Prepaying the entropic cost for allosteric regulation in KIX.
ABSTRACT: The kinase-inducible domain interacting (KIX) domain of the CREB binding protein (CBP) is capable of simultaneously binding two intrinsically disordered transcription factors, such as the mixed-lineage leukemia (MLL) and c-Myb peptides, at isolated interaction sites. In vitro, the affinity for binding c-Myb is approximately doubled when KIX is in complex with MLL, which suggests a positive cooperative binding mechanism, and the affinity for MLL is also slightly increased when KIX is first bound by c-Myb. Expanding the scope of recent NMR and computational studies, we explore the allosteric mechanism at a detailed molecular level that directly connects the microscopic structural dynamics to the macroscopic shift in binding affinities. To this end, we have performed molecular dynamics simulations of free KIX, KIX-c-Myb, MLL-KIX, and MLL-KIX-c-Myb using a topology-based G?-like model. Our results capture an increase in affinity for the peptide in the allosteric site when KIX is prebound by a complementary effector and both peptides follow an effector-independent folding-and-binding mechanism. More importantly, we discover that MLL binding lowers the entropic cost for c-Myb binding, and vice versa, by stabilizing the L12-G2 loop and the C-terminal region of the ?3 helix on KIX. This work demonstrates the importance of entropy in allosteric signaling between promiscuous molecular recognition sites and can inform the rational design of small molecule stabilizers to target important regions of conformationally dynamic proteins.
Project description:The KIX domain of CBP is a transcriptional coactivator. Concomitant binding to the activation domain of proto-oncogene protein c-Myb and the transactivation domain of the trithorax group protein mixed lineage leukemia (MLL) transcription factor lead to the biologically active ternary MLL?KIX?c-Myb complex which plays a role in Pol II-mediated transcription. The binding of the activation domain of MLL to KIX enhances c-Myb binding. Here we carried out molecular dynamics (MD) simulations for the MLL?KIX?c-Myb ternary complex, its binary components and KIX with the goal of providing a mechanistic explanation for the experimental observations. The dynamic behavior revealed that the MLL binding site is allosterically coupled to the c-Myb binding site. MLL binding redistributes the conformational ensemble of KIX, leading to higher populations of states which favor c-Myb binding. The key element in the allosteric communication pathways is the KIX loop, which acts as a control mechanism to enhance subsequent binding events. We tested this conclusion by in silico mutations of loop residues in the KIX?MLL complex and by comparing wild type and mutant dynamics through MD simulations. The loop assumed MLL binding conformation similar to that observed in the KIX?c-Myb state which disfavors the allosteric network. The coupling with c-Myb binding site faded, abolishing the positive cooperativity observed in the presence of MLL. Our major conclusion is that by eliciting a loop-mediated allosteric switch between the different states following the binding events, transcriptional activation can be regulated. The KIX system presents an example how nature makes use of conformational control in higher level regulation of transcriptional activity and thus cellular events.
Project description:Physical interaction between the transactivation domain (TAD) of the mixed-lineage leukemia protein (MLL) and the KIX domain of the cyclic-AMP response element binding protein (CREB) binding protein (CBP) is necessary for MLL-mediated transcriptional activation. We show by alanine-scanning mutagenesis that hydrophobic surface residues of KIX, especially L628, are energetically important for binding the MLL TAD. NMR studies of the KIX-L628A mutant suggest that L628 plays a crucial role in conformational transitions at the MLL binding site, necessary for high affinity interactions with MLL. Unexpectedly, MLL also binds to the c-Myb/phosphorylated kinase-inducible domain of CREB (pKID) site of KIX, highlighting the complex nature of interactions involving intrinsically disordered transcriptional activators.
Project description:The complex retrovirus human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia. Deregulation of cellular transcription is thought to be an important step for T-cell transformation caused by viral infection. HTLV-1 basic leucine zipper factor (HBZ) is one of the viral proteins believed to be involved in this process, as it deregulates the expression of numerous cellular genes. In the context of the provirus, HBZ represses HTLV-1 transcription, in part, by binding to the homologous cellular coactivators p300 and CBP. These coactivators play a central role in transcriptional regulation. In this study, we determined that HBZ binds with high affinity to the KIX domain of p300/CBP. This domain contains two binding surfaces that are differentially targeted by multiple cellular factors. We show that two ?XX?? motifs in the activation domain of HBZ mediate binding to a single surface of the KIX domain, the mixed-lineage leukemia (MLL) binding surface. Formation of this interaction inhibits binding of MLL to the KIX domain while enhancing the binding of the transcription factor c-Myb to the opposite surface of KIX. Consequently, HBZ inhibits transcriptional activation mediated by MLL and enhances activation mediated by c-Myb. CREB, which binds the same surface of KIX as c-Myb, also exhibited an increase in activity through HBZ. These results indicate that HBZ is able to alter gene expression by competing with transcription factors for the occupancy of one surface of KIX while enhancing the binding of factors to the other surface.
Project description:The KIX domain of the transcriptional coactivator CREB binding protein (CBP) co-operatively mediates interactions between transcription factors. Binding of the transcription factor mixed-lineage leukemia (MLL) induces the formation of a low-populated conformer of KIX that resembles the conformation of the KIX domain in the presence of a second transcription factor molecule. NMR spin relaxation studies have previously shown that allosteric coupling proceeds through a network of hydrophobic core residues that bridge the two binding sites. Here we describe high-resolution NMR solution structures of the binary complex of KIX with MLL and the ternary complex of KIX formed with MLL and phosphorylated kinase inducible domain of CREB (pKID) as a second ligand. We show that binding of pKID to the binary complex of KIX with MLL is accompanied by a defined repacking of the allosteric network in the hydrophobic core of the protein. Rotamer populations derived from methyl group (13)C chemical shifts reveal a dynamic contribution to the repacking process that is not captured by the structural coordinates and exemplify the dynamic nature of allosteric communication in the KIX domain.
Project description:The human T cell leukemia virus I basic leucine zipper protein (HTLV-1 HBZ) maintains chronic viral infection and promotes leukemogenesis through poorly understood mechanisms involving interactions with the KIX domain of the transcriptional coactivator CBP and its paralog p300. The KIX domain binds regulatory proteins at the distinct MLL and c-Myb/pKID sites to form binary or ternary complexes. The intrinsically disordered N-terminal activation domain of HBZ (HBZ AD) deregulates cellular signaling pathways by competing directly with cellular and viral transcription factors for binding to the MLL site and by allosterically perturbing binding of the transactivation domain of the hematopoietic transcription factor c-Myb. Crystal structures of the ternary KIX:c-Myb:HBZ complex show that the HBZ AD recruits two KIX:c-Myb entities through tandem amphipathic motifs (L/V)(V/L)DGLL and folds into a long ?-helix upon binding. Isothermal titration calorimetry reveals strong cooperativity in binding of the c-Myb activation domain to the KIX:HBZ complex and in binding of HBZ to the KIX:c-Myb complex. In addition, binding of KIX to the two HBZ (V/L)DGLL motifs is cooperative; the structures suggest that this cooperativity is achieved through propagation of the HBZ ?-helix beyond the first binding motif. Our study suggests that the unique structural flexibility and the multiple interaction motifs of the intrinsically disordered HBZ AD are responsible for its potency in hijacking KIX-mediated transcription pathways. The KIX:c-Myb:HBZ complex provides an example of cooperative stabilization in a transcription factor:coactivator network and gives insights into potential mechanisms through which HBZ dysregulates hematopoietic transcriptional programs and promotes T cell proliferation.
Project description:The mammalian CLOCK:BMAL1 transcription factor complex and its coactivators CREB-binding protein (CBP)/p300 and mixed-lineage leukemia 1 (MLL1) critically regulate circadian transcription and chromatin modification. Circadian oscillations are regulated by interactions of BMAL1's C-terminal transactivation domain (TAD) with the KIX domain of CBP/p300 (activating) and with the clock protein CRY1 (repressing) as well as by the BMAL1 G-region preceding the TAD. Circadian acetylation of Lys537 within the G-region enhances repressive BMAL1-TAD-CRY1 interactions. Here, we characterized the interaction of the CBP-KIX domain with BMAL1 proteins, including the BMAL1-TAD, parts of the G-region, and Lys537 Tethering the small compound 1-10 in the MLL-binding pocket of the CBP-KIX domain weakened BMAL1 binding, and MLL1-bound KIX did not form a ternary complex with BMAL1, indicating that the MLL-binding pocket is important for KIX-BMAL1 interactions. Small-angle X-ray scattering (SAXS) models of BMAL1 and BMAL1:KIX complexes revealed that the N-terminal BMAL1 G-region including Lys537 forms elongated extensions emerging from the bulkier BMAL1-TAD:KIX core complex. Fitting high-resolution KIX domain structures into the SAXS-derived envelopes suggested that the G-region emerges near the MLL-binding pocket, further supporting a role of this pocket in BMAL1 binding. Additionally, mutations in the second CREB-pKID/c-Myb-binding pocket of the KIX domain moderately impacted BMAL1 binding. The BMAL1(K537Q) mutation mimicking Lys537 acetylation, however, did not affect the KIX-binding affinity, in contrast to its enhancing effect on CRY1 binding. Our results significantly advance the mechanistic understanding of the protein interaction networks controlling CLOCK:BMAL1- and CBP-dependent gene regulation in the mammalian circadian clock.
Project description: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.
Project description:Forkhead box class O 3a (FOXO3a) is a transcription factor and tumor suppressor linked to longevity that determines cell fate through activating transcription of cell differentiation, survival, and apoptotic genes. Recruitment of the coactivator CBP/p300 is a crucial step in transcription, and we revealed that in addition to conserved region 3 (CR3) of FOXO3a, the C-terminal segment of CR2 (CR2C) binds CBP/p300 and contributes to transcriptional activity. CR2C and CR3 of FOXO3a interact with the KIX domain of CBP/p300 at both "MLL" and "c-Myb" binding sites simultaneously. A FOXO3a CR2C-CR3 peptide in complex with KIX exists in equilibrium between two equally populated conformational states, one of which has CR2C bound to the MLL site and CR3 bound to the c-Myb site, whereas in the other, CR2C and CR3 bind the c-Myb and MLL sites, respectively. This promiscuous interaction between FOXO3a and CBP/p300 is further supported by additional binding sites on CBP/p300, namely, the TAZ1 and TAZ2 domains. In functional studies, our structure-guided mutagenesis showed that both CR2C and CR3 are involved in the activation of certain endogenous FOXO3a target genes. Further, phosphorylation of S626, a known AMP-dependent protein kinase target in CR3, increased affinity for CBP/p300 and the phosphomimetic mutation enhanced transactivation of luciferase. These findings underscore the significance of promiscuous multivalent interactions and posttranslational modification in the recruitment of transcriptional coactivators, which may allow transcription factors to adapt to various gene-specific genomic and chromatin structures and respond to cell signals.
Project description:Molecular interactions between the tumor suppressor p53 and the transcriptional coactivators CBP/p300 are critical for the regulation of p53 transactivation and stability. The transactivation domain (TAD) of p53 binds directly to several CBP/p300 domains (TAZ1, TAZ2, NCBD, and KIX). Here we map the interaction between the p53 TAD and the CBP KIX domain using isothermal titration calorimetry and NMR spectroscopy. KIX is a structural domain in CBP/p300 that can simultaneously bind two polypeptide ligands, such as the activation domain of MLL and the kinase-inducible activation domain (pKID) of CREB, using distinct interaction surfaces. The p53 TAD consists of two subdomains (AD1 and AD2); peptides corresponding to the isolated AD1 and AD2 subdomains interact with KIX with relatively low affinity, but a longer peptide containing both subdomains binds KIX tightly. In the context of the full-length p53 TAD, AD1 and AD2 bind synergistically to KIX. Mapping of the chemical shift perturbations onto the structure of KIX shows that isolated AD1 and AD2 peptides bind to both the MLL and pKID sites. Spin-labeling experiments show that the complex of the full-length p53 TAD with KIX is disordered, with the AD1 and AD2 subdomains each interacting with both the MLL and pKID binding surfaces. Phosphorylation of the p53 TAD at Thr18 or Ser20 increases the KIX binding affinity. The affinity is further enhanced by simultaneous phosphorylation of Thr18 and Ser20, and the specificity of the interaction is increased. The p53 TAD simultaneously occupies the two distinct sites that have been identified on the CBP KIX domain and efficiently competes for these sites with other known KIX-binding transcription factors.
Project description:The viral oncoprotein Tax mediates transcriptional activation of human T-cell leukemia virus type 1 (HTLV-1). Both Tax and the cellular transcription factor CREB bind to viral cyclic AMP response elements (vCREs) located in the viral promoter. Tax and serine 133 phosphorylated CREB (pCREB) bound to the HTLV-1 promoter facilitate viral transcription via the recruitment of the large cellular coactivators CBP/p300. While the interaction between the phosphorylated kinase inducible domain (pKID) of pCREB and the KIX domain of CBP/p300 has been well characterized, the molecular interactions between KIX, full-length Tax, and pCREB have not been examined. Here we biochemically characterized the interaction between Tax and KIX in a physiologically relevant complex containing pCREB and vCRE DNA. Our data show that Tax and pCREB simultaneously and independently bind two distinct surfaces on the KIX domain: Tax binds KIX at the previously characterized mixed-lineage leukemia (MLL) protein interaction surface while pCREB binds KIX at the pKID-KIX interface. These results provide evidence for a model in which Tax and pCREB bind distinct surfaces of KIX for effective CBP/p300 recruitment to the HTLV-1 promoter. We also show that MLL competes with Tax for KIX binding, suggesting a novel mechanism of Tax oncogenesis in which normal MLL function is disrupted by Tax.