Structural insights into TAZ2 domain–mediated CBP/p300 recruitment by transactivation domain 1 of the lymphopoietic transcription factor E2A

Abstract

The E-protein transcription factors guide immune cell differentiation, with E12 and E47 (hereafter called E2A) being essential for B-cell specification and maturation. E2A and the oncogenic chimera E2A-PBX1 contain three transactivation domains (ADs), with AD1 and AD2 having redundant, independent, and cooperative functions in a cell-dependent manner. AD1 and AD2 both mediate their functions by binding to the KIX domain of the histone acetyltransferase paralogues CREB-binding protein (CBP) and E1A-binding protein P300 (p300). This interaction is necessary for B-cell maturation and oncogenesis by E2A-PBX1 and occurs through conserved ΦXXΦΦ motifs (with Φ denoting a hydrophobic amino acid) in AD1 and AD2. However, disruption of this interaction via mutation of the KIX domain in CBP/p300 does not completely abrogate binding of E2A and E2A-PBX1. Here, we determined that E2A-AD1 and E2A-AD2 also interact with the TAZ2 domain of CBP/p300. Characterization of the TAZ2:E2A-AD1(1–37) complex indicated that E2A-AD1 adopts an α-helical structure and uses its ΦXXΦΦ motif to bind TAZ2. Whereas this region overlapped with the KIX recognition region, key KIX-interacting E2A-AD1 residues were exposed, suggesting that E2A-AD1 could simultaneously bind both the KIX and TAZ2 domains. However, we did not detect a ternary complex involving E2A-AD1, KIX, and TAZ2 and found that E2A containing both intact AD1 and AD2 is required to bind to CBP/p300. Our findings highlight the structural plasticity and promiscuity of E2A-AD1 and suggest that E2A binds both the TAZ2 and KIX domains of CBP/p300 through AD1 and AD2.

Introduction

The innate and adaptive immune systems rely on the development and differentiation of hematopoietic stem cells to mature blood cells, including B and T lymphocytes, natural killer cells, and plasmacytoid dendritic cells (1–3). The generation of these immune cells, or lymphopoiesis, involves numerous intermediates, a progressive limitation of differentiation potential, and a coincident loss of the ability to self-renew (4). This process of lineage commitment is tightly regulated at the transcriptional level by complex regulatory networks comprising both lineage-specific and ubiquitous transcription factors (2, 5–7). One such set of transcription factors is the E-protein family, which comprise class I basic helix-loop-helix (bHLH)⁴ transcription factors that play essential roles in the development and specification of B- and T-lymphocytes (8–10).

Members of the E-protein family include the alternatively spliced isoforms E12 and E47 (also referred to collectively as E2A), HEB, and E2-2. Each family member contains a C-terminal bHLH domain responsible for E-protein dimerization and binding DNA at E-box CANNTG consensus sites in gene enhancer and/or promoter regions (11–17). In addition, the E-proteins possess three conserved activation domains, one of which (AD1) is positioned at the extreme N terminus, whereas the other two (AD2 and AD3) are more centrally located (Fig. 1A) (14, 15, 18, 19). These activation domains have been shown to display cooperative or independent transcriptional regulatory functions in a cell-specific manner (19–25). For example, AD1, AD2, and AD3 independently induce transcriptional activation but bind the same site of co-activators in a redundant manner, and when combined, AD1 and AD2 cooperate to display greater than additive gene induction (19, 21, 22, 26–28). Deletion of AD1 or AD2 in E2A abolishes B-lymphoid differentiation in a pre-B cell line, which is consistent with the observation that E2A plays a critical regulatory role at the earliest stages of B-lymphoid specification (21). The transcriptional regulatory role of these activation domains resides in their ability to recruit general transcriptional factors and coactivators, such as TFIID and the histone acetyltransferases SAGA, GCN5, PCAF, the paralogs CBP and p300, and corepressors such as ETO (19, 22, 23, 26, 29–31). Whereas AD3 allows the E-proteins to recruit TFIID to the core promoter by binding the TAFH domain of TAF4 (19), both AD1 and AD2 interact with CBP/p300 to enhance the acetyltransferase activity of this cofactor (26–28, 32). A conserved region within AD1 called the “p300/CBP and ETO target in E proteins” (PCET) motif, is also the target of the transcriptional repressor ETO, and competition between ETO and CBP/p300 for binding to the PCET motif has been proposed to be the mechanism underlying E-protein–mediated transcriptional silencing (31).

Figure 1.

Domain architecture of E2A and CBP/p300. A, schematic representation of E2A and the human histone acetyltransferase (HAT) p300, with the relevant domains (for E2A, activation domains AD1, AD2, and AD3 and the C-terminal DNA-binding bHLH domain; for p300, the TAZ1, KIX, and TAZ2 protein-protein interaction domains and the histone acetyltransferase catalytic domain) indicated. B, sequence alignment of the ΦXXΦΦ motifs of the ADs of E2A, HEB, FOXO3a, p53, and p63, where Φ represents a hydrophobic amino acid residue and X is any amino acid residue. Hydrophobic, acidic, and basic amino acid residues are colored yellow, red, and blue, respectively. Numbering is in accordance with the native protein sequence.

CBP/p300 are multimodular proteins in which several protein-protein interaction domains allow recruitment to enhancers and promoters via interactions with the activation domains of an array of transcription factors (Fig. 1A) (33–35). AD1 and AD2 of the E-proteins have been reported to bind the same surface of the KIX domain of CBP/p300 via their ΦXXΦΦ sequences (where Φ represents a hydrophobic amino acid, and X represents any other amino acid), which comprise the core PCET motif (Fig. 1B). This interaction has been shown to be particularly important for leukemia induction by the oncogenic protein E2A-PBX1 that arises from a t(1;19) chromosomal translocation (20, 27, 28). In addition to the KIX domain, the TAZ1 and TAZ2 domains of CBP/p300 have been shown to bind activation domains of several transcription factors, including B-Myb, C/EPBϵ, FOXO3a, HIF-1α, p53, p63, p73, STAT1, and STAT2, many of which also contain ΦXXΦΦ sequences (Fig. 1B) (36–47). Furthermore, several activation domains have displayed binding promiscuity to the KIX, TAZ1, and TAZ2 domains, presenting the opportunity for a multivalent mode of binding with transcription factors comprising multiple activation domains, with p53 representing the archetypical example (33, 38, 48, 49). Despite functional roles for the three activation domains being attributed to the transcriptional activity of E-proteins, their interactions with CBP/p300 have not been fully explored.

Here, we characterize a direct interaction between E2A-AD1 and the TAZ2 domain of CBP/p300 and elucidate the structure of a E2A-AD1(1–37):TAZ2 complex by NMR spectroscopy. The structure shows that residues throughout and adjacent to the helical PCET motif of E2A-AD1 interact with the TAZ2 domain in a manner reminiscent of activation subdomain 2 (AD2) of p53. Peptide microarray and mutagenesis revealed the requirement of both hydrophobic and electrostatic interactions in complex formation. These results provide a mechanistic rational for the cooperative manner in which the AD1 and AD2 domains of E2A induce gene expression.

Results

TAZ2 domain of CBP/p300 binds AD1 of E2A

Toward identifying which domains of CBP/p300 interact with E2A, and in particular its three activation domains (i.e. AD1, AD2, and AD3), an in vitro pulldown experiment was performed involving GB1-E2A fusion constructs that spanned the entire transactivation region of E2A (residues 1–483; Fig. 2 and Fig. S1). The KIX domain of CBP/p300 interacted modestly with E2A-AD1, and a significant interaction with E2A(1–483) was observed, which is consistent with previous studies (26–28) (Fig. 2). E2A did not significantly interact with TAZ1, but displayed a more pronounced ability to pull down the isolated TAZ2 domain, as indicated by the corresponding protein bands from pulldowns with E2A-AD1, E2A-AD2, and E2A(1–483) (Fig. 2). A quantitative assessment of the E2A:TAZ2 interaction by isothermal titration calorimetry indicated that the TAZ2 domain displayed a higher affinity for E2A-AD1 (i.e. K_d = 0.86 ± 0.04 μm) than for E2A-AD2 (K_d = 67 ± 7 μm) and that the former occurred with a 1:1 stoichiometric ratio (Fig. S2).

Figure 2.

E2A-AD1 binds the TAZ2 domain of CBP/p300. A, 15% SDS-PAGE analysis of the ability of various purified His₆-GB1-E2A fragments immobilized on IgG-agarose to pull down purified recombinant KIX, TAZ1, and TAZ2 domains of p300. The left lanes of each panel represent controls illustrating the migration of the recombinant isolated p300 domains. B, quantitative analysis of the His₆-GB1-E2A pulldowns presented in A. Significance between pulldown protein levels of TAZ1, TAZ2, and KIX by GB1 (negative control) and GB1-E2A constructs is indicated by brackets. Error bars, S.D. (**, p < 0.01; NS, not significant).

Analysis of the E2A-AD1 interaction with TAZ2 by NMR spectroscopy revealed poor dispersion of the backbone amide resonances of uniformly ¹⁵N-labeled E2A-AD1, which suggests that E2A is intrinsically disordered (Fig. 3A). Secondary structure propensity (50) analysis indicated that whereas E2A-AD1 is largely disordered, residues Lys¹⁴–Met²⁵ have elevated SSP scores between 0.1 and 0.35, suggesting that this region has residual α-helical structure (Fig. S3). The addition of saturating amounts of unlabeled TAZ2 domain caused chemical shift changes and increased spectral dispersion, in particular for the backbone amide groups of residues Lys¹⁴–Phe²⁶ and Leu⁹⁴ of E2A-AD1 (Fig. 3A (inset) and Fig. S4). Isothermal titration calorimetry measured the dissociation constants of TAZ2 binding for E2A-AD1 and E2A-AD1(1–37) to be 0.86 ± 0.04 and 4.1 ± 1.2 μm, respectively (Fig. S2). This is consistent with the chemical shift perturbation data of E2A-AD1, which indicates that the key TAZ2-interacting region of E2A-AD1 lies within E2A-AD1(1–37), whereas minor contributions from elsewhere in E2A-AD1 may lead to increased TAZ2 binding affinity for E2A-AD1 compared with E2A-AD1(1–37).

Figure 3.

The ΦXXΦΦ-containing region of E2A-AD1(1–37) adopts an ordered structure upon TAZ2 binding. A, the ¹H-¹⁵N HSQC of uniformly ¹⁵N-labeled E2A-AD1. Resonance assignments are indicated, and an asterisk denotes resonances that are due to impurities or could not be assigned due to spectral overlap. The inset indicates the weighted chemical shift change (Δδ = ((0.17Δδ_N)² + (Δδ_HN)²)^½ that each residue of E2A-AD1 undergoes upon the addition of saturating amounts of TAZ2. B, overlay of ¹H-¹⁵N HSQC spectra of uniformly ¹⁵N-labeled E2A-AD1(1–37) in the absence (black) and presence (red) of saturating amounts of TAZ2. Resonance assignments are indicated, and arrowed lines indicate the directional movement of resonances upon the addition of TAZ2. Resonances attributed to impurities are indicated with an asterisk. C, a plot of steady-state {¹H}-¹⁵N NOE values recorded at 14.1 teslas as a function of E2A-AD1(1–37) residue number when bound to TAZ2. Error bars, S.D.

The interaction of E2A-AD1(1–37) with TAZ2 was further characterized by NMR spectroscopy because it is more feasible to characterize smaller proteins. Similar to what was observed for E2A-AD1, the addition of saturating amounts of unlabeled TAZ2 domain to ¹⁵N-labeled E2A-AD1(1–37) caused large chemical shift changes and increased spectral dispersion for resonances corresponding to the backbone amide groups of Lys¹⁴–Phe²⁶ (Fig. 3B, red versus black spectra). Chemical shift analysis of backbone ¹H, ¹³C, and ¹⁵N chemical shifts of free and TAZ2-bound E2A-AD1(1–37) suggests that Asp¹³–Met²⁵ undergo a random coil–to–α-helix structural change upon TAZ2 binding (Fig. S5). Steady-state {¹H}-¹⁵N heteronuclear NOE analysis further indicated that when bound to the TAZ2 domain, E2A-AD1(1–37) adopted a more ordered structure between Lys¹⁴ and Leu²⁸ (i.e. {¹H}-¹⁵N NOE values >0.4), whereas the preceding N-terminal residues and the seven C-terminal residues displayed reduced {¹H}-¹⁵N NOE values suggestive of a disordered conformation in solution (Fig. 3C).

Structure of the E2A-AD1(1–37):TAZ2 complex

To investigate the molecular basis of the E2A-AD1(1–37):TAZ2 interaction, the structure of this complex was determined by NMR spectroscopy using 3408 NOE-derived distance restraints, including 197 intermolecular distance restraints, and 163 TALOS+-derived dihedral restraints. Structures were calculated from automated distance restraint weighting and assignment through CYANA and water refinement with CNS (51–53). The final ensemble of 20 low-energy structures of the 1:1 complex was well-defined (Fig. 4A) and displayed good overall structural statistics (Table 1), with root mean square deviation (r.m.s.d.) values of the ensemble core (E2A-AD1(1–37) residues Asp¹³–Pro²⁹ and TAZ2 residues Ser¹⁷²⁶–Lys¹⁸¹²) from the energy-minimized average structure for the backbone and heavy atoms of 0.51 ± 0.06 and 0.83 ± 0.06 Å, respectively.

Figure 4.

Structure of the E2A-AD1(1–37):TAZ2 complex. A, backbone superposition of the 20 lowest-energy structures of the E2A-AD1(1–37):TAZ2 complex, with the backbone atoms (N, C^α, and C′) of Asp¹³–Pro²⁹ of E2A-AD1(1–37) (pink) and Ser¹⁷²⁶–Lys¹⁸¹² of the TAZ2 domain (teal) displayed. B, backbone ribbon illustration of the lowest energy structure of the E2A-AD1(1–37):TAZ2 complex. The helices of the TAZ2 domain are labeled α1, α2, α3, and α4, and the N and C termini of E2A-AD1(1–37) and TAZ2 are indicated.

Table 1.

Structural statistics of the E2A-AD1:TAZ2 complex

Parameters	Values
	AD1	TAZ2
Distance restraints used for structure calculations
Intraresidue	172	621
Sequential (|i − j| = 1)	290	722
Medium Range (1 < |i − j| <5)	222	774
Long range (|i − j| ≥5)	1	409
Intermolecular	197
Total	3408
Hydrogen bond restraints	10	44
Dihedral restraints
ϕ	14	67
ψ	14	68
Energies (kcal mol−1)
Total	−3328 ± 93
Van der Waals	−758 ± 12
r.m.s.d. from experimental restraints
Distance constraints (Å)	0.021 ± 0.002
Dihedral angles (degrees)	0.418 ± 0.088
r.m.s.d. from idealized geometry
Bond lengths (Å)	0.015 ± 0.001
Bond angles (degrees)	0.973 ± 0.013
Global quality scores (raw/Z-score)
Verify3D	0.33/−2.09
Procheck (φ-ψ)	−0.17/−0.35
Procheck (all)	−0.33/−1.95
MolProbity clash score	16.88/−1.37
RPF validation
Recall	0.906
Precision	0.872
F-measure	0.889
Discriminating power	0.807
Ramachandran statistics (%)a
Residues in most favored regions	94.4
Residues in additionally allowed regions	4.6
Residues in generously allowed regions	0.2
Residues in disallowed regions	0.9
r.m.s.d. to mean structure (Å)a
Backbone atoms	0.51 ± 0.06
Heavy atoms	0.83 ± 0.06

^a Using converged regions, E2A-AD1 residues Asp¹³–Pro²⁹ and TAZ2 residues Ser¹⁷²⁶–Lys¹⁸¹².

The E2A-AD1(1–37) adopted an α-helix comprising amino acid residues Gly¹¹–Phe²⁶, whereas Pro²⁷–Pro²⁹ formed a short extended region (Fig. 4). The 10 N-terminal amino residues (Met¹–Val¹⁰) and eight C-terminal residues (Val³⁰–Pro³⁷) remained disordered and flexible and did not appreciably contact the TAZ2 domain, as indicated by the reduced {¹H}-¹⁵N NOE values (Fig. 3C) and lack of identifiable medium- and long-range NOEs. When compared with the structure of HEB-AD1 bound to the KIX domain (27), E2A-AD1(1–37) was extended by one helical turn at the N terminus (residues Gly¹¹–Lys¹⁴).

The TAZ2 domain comprised four α-helices (α1: Pro¹⁷²⁷–Gln¹⁷⁴⁷; α2: Pro¹⁷⁵⁶–Lys¹⁷⁶⁹; α3: Pro¹⁷⁸⁰–His¹⁷⁹⁵; α4: Pro¹⁸⁰⁴–Lys¹⁸¹⁰) separated by loops (Fig. 4), each of which adopted a HCCC-type Zn²⁺-coordinating conformation (54). Overall, the TAZ2 fold in the presence of E2A-AD1(1–37) described here was similar to that previously observed for this domain when bound to other proteins, including the E1A oncoprotein (backbone r.m.s.d of 1.9 Å), isolated p53-AD1 and -AD2 (backbone r.m.s.d. of 2.4 and 2.0 Å, respectively), the N-terminal region of p53 comprising both ADs (backbone r.m.s.d. of 1.8 Å), STAT1-AD (backbone r.m.s.d. of 1.5 Å), p63 (backbone r.m.s.d. of 2.0 Å), and p73 (backbone r.m.s.d. of 1.9 Å) (38, 39, 41, 42, 44, 47).

E2A-AD1:TAZ2 interface

The E2A-AD1 interactive surface on the TAZ2 domain involved a hydrophobic groove bounded by basic residues and covered ∼1260 Ų of solvent-accessible surface area (Fig. 5, A and B). The hydrophobic groove comprised the aliphatic region of Arg¹⁷³², Ile¹⁷³⁵, and Ala¹⁷³⁸ of α1; the aliphatic region of Lys¹⁷⁶⁰, Met¹⁷⁶¹, and Val¹⁷⁶⁴ of α2; and Pro¹⁷⁸⁰, Ile¹⁷⁸¹, Gln¹⁷⁸⁴, Leu¹⁷⁸⁵, Leu¹⁷⁸⁸, and Tyr¹⁷⁹¹ of α3 (Fig. 5C). Residues throughout the helical region of E2A-AD1, including Leu¹⁹, Phe²², Met²⁵, and Phe²⁶, and the residues Pro²⁷ and Leu²⁸ C-terminal to the helix made extensive nonpolar contacts with the TAZ2 domain (Fig. 5D). Within the E2A-AD1 helix, the side chain of Leu¹⁹ inserted into a cavity formed by Val¹⁷⁶⁴, Pro¹⁷⁸⁰, Ile¹⁷⁸¹, the aliphatic region of Gln¹⁷⁸⁴, and Leu¹⁷⁸⁵. The aromatic side chain of Phe²² from E2A-AD1 participated in van der Waals contacts with the aliphatic portion of Lys¹⁷⁶⁰, Met¹⁷⁶¹, and Val¹⁷⁶⁴. Met²⁵ interacted with Ala¹⁷³⁸ and Leu¹⁷⁸⁸, whereas Phe²⁶ associated with Ile¹⁷³⁵, Ala¹⁷³⁸, Ala¹⁷⁸⁷, Leu¹⁷⁸⁸, and the aliphatic region of Gln¹⁷⁸⁴. Beyond the helical region of E2A-AD1, Pro²⁷ formed nonpolar contacts with Ile¹⁷³⁵, whereas Leu²⁸ resided in hydrophobic pocket created by the aliphatic regions of Arg¹⁷³², Ile¹⁷³⁵, Leu¹⁷⁸⁸, and Tyr¹⁷⁹¹ of the TAZ2 domain.

Figure 5.

The E2A-AD1(1–37):TAZ2 interface. A, backbone ribbon representation of the lowest-energy structure of the E2A-AD1(1–37) (pink):TAZ2 (teal) complex onto which a transparent surface representation of the TAZ2 domain is also displayed. B, electrostatic surface representation of the E2A-AD1 binding face of TAZ2 domain, where blue and red shadings depict positively and negatively charged regions, respectively. C, expanded view of transparent surface of the TAZ2 domain displaying the backbone ribbon and residues involved in intermolecular van der Waals contacts with E2A-AD1(1–37). D, backbone ribbon representation of E2A-AD1(1–37) (pink) showing those residues making intermolecular van der Waals contacts on the surface of the TAZ2 domain.

Several electrostatic and hydrogen-bonding interactions involving residues in the helical region of E2A-AD1 complemented the extensive nonpolar network at the E2A-AD1:TAZ2 interface. Glu¹⁵ of E2A-AD1 was situated to form a salt bridge with His¹⁷⁶⁷ of α2 of TAZ2 (d_Oϵ*-Nδ1 = 3.6 ± 0.4 Å). Asp¹⁸ was positioned to participated in salt bridges with both Lys¹⁷⁶⁰ (d_Oδ*-Nζ = 3.1 ± 0.5 Å) and Arg¹⁷⁶³ (d_Oδ2-Nϵ = 4.5 ± 1.5 Å), whereas Asp²¹ was also in close proximity to Lys¹⁷⁶⁰ of TAZ2 (d_Oδ*-Nζ = 3.6 ± 0.9 Å).

Mutants traversing the helical region of E2A-AD1 attenuate TAZ2 binding

To assess the relative contribution of each residue of E2A-AD1 in TAZ2 recognition, the effects of E2A-AD1 alanine mutants were assessed by peptide microarray and isothermal titration calorimetry (Fig. 6). For the peptide microarray, HEB-AD1(11–28) peptides (which are identical to E2A-AD1 except for a Met²⁴ to Ala substitution), in which each position was individually substituted to alanine, were probed with recombinant His₆-GB1-TAZ2 (Fig. 6A). Compared with WT HEB-AD1(11–28), alanine substitutions at Leu¹⁹ and Phe²² led to significantly reduced signal, whereas substitutions at Leu¹⁶, Asp¹⁸, and Asp²¹ modestly reduced the signal. Complementary quantitative analysis by isothermal titration calorimetry indicated that the L19A and F22A substitutions both resulted in a ∼4-fold reduction in affinity (K_d of 17 and 16 μm, respectively) (Fig. 6B). Thus, whereas no single point mutation of E2A-AD1 completely abrogates TAZ2 binding, the E2A-AD1(1–37):TAZ2 structure, peptide microarray, and isothermal titration calorimetry indicate that Leu¹⁹ and Phe²² are key binding elements of the interaction.

Figure 6.

E2A-AD1 mutants impair TAZ2 binding. A, membranes spotted with HEB-AD1(11–28) peptides in which each residue had been individually substituted with alanine were probed with His₆-GB1-TAZ2. The sequence of HEB is indicated using E2A numbering, and each position of HEB-AD1(11–28) was mutated to alanine and spotted onto an array. The signal present from TAZ2 binding to each alanine mutant of HEB-AD1(11–28) is indicated as a spot below the sequence. B, the isothermal titration calorimetric dissociation constants determined for the interaction between WT, L19A, and F22A E2A-AD1(1–37) and the isolated TAZ2 domain. C and D, ribbon representation of the E2A-AD1(1–37):TAZ2 complex with a transparent TAZ2 surface showing the position of Leu¹⁹ (C) and Phe²² (D) of E2A-AD1 (pink) on the TAZ2 (teal) surface.

KIX and TAZ2 of CBP/p300 compete for E2A-AD1

The orientation of E2A-AD1 on the TAZ2 surface presented the possibility of a higher-order interaction with the KIX domain of CBP/p300, as several of the KIX-interactive residues, including the critical Leu²⁰ (20), were solvent-exposed, a scenario supported by the recent observation that full-length CBP/p300 displays intrinsic conformational flexibility (33, 55). To directly assess the formation of such a complex or whether KIX and TAZ2 displayed exclusive E2A-AD1 binding, an NMR-based displacement experiment was performed. The addition of unlabeled E2A-AD1(1–37) to uniformly ¹⁵N-labeled KIX resulted in significant chemical shift changes for a subset of backbone amide resonances in ¹⁵N-KIX spectra (Fig. 7A), consistent with the site-specific binding of E2A-AD1(1–37) to KIX (27). The subsequent addition of unlabeled TAZ2 to the sample caused those backbone resonances of uniformly ¹⁵N-labeled KIX to revert back toward chemical shift values consistent with the free form of the KIX domain (Fig. 7B). These results indicate that the TAZ2 and KIX domains of CBP/p300 can only interact with E2A-AD1 exclusive of one another.

Figure 7.

TAZ2 and KIX domains compete for E2A-AD1 in vitro. A, overlay of two selected regions from two-dimensional ¹H-¹⁵N HSQC spectra of 200 μm ¹⁵N-KIX in the absence (black) and presence (red) of 300 μm unlabeled E2A-AD1(1–37). B, overlay of the same two regions depicted in A after the addition of 500 μm unlabeled TAZ2. The direction of backbone amide resonance change is depicted by red and green arrows in A and B, respectively.

To further investigate a higher-order interaction between CBP and E2A, a pulldown experiment involving GB1-E2A(1–483) constructs and full-length FLAG-tagged CBP was performed and visualized using an anti-FLAG Western blotting (Fig. 8 and Fig. S6). Compared with WT, alanine substitutions in the AD1 region of E2A(1–483) that disrupt either TAZ2 binding (L19A and F22A) or KIX binding (L20A (20)) significantly reduced the ability of E2A(1–483) to pull down CBP. An alanine and proline substitution in AD2 of E2A(1–483) that disrupts KIX binding (L397A/I401P (28)) similarly displayed reduced pulldown of FLAG-CBP. Mutations in both AD1 and AD2 of E2A(1–483) were required to abrogate any observable interaction between GB1-E2A(1–483) and CBP.

Figure 8.

Mutation of AD1 or AD2 of E2A(1–483) reduces its ability to pull down CBP. A, immunoblotting of comparative pulldowns of FLAG-CBP by purified His₆-GB1-E2A(1–483) constructs immobilized on IgG-agarose. B, relative quantitative analysis of the Western blots performed in triplicate. ImageJ was used to measure the pixel density for each lane. The y axis values represent the ratio of FLAG-CBP pulldown by His₆-GB1-E2A(1–483) mutants to FLAG-CBP pulldown by His₆-GB1-E2A WT. Error bars, S.D. (n = 3; **, p < 0.0005; ***, p < 0.0001).

Discussion

The E2A activation domains are essential for lymphopoiesis and are involved in the onset of ALL through the oncogenic fusion protein E2A-PBX1 (56). In either situation, the recruitment of CBP/p300 is essential for E2A or E2A-PBX1 function. Here we investigate recruitment of CBP/p300 by E2A-AD1, which provides insight into the molecular mechanisms underlying lymphopoiesis and ALL.

Depending on the context, the activation domains of E2A function independently, redundantly, or cooperatively with each other (19, 21–28, 57). The KIX domain of CBP/p300 binds both E2A-AD1 and E2A-AD2 in a functionally redundant manner, with both E2A-AD1 and E2A-AD2 competing for the same site of KIX (27, 28). The additional interactions of TAZ2 with E2A-AD1 or E2A-AD2 that we describe (Fig. 2 and Fig. S2) raise the possibility that both AD1 and AD2 could simultaneously interact with the KIX and TAZ2 domains to allow for higher affinity association between CBP/p300 and E2A. This possibility is supported by our pulldowns of full-length CBP (Fig. 8), where intact AD1 and AD2 were required for maximum CBP/p300 pulldown and mutations in either the AD1 or AD2 region lessened the interaction by ∼50%. It is unclear what the binding preferences of E2A-AD1 and E2A-AD2 are to full-length CBP because E2A-AD1 and E2A-AD2 are able to bind both the KIX and TAZ2 domains. The ability of E2A to form a tight yet dynamic complex with CBP through multiple weaker interactions is likely essential for its transcriptional activity and provides rationale for how deletion of AD1 or AD2 abolishes B-lymphoid differentiation (21). This is reminiscent of p53, which also associates with multiple domains of CBP/p300 to form a tight, yet dynamic, complex (49).

The promiscuous interactions of intrinsically disordered proteins are recognized as key to their roles as protein interaction hubs (33, 58). This appears to be the case with E2A because E2A-AD1 and its nearly identical homologue HEB-AD1 (Fig. 1B) can complement and bind a variety of molecular surfaces, with high-resolution structures available of the KIX:HEB-AD1 and eTAFH:HEB-AD1 complexes. HEB-AD1 adopts an amphipathic α-helix from Lys¹⁴ to Phe²⁶ (renumbered according to the E2A sequence) when bound to KIX, a kinked α-helix spanning residues Asp¹³–Leu²⁰ and Ser²³–Phe²⁶ when bound to eTAFH, and E2A-AD1 adopts an amphipathic α-helix from Asp¹³ to Met²⁵ when bound to TAZ2. Leu¹⁶, Leu¹⁹, and Leu²⁰ form essential hydrophobic contacts with both eTAFH and KIX (27, 59), whereas Phe²² forms additional hydrophobic contacts with KIX and TAZ2 but not eTAFH. Although the entire PCET motif is involved in recognition of eTAFH, KIX, and TAZ2, Phe²⁶, Pro²⁷, and Leu²⁸ have additional hydrophobic contacts to TAZ2. Finally eTAFH, KIX, and TAZ2 have basic residues situated near the E2A-AD1–binding site, providing unique potential electrostatic contacts for Glu¹⁵, Asp¹⁸, and Asp²¹. The different modes of E2A-AD1 binding to eTAFH, KIX, and TAZ2 are highlighted by the importance of Leu²⁰, which is essential for the HEB:KIX and HEB:eTAFH interactions (27, 59) but dispensable for binding to TAZ2, as it is facing solvent and alanine substitutions are nonperturbing to a pulldown experiment (Figs. 5 and 6).

The TAZ2 domain of CBP/p300 is promiscuous and interacts with many intrinsically disordered proteins, with structures available for TAZ2 in complex with activation domains from E1A, C/EBPϵ, p53, p63, p73, and STAT1 (36, 38, 39, 41, 42, 47). Highlighting the promiscuity of the TAZ2 domain, E1A, p73, and STAT1 bind TAZ2 in part to a hydrophobic groove formed by α1, α2, and α3, whereas p53, p63, and E2A-AD1 adopt an α-helical structure when they bind to this same region of TAZ2 (Fig. 9). The adenoviral protein E1A has been shown to decrease E2A transcriptional activity (60), and the observation that E2A-AD1 and E1A bind overlapping sites of TAZ2 suggests that E1A inhibits E2A function through direct competition for TAZ2. Interestingly, although E2A-AD1, p53, and p63 bind the same region of TAZ2 as an α-helix, the peptides have differing orientations (Figs. 9 and 10). Despite the opposite orientations of p53 and E2A-AD1, Phe²² and Phe²⁶ of E2A-AD1 occupy similar positions as Phe⁵⁴ and Ile⁵⁰, respectively (Fig. 10). These residues all face TAZ2, suggesting that an amphipathic helix is key for binding to the α1, α2, and α3 binding surface of TAZ2. The importance of these hydrophobic residues is confirmed by our mutagenesis studies in which mutation of Phe²² or Phe²⁶ to alanine decreased the signal observed in a microarray, whereas mutation of Ile⁵⁰ and Phe⁵⁴ of p53 weakens binding of p53-AD2 to TAZ2 by ∼3-fold and 2-fold, respectively (44). Other than the presence of a ΦXXΦΦ sequence and being acidic with some hydrophobic residues, the sequences of TAZ2-binding activation domains are quite divergent, and TAZ2 appears to be able to accommodate a variety of peptide sequences, orientations, and secondary structure content.

Figure 9.

Transcription factors adopt diverse structures and orientations when interacting with Taz2. A, backbone ribbon representation of p53 (5HPD; orange (38)), E2A-AD1(1–37) (pink), and STAT1 (2KA6; yellow (47)) in complex with TAZ2, which is shown as a teal surface. In B, the binding orientations of E2A-AD1(1–37) (pink), E1A (2KJE; blue (42)), p63 (6FGN; orange), and p73 (6FGS; yellow (39)) are shown.

Figure 10.

E2A-AD1 and p53 bind TAZ2 with opposite orientations. A, ribbon representation of a superposition of the E2A-AD1(1–37):TAZ2 and p53:TAZ2 (38) complexes, with only TAZ2 (teal), E2A-AD1(1–37) (pink), and p53 (yellow) shown. The side chains of Phe²² and Phe²⁶ of E2A-AD1 as well as Ile⁵⁰ and the Phe⁵⁴ of p53 are displayed as sticks. B, sequence alignment of E2A-AD1 and p53. The sequence of p53 is reversed with respect to E2A-AD1, and the residues highlighted in A are colored red.

The orientation of an amphipathic α-helix bound to TAZ2 likely depends on the specific interactions of polar residues and neighboring sequences with TAZ2. For E2A-AD1, Asp¹⁸ and Asp²¹ are likely involved in a salt bridge to the side chain of Lys¹⁷⁶⁰ (N^ζ–O^δ distance of 3.1 ± 0.53 and 3.6 ± 0.9 Å, respectively), whereas Phe²⁶, Pro²⁷, and Leu²⁸ all participate in further hydrophobic contacts to TAZ2. For p53, a similar situation occurs where Glu¹¹ and Glu¹⁷ contact the guanidinium group of Arg¹⁷³¹ and multiple other residues engage in specific polar contacts to TAZ2 (41). In support of this, substitution of Asp¹⁸ and Asp²¹ with alanine decreases TAZ2 binding in a peptide microarray (Fig. 6). Given that acidic residues influence E2A-AD1 affinity for TAZ2, phosphorylation of E1A (e.g. at Thr¹², Ser¹⁷, and Ser²³) may enhance the affinity of E2A-AD1 to TAZ2 by forming salt bridges to nearby basic residues of TAZ2. A similar situation has been observed with p53, which has a graded enhancement of affinity for CBP/p300 upon phosphorylation (61). Overall, TAZ2 is able to accommodate a wide variety of peptides through a complex interplay of hydrophobic and electrostatic interactions, and phosphorylation may be a common method to regulate protein association with TAZ2.

Experimental procedures

Plasmid preparation

All constructs were cloned into a pET21a(+) vector using BamHI and XhoI restriction enzymes downstream of sequences coding for a His₆ tag, the B1 domain of protein G (GB1), and either a thrombin (human E2A(1–37) (hereafter referred to as E2A-AD1(1–37)), CBP(346–440) (TAZ1), and p300(1723–1812) (TAZ2)) or tobacco etch virus (human E2A(1–100) (E2A-AD1), E2A(101–300) (E2A-AD3), E2A(301–483) (E2A-AD2), and E2A(1–483)) protease recognition sequence. A modified TAZ2 construct in which cysteine residues not involved in zinc coordination were substituted for alanine (i.e. C1738A, C1746A, C1789A, and C1790A) was generated based on its reported enhanced stability (41, 54). E2A-AD1(1–37) mutants were generated using the QuikChange site-directed mutagenesis kit (Stratagene), with His₆-GB1-E2A-AD1(1–37) as a template. E2A(1–483) mutagenesis was performed using QuikChange II site-directed mutagenesis kit (Agilent Technologies, 200523) with His₆-GB1-E2A(1–483) as a template. The fidelity of all constructs was verified by DNA sequencing. Plasmids were subsequently transformed into Escherichia coli BL21(DE3) cells for recombinant protein expression.

Protein expression and purification

The isolated KIX domain (residues 586–673 of CBP) was expressed and purified as described previously (27). E. coli BL21(DE3) cells harboring the His₆-GB1-TAZ1 and His₆-GB1-TAZ2 encoding plasmids were grown in lysogeny broth or ¹⁵N- or ¹³C/¹⁵N-enriched M9 medium at 37 °C supplemented with 100 μm ZnCl₂. Protein expression was induced at an optical density of 0.6 at 600 nm by adding isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mm. Growth was continued overnight at 23 °C with shaking. Harvested cell pellets were lysed by sonication in denaturing binding buffer (20 mm Tris-HCl, pH 8.0, 250 mm NaCl, 8 m urea, 10 mm β-mercaptoethanol, 10 μm ZnCl₂), clarified by centrifugation, and applied to Ni²⁺-affinity resin (GE Healthcare). Upon extensive washes with denaturing binding buffer containing 10 mm imidazole, protein constructs were refolded on-column by application of native Ni²⁺ binding buffer (20 mm Tris-HCl, pH 8.0, 250 mm NaCl, 10 mm β-mercaptoethanol, 10 μm ZnCl₂) and subsequently eluted in the same buffer containing 300 mm imidazole. The elution fractions were pooled, and β-mercaptoethanol and ZnCl₂ were added to final concentrations of 40 mm and 4-fold excess relative protein, respectively. Thrombin, at 1 unit/50 nmol of protein, was added, and the samples were dialyzed overnight in buffer A (20 mm HEPES, pH 8.0, 50 mm NaCl, 5 mm β-mercaptoethanol, 10 μm ZnCl₂). The cleaved, refolded TAZ1 or TAZ2 constructs were separated from the His₆-GB1 fragments via Fast Flow SP Sepharose cation chromatography (GE Healthcare) using buffer A as a wash buffer and eluted with buffer A containing 500 mm NaCl.

Expression of all His₆-GB1-E2A constructs was induced in transformed E. coli BL21(DE3) cells with 0.5 mm IPTG at an optical density of ∼1 at 600 nm. Growth was continued for an additional 4 h at 37 °C with shaking. Expression of His₆-GB1-E2A(1–483) L397A/I401P mutants was performed as described except that after induction with IPTG, growth was continued for an additional 16 h at 23 °C with shaking. Purification of His₆-GB1-E2A(1–483), E2A-AD1, and E2A-AD2 was performed as described above for the TAZ domains, with the exception that ZnCl₂ was excluded from all buffers. Purification of WT and mutant E2A-AD1(1–37) constructs was performed as described previously (28).

For uniformly ¹³C- and/or ¹⁵N-labeled NMR samples, HPLC was used to purify TAZ2 or E2A-AD1(1–37) on a C₁₈ reverse phase column with a water:acetonitrile gradient with 0.05% TFA. Fractions containing protein were pooled, lyophilized, and stored at −20 °C. The integrity of each protein sample was verified by SDS-PAGE analysis, MS, and NMR spectroscopy.

In vitro pulldown experiments

His₆-GB1, His₆-GB1-E2A-AD1, His₆-GB1-E2A-AD3, His₆-GB1-E2A-AD2, and His₆-GB1-E2A(1–483) were incubated with 20 μl of 50% IgG agarose slurry (GE Healthcare) for 15 min in assay buffer (20 mm MES, pH 6.5, 100 mm NaCl). After two washes with assay buffer, 1 ml of 20 μm KIX, TAZ1, or TAZ2 was added to the beads and left for 30 min with gentle agitation. After incubation, the beads were washed three times with assay buffer and resuspended in SDS-PAGE loading buffer. KIX, TAZ1, and TAZ2 binding to the His₆-GB1-E2A constructs were visualized using SDS-PAGE. For TAZ1 pulldown experiments, the assay buffer included 5 mm β-mercaptoethanol, and for TAZ2 pulldown experiments, the assay buffer included 5 mm β-mercaptoethanol and 10 μm ZnCl₂. All experiments were done in duplicate.

Cell culture and cell lysis

HEK 293T cells were seeded at 0.8 × 10⁶ cells/well in a 6-well culture plate. The following day, the cells were transfected with 2 μg of a FLAG-CBP plasmid using jetPRIME transfection reagent (Polyplus, 114-01) according to the manufacturer's protocol. After 24 h, the transfection medium was exchanged for cell growth medium. 48 h after transfection, the cells were washed twice with PBS and lysed for 10 min at room temperature with gentle agitation in Nonidet P-40 buffer (50 mm Tris pH 8.0, 150 mm NaCl, 1.0% Nonidet P-40) plus protease inhibitors (Thermo Fisher Scientific, A32965). The lysates were sonicated briefly and clarified by centrifugation.

Immunoblotting

In vitro pulldown experiments were performed as described above with the following modifications: (i) His₆-GB1 and His₆-GB1-E2A(1–483) WT and mutant constructs were incubated with 5 μl of 50% IgG-agarose slurry (GE Healthcare) for 30 min in Nonidet P-40 buffer; (ii) 250 μl of FLAG-CBP–transfected HEK 293T cell lysate was added to the beads and left for 3 h with gentle agitation; and (iii) all washes were done with Nonidet P-40 buffer. The pulldown experiments were loaded onto 6% Tris-glycine gels and separated by SDS-PAGE. The proteins were transferred to nitrocellulose membranes using a Bio-Rad wet electroblotting system for 16 h at 20 V at 4 °C. Membranes were blocked for 1 h at room temperature with 5% skimmed milk in TBS plus 0.1% Tween 20 (TBST) and subsequently incubated for 1 h at room temperature with horseradish peroxidase–conjugated α-FLAG antibody (Sigma–Aldrich, A8592) diluted 1:5000 in TBST. Membranes were then washed four times with TBST, stained with Immobilin Forte Western HRP substrate (EMD Millipore, WBLUF0100), and exposed to X-ray films. The pulldown experiments and immunoblotting were performed in triplicate, and ImageJ was used to quantify the X-ray films.

Isothermal titration calorimetry

Experiments were carried out at 30 °C in 20 mm MES, pH 6.5, 5 mm β-mercaptoethanol and 10 μm ZnCl₂, using 100–200 μm TAZ2 in the cell and 1–2 mm E2A construct in the syringe (E2A-AD1, E2A-AD2, E2A-AD1(1–37), E2A-AD1(1–37)L19A, or E1A-AD1(1–37)F22A). Experiments were performed on VP-ITC or iTC200 calorimeters (MicroCal). Thermograms were fit to a one-site binding model using MicroCal Origin 7.0 software. All experiments were collected in at least duplicate.

Peptide microarray

Peptide microarrays were synthesized through automatic SPOT synthesis with a MultiPep automated peptide synthesis system (Intavis) using 9-fluorenylmethyloxycarbonyl chemistry (62). HEB-AD1–derived peptides were synthesized onto continuous cellulose membranes to generate strip arrays, which were hydrated with ethanol, washed with assay buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl), and blocked overnight in assay buffer containing 2% milk. Following washes with assay buffer containing 0.5% Tween 20, the arrays were incubated for 4 h with 5–10 μg/ml His₆-GB1-TAZ2 in 50 mm Tris-HCl, pH 7.4, in assay buffer containing 2% skim milk, washed with assay buffer, and incubated for 1 h with rabbit polyclonal antibody against the His₆ tag conjugated to horseradish peroxidase (Abcam) diluted in assay buffer containing 2% skim milk. After a final wash with assay buffer, luminata horseradish peroxidase substrate (Millipore) was applied to the arrays, and X-ray film was used to detect the signal.

NMR spectroscopy and structure calculation

Lyophilized recombinant TAZ2 was reconstituted for NMR studies as described previously (54). For the assignment of E2A-AD1, a 1 mm sample of ¹³C/¹⁵N-labeled E2A-AD1 in 20 mm MES, 50 mm NaCl, and 5% D₂O adjusted to pH 6.5 was prepared, and data were collected at 25 °C. For structural analysis of the E2A-AD1(1–37):TAZ2 complex, two samples were prepared containing either 1 mm uniformly ¹³C/¹⁵N-labeled TAZ2 with 3 mm E2A-AD1(1–37) or 2 mm TAZ2 with 1.4 mm uniformly ¹³C/¹⁵N-labeled E2A-AD1(1–37) in 20 mm MES, 5 mm β-mercaptoethanol, 100 μm ZnCl₂, and 5% D₂O adjusted to pH 6.5 without accounting for deuterium isotope effects. Resonance assignments of E2A-AD1, E2A-AD1(1–37), and TAZ2 were carried out using standard multidimensional heteronuclear NMR experiments. Distance restraints for the E2A-AD1(1–37):TAZ2 complex were determined using three-dimensional ¹⁵N NOESY-HSQC and both aliphatic and aromatic ¹³C NOESY-HSQC experiments, all with a 100-ms mixing time. The validity of intermolecular restraints was confirmed using ¹²C/¹⁴N-filtered ¹⁵N-edited and ¹²C/¹⁴N-filtered ¹³C-edited NOESY spectra. All NMR experiments involving E2A-AD1(1–37) were collected at 15 °C on 500-, 600-, or 800-MHz Varian INOVA spectrometers equipped with triple resonance cryoprobes. NMR data were processed using NMRPipe (63) and analyzed using CCPNMR Analysis (64).

NOESY peaks lists were exported from CCPNMR Analysis and used in CYANA for automatic NOE assignment and distance restraint calibration (52, 53). Dihedral angle restraints based on chemical shifts were generated using TALOS+, with angle error set to 2 times the error output of TALOS+ (65). After confirming a correct TAZ2 fold using only NOE-based distance and dihedral angle restraints, hydrogen bond restraints (1.8 ≤ d_OH ≤ 2.2 Å; 2.7 ≤ d_ON ≤ 3.2 Å) were applied to helical regions of the E2A-AD1(1–37):TAZ2 complex, whereas zinc coordination restraints were applied to known Zn²⁺-coordinating residues (54). Using CYANA, an initial ensemble of the lowest-energy 20 models was retained from 100 generated models. These 20 models were further energy-minimized in explicit water using fmcGUI and CNS (51). The final ensemble of 20 models was validated using PROCHECK-NMR (66), and the recall, precision, F-measure, and discriminating power scores of the final ensemble of the E2A-AD1(1–37):TAZ2 complex were calculated using CCPNMR Analysis (64, 67). The protein structure validation software suite was used to determine the ordered residues and evaluate the quality of the final E2A-AD1(1–37):TAZ2 ensemble (68). The ensemble of the 20 lowest-energy structural models was deposited to the Protein Data Bank (accession no. 2MH0), whereas ¹H, ¹³C, and ¹⁵N chemical shifts and restraints were deposited to the BMRB (accession no. 19610).

For the NMR-based experiment assessing the ability of TAZ2 and KIX to compete for binding to E2A-AD1, a sample of 200 μm ¹⁵N-labeled KIX and 300 μm unlabeled E2A-AD1(1–37) in 20 mm MES, pH 6.8, 5 mm β-mercaptoethanol, 10 μm ZnCl₂, 95% H₂O/5% D₂O was used. Unlabeled TAZ2 was subsequently added to the sample to a final concentration of 500 μm.

Data availability

Chemical shift assignments for the E2A-AD1(1–37):TAZ2 complex and E2A-AD1 have been deposited in the BioMagResBank (accession numbers 19610 and 50196, respectively). The atomic coordinates (accession code 2MH0) have been deposited in the Protein Data Bank.

Author contributions

M. R. L., A. C. K., S. C., G. S. B., D. P. L., D. N. L., and S. P. S. conceptualization; M. R. L., A. D. B., A. C. K., J. E. F., and D. N. L. investigation; M. R. L., A. C. K., J. E. F., G. S. B., D. N. L., and S. P. S. writing-original draft; M. R. L., A. D. B., A. C. K., D. N. L., and S. P. S. writing-review and editing; A. D. B. and K. M. formal analysis; S. C., G. S. B., D. P. L., and S. P. S. and D. N. L. supervision; K. M. and J. E. F. methodology; D. P. L. and S. P. S. funding acquisition.