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Intrinsically disordered proteins (IDPs) frequently function in protein interaction networks that regulate crucial cellular signaling pathways. Many IDPs undergo transitions from disordered conformational ensembles to folded structures upon binding to their cellular targets. Several possible binding mechanisms for coupled folding and binding have been identified: folding of the IDP after association with the target (“induced fit”), or binding of a prefolded state in the conformational ensemble of the IDP to the target protein (“conformational selection”), or some combination of these two extremes. The interaction of the intrinsically disordered phosphorylated kinase-inducible domain (pKID) of the cAMP-response element binding (CREB) protein with the KIX domain of a general transcriptional coactivator CREB-binding protein (CBP) provides an example of the induced-fit mechanism. Here we show by NMR relaxation dispersion experiments that a different intrinsically disordered ligand, the transactivation domain of the transcription factor c-Myb, interacts with KIX at the same site as pKID but via a different binding mechanism that involves elements of conformational selection and induced fit. In contrast to pKID, the c-Myb activation domain has a strong propensity for spontaneous helix formation in its N-terminal region, which binds to KIX in a predominantly folded conformation. The C-terminal region of c-Myb exhibits a much smaller helical propensity and likely folds via an induced-fit process after binding to KIX. We propose that the intrinsic secondary structure propensities of pKID and c-Myb determine their binding mechanisms, consistent with their functions as inducible and constitutive transcriptional activators.

B-cell non-Hodgkin’s lymphoma comprises biologically and clinically distinct diseases the pathogenesis of which is associated with genetic lesions affecting oncogenes and tumour-suppressor genes. We report here that the two most common types—follicular lymphoma and diffuse large B-cell lymphoma—harbour frequent structural alterations inactivating CREBBP and, more rarely, EP300 , two highly related histone and non-histone acetyltransferases (HATs) that act as transcriptional co-activators in multiple signalling pathways. Overall, about 39% of diffuse large B-cell lymphoma and 41% of follicular lymphoma cases display genomic deletions and/or somatic mutations that remove or inactivate the HAT coding domain of these two genes. These lesions usually affect one allele, suggesting that reduction in HAT dosage is important for lymphomagenesis. We demonstrate specific defects in acetylation-mediated inactivation of the BCL6 oncoprotein and activation of the p53 tumour suppressor. These results identify CREBBP/EP300 mutations as a major pathogenetic mechanism shared by common forms of B-cell non-Hodgkin’s lymphoma, with direct implications for the use of drugs targeting acetylation/deacetylation mechanisms. CREBBP and EP300 mutations in B-cell lymphoma In three different subtypes of B-cell lymphomas, two papers report frequent somatic mutations in the genes CREBBP and EP300 , which are present in primary tumours or acquired at relapse. These genes encode related acetyltransferases that mainly function to regulate gene expression by acetylating histones and other transcriptional regulators. The mutations disrupt these activities and thus alter chromatin regulation of gene expression, as well as proliferation and potentially the response to anticancer drugs. These studies may provide a rationale for the use of histone deacetylase inhibitors in certain B-cell lymphomas. In three different subtypes of B-cell lymphomas, two papers now report frequent somatic mutations in CREBBP and EP300 , present in primary tumours or acquired at relapse. These genes encode related acetyltransferases that mainly function to regulate gene expression by acetylating histones and other transcriptional regulators. The mutations found inactivate these activities and thus alter chromatin regulation of gene expression, as well as proliferation and potentially the response to therapeutic drugs.

Type I IFNs are key cytokines mediating innate antiviral immunity. cGMP-AMP synthase, ritinoic acid-inducible protein 1 (RIG-I)–like receptors, and Toll-like receptors recognize microbial double-stranded (ds)DNA, dsRNA, and LPS to induce the expression of type I IFNs. These signaling pathways converge at the recruitment and activation of the transcription factor IRF-3 (IFN regulatory factor 3). The adaptor proteins STING (stimulator of IFN genes), MAVS (mitochondrial antiviral signaling), and TRIF (TIR domain-containing adaptor inducing IFN-β) mediate the recruitment of IRF-3 through a conserved pLxIS motif. Here we show that the pLxIS motif of phosphorylated STING, MAVS, and TRIF binds to IRF-3 in a similar manner, whereas residues upstream of the motif confer specificity. The structure of the IRF-3 phosphomimetic mutant S386/396E bound to the cAMP response element binding protein (CREB)-binding protein reveals that the pLxIS motif also mediates IRF-3 dimerization and activation. Moreover, rotavirus NSP1 (nonstructural protein 1) employs a pLxIS motif to target IRF-3 for degradation, but phosphorylation of NSP1 is not required for its activity. These results suggest a concerted mechanism for the recruitment and activation of IRF-3 that can be subverted by viral proteins to evade innate immune responses.

CREBBP and EP300 mutations in B-cell lymphoma In three different subtypes of B-cell lymphomas, two papers report frequent somatic mutations in the genes CREBBP and EP300 , which are present in primary tumours or acquired at relapse. These genes encode related acetyltransferases that mainly function to regulate gene expression by acetylating histones and other transcriptional regulators. The mutations disrupt these activities and thus alter chromatin regulation of gene expression, as well as proliferation and potentially the response to anticancer drugs. These studies may provide a rationale for the use of histone deacetylase inhibitors in certain B-cell lymphomas. In three different subtypes of B-cell lymphomas, two papers now report frequent somatic mutations in CREBBP and EP300 , present in primary tumours or acquired at relapse. These genes encode related acetyltransferases that mainly function to regulate gene expression by acetylating histones and other transcriptional regulators. The mutations found inactivate these activities and thus alter chromatin regulation of gene expression, as well as proliferation and potentially the response to therapeutic drugs. Relapsed acute lymphoblastic leukaemia (ALL) is a leading cause of death due to disease in young people, but the biological determinants of treatment failure remain poorly understood. Recent genome-wide profiling of structural DNA alterations in ALL have identified multiple submicroscopic somatic mutations targeting key cellular pathways 1 , 2 , and have demonstrated substantial evolution in genetic alterations from diagnosis to relapse 3 . However, DNA sequence mutations in ALL have not been analysed in detail. To identify novel mutations in relapsed ALL, we resequenced 300 genes in matched diagnosis and relapse samples from 23 patients with ALL. This identified 52 somatic non-synonymous mutations in 32 genes, many of which were novel, including the transcriptional coactivators CREBBP and NCOR1 , the transcription factors ERG , SPI1 , TCF4 and TCF7L2 , components of the Ras signalling pathway, histone genes, genes involved in histone modification ( CREBBP and CTCF) , and genes previously shown 1 , 2 to be targets of recurring DNA copy number alteration in ALL. Analysis of an extended cohort of 71 diagnosis–relapse cases and 270 acute leukaemia cases that did not relapse found that 18.3% of relapse cases had sequence or deletion mutations of CREBBP , which encodes the transcriptional coactivator and histone acetyltransferase CREB-binding protein (CREBBP, also known as CBP) 4 . The mutations were either present at diagnosis or acquired at relapse, and resulted in truncated alleles or deleterious substitutions in conserved residues of the histone acetyltransferase domain. Functionally, the mutations impaired histone acetylation and transcriptional regulation of CREBBP targets, including glucocorticoid responsive genes. Several mutations acquired at relapse were detected in subclones at diagnosis, suggesting that the mutations may confer resistance to therapy. These results extend the landscape of genetic alterations in leukaemia, and identify mutations targeting transcriptional and epigenetic regulation as a mechanism of resistance in ALL.

An important component of the activity of p53 as a tumor suppressor is its interaction with the transcriptional coactivators cyclic-AMP response element-binding protein (CREB)-binding protein (CBP) and p300, which activate transcription of p53-regulated stress response genes and stabilize p53 against ubiquitin-mediated degradation. The highest affinity interactions are between the intrinsically disordered N-terminal transactivation domain (TAD) of p53 and the TAZ1 and TAZ2 domains of CBP/p300. The NMR spectra of simple binary complexes of the TAZ1 and TAZ2 domains with the p53TAD suffer from exchange broadening, but innovations in construct design and isotopic labeling have enabled us to obtain high-resolution structures using fusion proteins, uniformly labeled in the case of the TAZ2–p53TAD fusion and segmentally labeled through transintein splicing for the TAZ1–p53TAD fusion. The p53TAD is bipartite, with two interaction motifs, termed AD1 and AD2, which fold to form short amphipathic helices upon binding to TAZ1 and TAZ2 whereas intervening regions of the p53TAD remain flexible. Both the AD1 and AD2 motifs bind to hydrophobic surfaces of the TAZ domains, with AD2 making more extensive hydrophobic contacts consistent with its greater contribution to the binding affinity. Binding of AD1 and AD2 is synergistic, and structural studies performed with isolated motifs can be misleading. The present structures of the full-length p53TAD complexes demonstrate the versatility of the interactions available to an intrinsically disordered domain containing bipartite interaction motifs and provide valuable insights into the structural basis of the affinity changes that occur upon stress-related posttranslational modification.

Single-molecule FRET is used to examine how an intrinsically disordered protein, the adenovirus E1A oncoprotein, interacts with two different protein partners (the pocket domain of pRb and the TAZ2 domain of CBP/p300); the biophysical behaviour of E1A depends on whether the N-terminal region and/or the CR2 region of E1A is free to interact with potential protein partners or whether they are ‘masked’ (that is, via their absence or a pre-existing interaction with another protein partner). Regulated allostery in a disordered hub protein Cellular function relies on intricate signalling networks incorporating protein signalling hubs that interact with several partners in order to modulate multiple downstream signals. This study of one such hub, the adenovirus early region 1A (E1A) oncoprotein, uses novel single molecule FRET measurements to overcome aggregation problems and to directly probe the allosteric interactions of E1A with two key partners. The results reveal a striking cooperativity modulation, regulated by multiple interactions and availability of E1A interaction motifs. Such a modulation of allosteric interactions may be a common feature of intrinsically disordered hub proteins. Allostery is an intrinsic property of many globular proteins and enzymes that is indispensable for cellular regulatory and feedback mechanisms. Recent theoretical 1 and empirical 2 observations indicate that allostery is also manifest in intrinsically disordered proteins, which account for a substantial proportion of the proteome 3 , 4 . Many intrinsically disordered proteins are promiscuous binders that interact with multiple partners and frequently function as molecular hubs in protein interaction networks. The adenovirus early region 1A (E1A) oncoprotein is a prime example of a molecular hub intrinsically disordered protein 5 . E1A can induce marked epigenetic reprogramming of the cell within hours after infection, through interactions with a diverse set of partners that include key host regulators such as the general transcriptional coactivator CREB binding protein (CBP), its paralogue p300, and the retinoblastoma protein (pRb; also called RB1) 6 , 7 . Little is known about the allosteric effects at play in E1A–CBP–pRb interactions, or more generally in hub intrinsically disordered protein interaction networks. Here we used single-molecule fluorescence resonance energy transfer (smFRET) to study coupled binding and folding processes in the ternary E1A system. The low concentrations used in these high-sensitivity experiments proved to be essential for these studies, which are challenging owing to a combination of E1A aggregation propensity and high-affinity binding interactions. Our data revealed that E1A–CBP–pRb interactions have either positive or negative cooperativity, depending on the available E1A interaction sites. This striking cooperativity switch enables fine-tuning of the thermodynamic accessibility of the ternary versus binary E1A complexes, and may permit a context-specific tuning of associated downstream signalling outputs. Such a modulation of allosteric interactions is probably a common mechanism in molecular hub intrinsically disordered protein function.

A classical dogma of molecular biology dictates that the 3D structure of a protein is necessary for its function. However, a considerable fraction of the human proteome, although functional, does not adopt a defined folded state under physiological conditions. These intrinsically disordered proteins tend to fold upon binding to their partners with a molecular mechanism that is elusive to experimental characterization. Indeed, although many hypotheses have been put forward, the functional role (if any) of disorder in these intrinsically denatured systems is still shrouded in mystery. Here, we characterize the structure of the transition state of the binding-induced folding in the reaction between the KIX domain of the CREB-binding protein and the transactivation domain of c-Myb. The analysis, based on the characterization of a series of conservative site-directed mutants, reveals a very high content of native-like structure in the transition state and indicates that the recognition between KIX and c-Myb is geometrically precise. The implications of our results in the light of previous work on intrinsically unstructured systems are discussed.

Many proteins that are important to cellular signalling are intrinsically disordered, as they only fold after binding with signalling partners. In this paper the molecular interactions governing such proteins' coupled folding and binding process, and how they differ radically from interactions between pre-folded proteins are described. Protein folding and binding are analogous processes, in which the protein ‘searches’ for favourable intramolecular or intermolecular interactions on a funnelled energy landscape 1 , 2 . Many eukaryotic proteins are disordered under physiological conditions, and fold into ordered structures only on binding to their cellular targets 3 , 4 , 5 , 6 . The mechanism by which folding is coupled to binding is poorly understood, but it has been hypothesized on theoretical grounds that the binding kinetics may be enhanced by a ‘fly-casting’ effect, where the disordered protein binds weakly and non-specifically to its target and folds as it approaches the cognate binding site 7 . Here we show, using NMR titrations and 15 N relaxation dispersion, that the phosphorylated kinase inducible activation domain (pKID) of the transcription factor CREB forms an ensemble of transient encounter complexes on binding to the KIX domain of the CREB binding protein. The encounter complexes are stabilized primarily by non-specific hydrophobic contacts, and evolve by way of an intermediate to the fully bound state without dissociation from KIX. The carboxy-terminal helix of pKID is only partially folded in the intermediate, and becomes stabilized by intermolecular interactions formed in the final bound state. Future applications of our method will provide new understanding of the molecular mechanisms by which intrinsically disordered proteins perform their diverse biological functions.

Background CREB-dependent transcription necessary for long-term memory is driven by interactions with CREB-binding protein (CBP), a multi-domain protein that binds numerous transcription factors potentially affecting expression of thousands of genes. Identifying specific domain functions for multi-domain proteins is essential to understand processes such as cognitive function and circadian clocks. We investigated the function of the CBP KIX domain in hippocampal memory and gene expression using CBP KIX/KIX mice with mutations that prevent phospho-CREB (Ser133) binding. Results We found that CBP KIX/KIX mice were impaired in long-term memory, but not learning acquisition or short-term memory for the Morris water maze. Using an unbiased analysis of gene expression in the dorsal hippocampus after training in the Morris water maze or contextual fear conditioning, we discovered dysregulation of CREB, CLOCK, and BMAL1 target genes and downregulation of circadian genes in CBP KIX/KIX mice. Given our finding that the CBP KIX domain was important for transcription of circadian genes, we profiled circadian activity and phase resetting in CBP KIX/KIX mice. CBP KIX/KIX mice exhibited delayed activity peaks after light offset and longer free-running periods in constant dark. Interestingly, CBP KIX/KIX mice displayed phase delays and advances in response to photic stimulation comparable to wildtype littermates. Thus, this work delineates site-specific regulation of the circadian clock by a multi-domain protein. Conclusions These studies provide insight into the significance of the CBP KIX domain by defining targets of CBP transcriptional co-activation in memory and the role of the CBP KIX domain in vivo on circadian rhythms. Graphical abstract

The transcription factor c-Myb promotes the proliferation of hematopoietic cells by interacting with the KIX domain of CREB-binding protein; however, its aberrant expression causes leukemia. Therefore, inhibitors of the c-Myb–KIX interaction are potentially useful as antitumor drugs. Since the intrinsically disordered transactivation domain (TAD) of c-Myb binds KIX via a conformational selection mechanism where helix formation precedes binding, stabilizing the helical structure of c-Myb TAD is expected to increase the KIX-binding affinity. Here, to develop an inhibitor of the c-Myb–KIX interaction, we designed mutants of the c-Myb TAD peptide fragment where the helical structure is stabilized, based on theoretical predictions using AGADIR. Three of the four initially designed peptides each had a different Lys-to-Arg substitution on the helix surface opposite the KIX-binding interface. Furthermore, the triple mutant with three Lys-to-Arg substitutions, named RRR, showed a high helical propensity and achieved three-fold higher affinity to KIX than the wild-type TAD with a dissociation constant of 80 nM. Moreover, the RRR inhibitor efficiently competed out the c-Myb–KIX interaction. These results suggest that stabilizing the helical structure based on theoretical predictions, especially by conservative Lys-to-Arg substitutions, is a simple and useful strategy for designing helical peptide inhibitors of protein–protein interactions.