any one of the CBP domains (through AD2) to form a ternary complex. Phosphorylation of p53 at T18 impairs binding to HDM2 and enhances affinity for the CBP KIX domain. Multisite phosphorylation of the p53 TAD at S15, T18, and S20 leads to increased affinity for the TAZ1 and KIX domains of CBP. These observations suggest a mechanism whereby HDM2 and CBP/p300 function synergistically to regulate the p53 response. In unstressed cells, CBP/p300, HDM2 and p53 form a ternary complex that promotes polyubiquitination and degradation of p53. After cellular stress and DNA damage, p53 becomes phosphorylated at T18 and other residues in the AD1 region, releases HDM2 and binds preferentially to CBP/p300, leading to stabilization and activation of p53.p53 transactivation domain ͉ phosphorylation ͉ protein-protein interaction ͉ transcriptional coactivator ͉ tumor suppressor T he p53 tumor suppressor is activated as a transcriptional regulator in response to DNA damage, leading to the arrest of cell growth and apoptosis. p53 is a modular protein that binds DNA as a tetramer; each subunit contains an N-terminal transactivation domain (TAD), proline-rich domain, core DNA binding domain, tetramerization domain, and C-terminal regulatory domain. In the absence of cellular stress, p53 binds target promoters in an inactive latent state and recruits HDM2 (the human homolog of mouse double minute 2, MDM2) to chromatin (1, 2). HDM2 functions as a ubiquitin E3 ligase that maintains p53 at low levels by continuous proteasomal degradation (3). DNA damage initiates a cascade of phosphorylation and acetylation events at multiple sites on p53 (Fig. 1A), resulting in stabilization and enhancement of p53 transcriptional activity (4-7). In particular, phosphorylation at threonine-18 (T18) helps stabilize p53 by inhibiting binding to HDM2 (8, 9), whereas phosphorylation of serines 15 and 20 (S15, S20) enhances recruitment of the general transcriptional coactivators and acetylases, CREB binding protein (CBP) and p300 (10-12). S15 must be phosphorylated before phosphorylation can occur at T18 and S20 (13).CBP and p300 play a central role in regulation of p53 stability and the response to genotoxic stress (14-16). In unstres...

The activity and stability of the tumor suppressor p53 is regulated by interactions with key cellular proteins such as MDM2 and CBP/p300. The transactivation domain (TAD) of p53 contains two subdomains (AD1 and AD2) and interacts directly with the N-terminal domain of MDM2 and with several domains of CBP/p300. Here we report the NMR structure of the full-length p53 TAD in complex with the nuclear coactivator binding domain (NCBD) of CBP. Both the p53 TAD and NCBD are intrinsically disordered and fold synergistically upon binding, as evidenced by the observed increase in helicity and increased dispersion of the amide proton resonances. The p53 TAD folds to form a pair of helices (denoted Pα1 and Pα2), which extend from Phe19 to Leu25 and Pro47 to Trp53, respectively. In the complex, the NCBD forms a bundle of three helices (Cα1: residues 2066-2075, Cα2: residues 2081-2092, and Cα3: residues 2095-2105) with a hydrophobic groove into which the p53 helices Pα1 and Pα2 dock. The polypeptide chain between the p53 helices remains flexible and makes no detectable intermolecular contacts with the NCBD. Complex formation is driven largely by hydrophobic contacts that form a stable intermolecular hydrophobic core. A salt bridge between D49 of p53 and R2105 of NCBD may contribute to the binding specificity. The structure provides the first insights into simultaneous binding of the AD1 and AD2 motifs to a target protein.The p53 tumor suppressor acts as a hub in signal transduction networks that mediate the cellular response to stress, leading to cell-cycle arrest, senescence, or apoptosis (1,2). Due to its role in determining cell fate, p53 is tightly controlled by numerous regulatory proteins that include MDM2, MDMX, CBP/p300, and various kinases. In unstressed cells, p53 is maintained at extremely low levels through interactions with the ubiquitin E3 ligase MDM2 (3,4). This interaction results in ubiquitination and proteasomal degradation of p53 and also blocks interactions with the basal transcriptional proteins (5,6). MDMX, which is highly homologous to MDM2 but lacks ubiquitin ligase activity, also negatively regulates p53 and inhibits its transactivation function (7,8). Upon cellular stress, specific kinases are activated which phosphorylate the N-terminal region of p53. Phosphorylation facilitates release from MDM2 and enhances binding to the general transcriptional coactivators CBP and p300 (9-13).CBP and p300 function as scaffolds for the recruitment and assembly of the transcriptional machinery and modify both chromatin and transcription factors through their intrinsic † This work was supported by grant CA96865 from the National Institutes of Health. C.W.L. was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2004-214-C00207 (Figure S1), CD spectra of the free proteins and the complexes ( Figure S2), graphs of Cα secondary chemical shifts for each component of the complex ( Figure S3) and a graph showing the locations of paramagne...

Transcriptional activation is mediated by large protein complexes assembled on target gene promoter regions. These complexes contain activators and coactivators of transcription as well as elements of the basal transcription machinery (1). The specificity, timing, and degree of transcriptional activation depend not only on which proteins form this complex but also on how they interact with each other. At the simplest level, direct interactions involved in complex formation provide a mechanism for recruiting specific functionalities to a promoter sequence. However, in some cases, binding events also give rise to allosteric effects on binding or enzymatic activity, providing another means by which transcriptional activity can be modulated.A transcriptional coactivator that may be allosterically regulated by simultaneous interactions with more than one target protein is the cAMP-response element (CREB) 1 -binding protein (CBP), as well as its paralog p300. In addition to its catalytic histone acetyltransferase domain, CBP is composed of several protein-binding modules that serve to bridge genespecific transcription factors with components of the basal transcription complex (2). CBP domains such as KIX, CH1, and CH3 recognize a diverse range of protein sequences that in some cases appear to share a general structural motif in the bound state. For example, the KIX domain binds an amphipathic helix formed by either the phosphorylated kinaseinducible domain (pKID) of CREB or the transactivation domain of c-Myb (3, 4). Although both of these ligands bind to the shallow hydrophobic groove formed between KIX helices ␣ 1 and ␣ 3 , the high affinity of CREB for CBP is mediated by critical interactions involving the pKID phosphoryl group and is therefore inducible, whereas the c-Myb activation domain binds constitutively with somewhat lower affinity (5, 6).It has been proposed that CBP-mediated transcriptional activation can be enhanced by cooperative interactions between CBP and more than one DNA-bound transcriptional activator (7-9). Since the nuclear concentration of CBP is thought to be limiting (10 -12), the presence of multiple protein-binding domains on CBP, together with the high affinity of CBP for the activators involved, should sequester a larger proportion of CBP to specific promoter sites and thereby provide a mechanism for transcriptional synergy by multiple activators. There are a number of cases where synergistic increases in transcription have been shown to occur when multiple CBP-binding activators bind to the same gene promoter. Examples include cooperative transcriptional activation by CREB and the serum response factor (13, 14) as well as enhancement of c-Myb transcriptional activation by core binding factor (15, 16), CCAAT/

The adenovirus early region 1A (E1A) oncoprotein mediates cell transformation by deregulating host cellular processes and activating viral gene expression by recruitment of cellular proteins that include cyclic-AMP response element binding (CREB) binding protein (CBP)/p300 and the retinoblastoma protein (pRb). While E1A is capable of independent interaction with CBP/p300 or pRb, simultaneous binding of both proteins is required for maximal biological activity. To obtain insights into the mechanism by which E1A hijacks the cellular transcription machinery by competing with essential transcription factors for binding to CBP/p300, we have determined the structure of the complex between the transcriptional adaptor zinc finger-2 (TAZ2) domain of CBP and the conserved region-1 (CR1) domain of E1A. The E1A CR1 domain is unstructured in the free state and upon binding folds into a local helical structure mediated by an extensive network of intermolecular hydrophobic contacts. By NMR titrations, we show that E1A efficiently competes with the N-terminal transactivation domain of p53 for binding to TAZ2 and that pRb interacts with E1A at 2 independent sites located in CR1 and CR2. We show that pRb and the CBP TAZ2 domain can bind simultaneously to the CR1 site of E1A to form a ternary complex and propose a structural model for the pRb:E1A:CBP complex on the basis of published x-ray data for homologous binary complexes. These observations reveal the molecular basis by which E1A inhibits p53-mediated transcriptional activation and provide a rationale for the efficiency of cellular transformation by the adenoviral E1A oncoprotein.CBP/p300 ͉ NMR ͉ protein structure ͉ transcriptional coactivator ͉ retinoblastoma protein T he adenovirus early region 1A (E1A) protein plays a central function in viral infection by activating genes required for viral replication and by deregulating the host cell cycle to force entry into S phase and repress differentiation (1-3). E1A deregulates the cell cycle through interactions with key cellular proteins, including the general transcriptional coactivators cyclic-AMP response element binding (CREB) binding protein (CBP) and p300 (4, 5) and the retinoblastoma protein (pRb) (6), which lead to epigenetic modifications and reprogramming of host cell transcriptional processes (7). Binding of E1A to pRb displaces the E2F transcription factors, thereby relieving E2F repression and resulting in premature S-phase entry and transcriptional activation of E2F-regulated genes (8-10). Association of E1A with CBP/p300 results in global hypoacetylation of K18 of histone H3 and may be linked to the ability of E1A to induce oncogenic transformation (11). E1A is a multifunctional protein (12) composed of 4 conserved regions (CR1-CR4, Fig. 1A). E1A uses both CR1 and CR2 for interaction with pRb (13,14). CR2 contains a characteristic pRb recognition motif, LX-CXE, which binds with high affinity to the B cyclin fold domain of the pRb pocket region (15, 16). The CR1 region of E1A binds at the interface of the A and B ...

CBP/p300 transcriptional coactivators mediate gene expression by integrating cellular signals through interactions with multiple transcription factors. To elucidate the molecular and structural basis for CBP-dependent gene expression, we determined structures of the CBP TAZ1 and TAZ2 domains in complex with the transactivation domains (TADs) of signal transducer and activator of transcription 2 (STAT2) and STAT1, respectively. Despite the topological similarity of the TAZ1 and TAZ2 domains, subtle differences in helix packing and surface grooves constitute major determinants of target selectivity. Our results suggest that TAZ1 preferentially binds long TADs capable of contacting multiple surface grooves simultaneously, whereas smaller TADs that are restricted to a single contiguous binding surface form complexes with TAZ2. Complex formation for both STAT TADs involves coupled folding and binding, driven by intermolecular hydrophobic and electrostatic interactions. Phosphorylation of S727, required for maximal transcriptional activity of STAT1, does not enhance binding to any of the CBP domains. Because the different STAT TADs recognize different regions of CBP/p300, there is a potential for multivalent binding by STAT heterodimers that could enhance the recruitment of the coactivators to promoters.