TAZ1 Domain Research Articles

Quantitative Analysis of Protein-Ligand Interactions by NMR

Determination of affinities and binding sites involved in protein-ligand interactions is essential for understanding molecular mechanisms in biological systems. We combined singular value decomposition and global analysis of NMR chemical shift perturbations caused by protein-protein interactions to determine the number and location of binding sites on the protein surface and to measure the binding affinities. This review focuses on the details of the quantitative analysis of NMR titration data. As an example, the study on the interactions of the intrinsically disordered AD2 subdomain of the tumor suppressor p53 with the TAZ2 domain of the transcriptional coactivator CBP is described.

Interaction of the transactivation domain of B-Myb with the TAZ2 domain of the coactivator p300: molecular features and properties of the complex.

The transcription factor B-Myb is a key regulator of the cell cycle in vertebrates, with activation of transcription involving the recognition of specific DNA target sites and the recruitment of functional partner proteins, including the coactivators p300 and CBP. Here we report the results of detailed studies of the interaction between the transactivation domain of B-Myb (B-Myb TAD) and the TAZ2 domain of p300. The B-Myb TAD was characterized using circular dichroism, fluorescence and NMR spectroscopy, which revealed that the isolated domain exists as a random coil polypeptide. Pull-down and spectroscopic experiments clearly showed that the B-Myb TAD binds to p300 TAZ2 to form a moderately tight (Kd ∼1.0–10 µM) complex, which results in at least partial folding of the B-Myb TAD. Significant changes in NMR spectra of p300 TAZ2 suggest that the B-Myb TAD binds to a relatively large patch on the surface of the domain (∼1200 Å2). The apparent B-Myb TAD binding site on p300 TAZ2 shows striking similarity to the surface of CBP TAZ2 involved in binding to the transactivation domain of the transcription factor signal transducer and activator of transcription 1 (STAT1), which suggests that the structure of the B-Myb TAD-p300 TAZ2 complex may share many features with that reported for STAT1 TAD-p300 TAZ2.

Quantitative Analysis of Multisite Protein–Ligand Interactions by NMR: Binding of Intrinsically Disordered p53 Transactivation Subdomains with the TAZ2 Domain of CBP

Determination of affinities and binding sites involved in protein-ligand interactions is essential for understanding molecular mechanisms in biological systems. Here we combine singular value decomposition and global analysis of NMR chemical shift perturbations caused by protein-protein interactions to determine the number and location of binding sites on the protein surface and to measure the binding affinities. Using this method we show that the isolated AD1 and AD2 binding motifs, derived from the intrinsically disordered N-terminal transactivation domain of the tumor suppressor p53, both interact with the TAZ2 domain of the transcriptional coactivator CBP at two binding sites. Simulations of titration curves and line shapes show that a primary dissociation constant as small as 1-10 nM can be accurately estimated by NMR titration methods, provided that the primary and secondary binding processes are coupled. Unexpectedly, the site of binding of AD2 on the hydrophobic surface of TAZ2 overlaps with the binding site for AD1, but AD2 binds TAZ2 more tightly. The results highlight the complexity of interactions between intrinsically disordered proteins and their targets. Furthermore, the association rate of AD2 to TAZ2 is estimated to be 1.7 × 10(10) M(-1) s(-1), approaching the diffusion-controlled limit and indicating that intrinsic disorder plus complementary electrostatics can significantly accelerate protein binding interactions.

1PT158 天然変成蛋白質p53TADによるCBP TAZ2への極めて速い結合反応(日本生物物理学会第50回年会(2012年度))

1PT158 天然変成蛋白質p53TADによるCBP TAZ2への極めて速い結合反応(日本生物物理学会第50回年会(2012年度))

Disrupting the CH1 Domain Structure in the Acetyltransferases CBP and p300 Results in Lean Mice with Increased Metabolic Control

Opposing activities of acetyltransferases and deacetylases help regulate energy balance. Mice heterozygous for the acetyltransferase CREB binding protein (CBP) are lean and insulin sensitized, but how CBP regulates energy homeostasis is unclear. In one model, the main CBP interaction with the glucagon-responsive factor CREB is not limiting for liver gluconeogenesis, whereas a second model posits that Ser436 in the CH1 (TAZ1) domain of CBP is required for insulin and the antidiabetic drug metformin to inhibit CREB-mediated liver gluconeogenesis. Here we show that conditional knockout of CBP in liver does not decrease fasting blood glucose or gluconeogenic gene expression, consistent with the first model. However, mice in which the CBP CH1 domain structure is disrupted by deleting residues 342-393(ΔCH1) are lean and insulin sensitized, as are p300ΔCH1 mutants. CBP(ΔCH1/ΔCH1) mice remain metformin responsive. An intact CH1 domain is thus necessary for normal energy storage, but not for the blood glucose-lowering actions of insulin and metformin.

Structure of p300 bound to MEF2 on DNA reveals a mechanism of enhanceosome assembly

Transcription co-activators CBP and p300 are recruited by sequence-specific transcription factors to specific genomic loci to control gene expression. A highly conserved domain in CBP/p300, the TAZ2 domain, mediates direct interaction with a variety of transcription factors including the myocyte enhancer factor 2 (MEF2). Here we report the crystal structure of a ternary complex of the p300 TAZ2 domain bound to MEF2 on DNA at 2.2Å resolution. The structure reveals three MEF2:DNA complexes binding to different sites of the TAZ2 domain. Using structure-guided mutations and a mammalian two-hybrid assay, we show that all three interfaces contribute to the binding of MEF2 to p300, suggesting that p300 may use one of the three interfaces to interact with MEF2 in different cellular contexts and that one p300 can bind three MEF2:DNA complexes simultaneously. These studies, together with previously characterized TAZ2 complexes bound to different transcription factors, demonstrate the potency and versatility of TAZ2 in protein–protein interactions. Our results also support a model wherein p300 promotes the assembly of a higher-order enhanceosome by simultaneous interactions with multiple DNA-bound transcription factors.

Investigation of the interaction between CREB-binding protein and STAT4/STAT6

Coactivator CBP (CREB-binding protein) has been implicated in the regulation of transcription for all signal transducer and activator of transcription factors (STATs); however, the mechanism remains unclear. Using yeast two-hybrid screening and immunoprecipitation techniques, we investigated the direct interaction of CBP with STAT4 and STAT6. The full-length CBP and five fragments of CBP (residues 1-436, 529-1200, 1-697, 967-1574 and 1678-2175) were constructed using pGBKT7 vectors, while STAT4, STAT6 and N-terminal deleted STAT4 were constructed using pGADT7 vectors. It was found that STAT4, but not STAT6, interacted directly with the 1678-2175 fragment of CBP containing the ZZ, TAZ2 and SID domain. The N-terminal of STAT4 plays an important role in this interaction since N-terminal deleted STAT4 failed to bind to any CBP fragment. The results were confirmed by immunoprecipitation using HA-tagged STAT4 or STAT6 and c-Myc tagged CBP. This work will contribute to our understanding of the mechanisms of Th cytokine imbalance.

Graded enhancement of p53 binding to CREB-binding protein (CBP) by multisite phosphorylation

The transcriptional activity of p53 is regulated by a cascade of posttranslational modifications. Although acetylation of p53 by CREB-binding protein (CBP)/p300 is known to be indispensable for p53 activation, the role of phosphorylation, and in particular multisite phosphorylation, in activation of CBP/p300-dependent p53 transcriptional pathways remains unclear. We investigated the role of single site and multiple site phosphorylation of the p53 transactivation domain in mediating its interaction with CBP and with the ubiquitin ligase HDM2. Phosphorylation at Thr18 functions as an on/off switch to regulate binding to the N-terminal domain of HDM2. In contrast, binding to CBP is modulated by the extent of p53 phosphorylation; addition of successive phosphoryl groups enhances the affinity for the TAZ1, TAZ2, and KIX domains of CBP in an additive manner. Activation of p53-dependent transcriptional pathways requires that p53 compete with numerous cellular transcription factors for binding to limiting amounts of CBP/p300. Multisite phosphorylation represents a mechanism for a graded p53 response, with each successive phosphorylation event resulting in increasingly efficient recruitment of CBP/p300 to p53-regulated transcriptional programs, in the face of competition from cellular transcription factors. Multisite phosphorylation thus acts as a rheostat to enhance binding to CBP/p300 and provides a plausible mechanistic explanation for the gradually increasing p53 response observed following prolonged or severe genotoxic stress.

Ras signaling requires dynamic properties of Ets1 for phosphorylation-enhanced binding to coactivator CBP

Ras/MAPK signaling is often aberrantly activated in human cancers. The downstream effectors are transcription factors, including those encoded by the ETS gene family. Using cell-based assays and biophysical measurements, we have determined the mechanism by which Ras/MAPK signaling affects the function of Ets1 via phosphorylation of Thr38 and Ser41. These ERK2 phosphoacceptors lie within the unstructured N-terminal region of Ets1, immediately adjacent to the PNT domain. NMR spectroscopic analyses demonstrated that the PNT domain is a four-helix bundle (H2-H5), resembling the SAM domain, appended with two additional helices (H0-H1). Phosphorylation shifted a conformational equilibrium, displacing the dynamic helix H0 from the core bundle. The affinity of Ets1 for the TAZ1 (or CH1) domain of the coactivator CBP was enhanced 34-fold by phosphorylation, and this binding was sensitive to ionic strength. NMR-monitored titration experiments mapped the interaction surfaces of the TAZ1 domain and Ets1, the latter encompassing both the phosphoacceptors and PNT domain. Charge complementarity of these surfaces indicate that electrostatic forces act in concert with a conformational equilibrium to mediate phosphorylation effects. We conclude that the dynamic helical elements of Ets1, appended to a conserved structural core, constitute a phospho-switch that directs Ras/MAPK signaling to downstream changes in gene expression. This detailed structural and mechanistic information will guide strategies for targeting ETS proteins in human disease.

3P040 NMRによる天然変性蛋白質p53の転写活性化サブドメインとCBP TAZ2との相互作用解析(蛋白質-構造機能相関,第48回日本生物物理学会年会)

3P040 NMRによる天然変性蛋白質p53の転写活性化サブドメインとCBP TAZ2との相互作用解析(蛋白質-構造機能相関,第48回日本生物物理学会年会)

Structure of the Taz2 domain of p300: insights into ligand binding

CBP and its paralog p300 are histone acetyl transferases that regulate gene expression by interacting with multiple transcription factors via specialized domains. The structure of a segment of human p300 protein (residues 1723-1836) corresponding to the extended zinc-binding Taz2 domain has been investigated. The crystal structure was solved by the SAD approach utilizing the anomalous diffraction signal of the bound Zn ions. The structure comprises an atypical helical bundle stabilized by three Zn ions and closely resembles the solution structures determined previously for shorter peptides. Residues 1813-1834 from the current construct form a helical extension of the C-terminal helix and make extensive crystal-contact interactions with the peptide-binding site of Taz2, providing additional insights into the mechanism of the recognition of diverse transactivation domains (TADs) by Taz2. On the basis of these results and molecular modeling, a hypothetical model of the binding of phosphorylated p53 TAD1 to Taz2 has been proposed.

Crystal structure of the Taz2 domain of p300 transcriptional coactivator

Crystal structure of the Taz2 domain of p300 transcriptional coactivator

Cooperative regulation of p53 by modulation of ternary complex formation with CBP/p300 and HDM2.

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.

Molecular basis of the interactions between the p73 N terminus and p300: Effects on transactivation and modulation by phosphorylation

The transcription factor p73 belongs to the p53 family of proteins and can transactivate a number of target genes in common with p53. Here, we characterized the interaction of the p73 N terminus with four domains of the transcriptional coactivator p300 and with the negative regulator Mdm2 by using biophysical and cellular measurements. We found that, like p53, the N terminus of p73 contained two distinct transactivation subdomains, comprising residues 10-30 and residues 46-67. The p73 N terminus bound weakly to the Taz1, Kix, and IBiD domains of p300 but with submicromolar affinity for Taz2, in contrast to previous reports. We found weaker binding of the p73 N terminus to the p300 domains in vitro correlated with a significant decrease in transactivation activity in a cell line for the QS and T14A mutants, and tighter binding of the phosphomimetic T14D in vitro correlated with an increase in vivo. Further, we found that phosphorylation of T14 increased the affinity of the p73 N terminus for Taz2 10-fold. The phosphomimetic p73alpha T14D caused increased levels of transactivation.

Structural basis for recruitment of CBP/p300 coactivators by STAT1 and STAT2 transactivation domains

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.

Structural Basis for p300 Taz2-p53 TAD1 Binding and Modulation by Phosphorylation

Coactivators CREB-binding protein and p300 play important roles in mediating the transcriptional activity of p53. Until now, however, no detailed structural information has been available on how any of the domains of p300 interact with p53. Here, we report the NMR structure of the complex of the Taz2 (C/H3) domain of p300 and the N-terminal transactivation domain of p53. In the complex, p53 forms a short alpha helix and interacts with the Taz2 domain through an extended surface. Mutational analyses demonstrate the importance of hydrophobic residues for complex stabilization. Additionally, they suggest that the increased affinity of Taz2 for p53(1-39) phosphorylated at Thr(18) is due in part to electrostatic interactions of the phosphate with neighboring arginine residues in Taz2. Thermodynamic experiments revealed the importance of hydrophobic interactions in the complex of Taz2 with p53 phosphorylated at Ser(15) and Thr(18).

Two Distinct Motifs within the p53 Transactivation Domain Bind to the Taz2 Domain of p300 and Are Differentially Affected by Phosphorylation

The tumor suppressor p53 functions as a transcriptional activator for many genes, including several key genes involved in cell cycle arrest and apoptosis. Following DNA damage-induced stress, p53 undergoes extensive posttranslational modification, resulting in increased stability and activity. Two critical cofactors for p53-mediated transactivation are the histone acetyltransferase paralogues CREB-binding protein (CBP) and p300. The N-terminal transactivation domain of p53 interacts with several domains of CBP/p300, including the Taz2 domain. Here, we report the effects of specific p53 phosphorylations on its interaction with the Taz2 domain of p300. Using a competitive fluorescence anisotropy assay, we determined that monophosphorylation of p53 at Ser(15) or Thr(18) increased the affinity of p53(1-39) for Taz2, and diphosphorylations at Ser(15) and Ser(37) or Thr(18) and Ser(20) further increased the affinity. In addition, we identified a second binding site for Taz2 within p53 residues 35-59. This second site bound Taz2 with a similar affinity as the first site, but the binding was unaffected by phosphorylation. Thus, p53 posttranslational modification modulates only one of the two binding sites for p300 Taz2. Further investigation of Taz2 binding to p53(1-39) or p53(35-59) by isothermal titration calorimetry indicated that upon complex formation, the change in heat capacity at constant pressure, DeltaC(p), was negative for both sites, suggesting the importance of hydrophobic interactions. However, the more negative value of DeltaC(p) for Taz2 binding to the first (-330 cal/(mol.K)) compared to the second site (-234 cal/(mol.K)) suggests that the importance of nonpolar and polar interactions differs between the two sites.

Biochemical and NMR Mapping of the Interface between CREB-binding Protein and Ligand Binding Domains of Nuclear Receptor: BEYOND THE LXXLL MOTIF

Biochemical and NMR Mapping of the Interface between CREB-binding Protein and Ligand Binding Domains of Nuclear Receptor: BEYOND THE LXXLL MOTIF

CBP/p300 TAZ1 Domain Forms a Structured Scaffold for Ligand Binding,

The transcriptional coactivator protein CBP and its paralog p300 each contain two homologous zinc-containing TAZ domains, which constitute the interaction sites for a number of transcription factors. Previous reports of the three-dimensional structures of TAZ1 in complex with binding partners and of the isolated CBP TAZ2 domain show a distinctive topology composed of four amphipathic helices, organized by three zinc-binding clusters with HCCC-type coordination. The isolated CBP TAZ2 domain forms a stable three-dimensional structure in solution, but a recent report [Dial, R., Sun, Z., and Freedman, S. J. (2003) Biochemistry 42, 9937] suggested that the isolated p300 TAZ1 domain lacks a well-defined structure and behaves like a molten globule, even in the presence of Zn(2+), and that the formation of a stable three-dimensional structure requires binding of a protein partner. In marked contrast to this result, we find that both the CBP and p300 TAZ domains in the presence of stoichiometric concentrations of Zn(2+) adopt a well-defined structure in solution in the absence of binding partners. We have determined the three-dimensional structure of the isolated CBP TAZ1 domain by NMR methods and show that it has the same structure in the presence and absence of binding partners. This is an important finding: whether the free TAZ1 domain forms a folded structure or behaves as a molten globule will have a significant bearing on the mechanism of protein-protein recognition. Although TAZ1 and TAZ2 share many structural similarities, there is a major structural difference: the fourth helix is oriented in opposite directions in the TAZ1 and TAZ2 domains. The structure of the free TAZ1 domain suggests that this difference is an inherent feature that determines binding specificity and facilitates discrimination between different subsets of transcription factors by the two TAZ domains.

ZZ Domain of CBP: an Unusual Zinc Finger Fold in a Protein Interaction Module

CREB-binding protein (CBP) is a large, multi-domain protein that provides a multitude of binding sites for transcriptional coactivators. The site of interaction of the tumor suppressor p53 and the oncoprotein E1A with CBP/p300 has been identified with the third cysteine-histidine-rich (CH3) domain, which incorporates two zinc-binding motifs, ZZ and TAZ2. We show that these two domains fold independently and do not interact in solution. Our experiments demonstrate conclusively that the interaction of p53 and E1A with the CH3 domain resides exclusively in the TAZ2 domain, with no contribution from the ZZ domain. We report also the three-dimensional solution structure of the ZZ domain of murine CBP. The 52 residue ZZ domain contains two twisted antiparallel beta-sheets and a short alpha-helix, and binds two zinc ions. The identity of the zinc coordinating ligands was resolved unambiguously using NMR spectroscopy of the ZZ domain substituted with (113)Cd. One zinc ion is coordinated tetrahedrally via two CXXC motifs to four cysteine side-chains, and the second zinc ion is coordinated tetrahedrally by a third CXXC motif, together with an unusual HXH motif coordinating via the N(epsilon2) atom of His40 and the N(delta1) atom of His-42. The first zinc cluster of the ZZ domain is strictly conserved, whereas the second zinc cluster shows variability in the position of the two histidine residues, reflecting the wide variety of molecules that incorporate ZZ domains. The structure of the ZZ domain shows that it belongs to the family of cross-brace zinc finger motifs that include the PHD, RING, and FYVE domains; however, its biological function is unclear. Mapping of the positions of conserved residues onto the calculated structures reveals a face containing exposed aromatic and hydrophobic side-chains, while the opposite face contains a series of conserved charged or hydrophilic groups. These homologies suggest that the ZZ domain is involved in ligand binding or molecular scaffolding, with specificity provided by the variability of the sequence that contains the helix in the murine CPB ZZ domain structure.