Identification and structural characterization of a CBP/p300-binding domain from the ETS family transcription factor GABP alpha

Abstract

Using NMR spectroscopy, we identified and characterized a previously unrecognized structured domain near the N-terminus (residues 35-121) of the ETS family transcription factor GABP alpha. The monomeric domain folds as a five-stranded beta-sheet crossed by a distorted helix. Although globally resembling ubiquitin, the GABP alpha fragment differs in its secondary structure topology and thus appears to represent a new protein fold that we term the OST (On-SighT) domain. The surface of the GABP alpha OST domain contains two predominant clusters of negatively-charged residues suggestive of electrostatically driven interactions with positively-charged partner proteins. Following a best-candidate approach to identify such a partner, we demonstrated through NMR-monitored titrations and glutathione S-transferase pulldown assays that the OST domain binds to the CH1 and CH3 domains of the co-activator histone acetyltransferase CBP/p300. This provides a direct structural link between GABP and a central component of the transcriptional machinery.

Figure 1

(a) Cartoon of GABP domain organization. Boundaries are predicted or correspond to fragments used for studies of the individual domains. (b) The close superimposition of corresponding peaks in the ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled GABPα³⁵⁻¹²¹ (black) and GABPα¹⁻³²⁰ (red) demonstrates that the OST domain adopts an independently folded structure. The additional well-dispersed peaks in the spectrum of GABPα¹⁻³²⁰ arise from the PNT domain, whereas those clustered near ∼8.0 to 8.6 ppm in ¹H correspond to residues with predominantly random coil conformations. Assignments are given for GABPα³⁵⁻¹²¹, and aliased peaks are denoted with an asterisk. (c) “Beads-on-a-string” model of full-length GABPαshowing the 3 structured domains (PNT, 1SXD.pdb; ETS, 1AWC.pdb) along with all remaining residues in extended conformations. Methods: Genes encoding GABPα³⁵⁻¹²¹, GABPα¹⁻¹⁶⁹, and GABPα¹⁻³²⁰ were PCR-amplified from the full-length murine GABPαDNA (Genbank 34328119) and inserted in the vector pET28a (Novagen) for expression with an N-terminal His₆-tag and thrombin cleavage site. A codon for non-native Trp122 was added to the GABPα³⁵⁻¹²¹ gene to facilitate concentration measurements by absorbance spectroscopy. His₆-GABPα^168−256 was previously described. The GABP constructs were expressed in E. coli BL21(λDE3) cells grown at 37 °C in LB or minimal M9T medium containing 1 g/l ¹⁵NH₄Cl for uniform ¹⁵N-labeling, 1 g/l ¹⁵NH₄Cl and 3 g/l ¹³C₆-glucose for uniform ¹³C/¹⁵N-labeling, and 0.3 g/l ¹³C₆-glucose and 2.7 g/l ¹²C₆-glucose for non-random 10% ¹³C-labeling (Stable Spectral Isotopes). After induction with 1 mM IPTG at OD₆₀₀ ∼ 1 and growth for 4 hours at 37 °C, the cells were harvested by centrifugation. The cell pellets were resuspended in buffer A (5 mM imidazole, 50 mM HEPES (pH 7.5), 500 mM NaCl, 5 % glycerol) and lysed by passage through a French press three times at 10,000 psi, followed by 15 min of sonication on ice. The lysate was cleared by centrifugation, and the supernatant loaded onto a Ni-NTA column (GE Healthcare), pre-equilibrated with buffer A. The column was washed with 10 column volumes of buffer A plus 60 mM imidazole, and then the bound proteins were eluted with buffer A plus 250 mM imidazole. Following SDS-PAGE analysis, the appropriate fractions were pooled and dialyzed overnight in 20 mM sodium phosphate (pH 7.2) and 20 mM NaCl with a few crystals of thrombin (Roche) for the cleavage of the N-terminal His₆-tag. Thrombin and the cleaved His₆-tag were removed by incubation with ρ-aminobenzamidine beads (Sigma) and TALON metal affinity resin (BD Biosciences) at room temperature with mild shaking for 15 min, followed by centrifugation. The purified proteins, with an N-terminal Gly-Ser-His remaining from the cleavage site, were concentrated to ∼ 1 to 1.5 mM in NMR sample buffer (20 mM sodium phosphate (pH 7.0), 50 mM NaCl, 2 mM DTT, ∼10 % D₂O) using a 5 kDa cut-off Amicon Ultrafiltration device (Millipore). Spectra were recorded at 30 °C using Varian 500 MHz Unity, 600 MHz Inova, or 800 MHz Inova NMR spectrometers equipped with a triple resonance gradient probes, and analyzed using NMRpipe and Sparky. Main-chain and aliphatic side-chain ¹H, ¹³C, and ¹⁵N resonances were assigned using standard triple resonance correlation experiments. Resonances from aromatic side-chains were assigned using ¹H-¹³C HSQC, CβHδand CβHεexperiments. Stereospecific assignments of prochiral H^β,β signals were obtained from HNHB and short mixing time (30 msec) ¹⁵N-TOCSY-HSQC spectra, of Asn and Gln ¹⁵NH₂ groups from a EZ-HMQC-NH₂ spectrum, and of Val and Leu methyl groups from a constant time ¹H-¹³C HSQC spectrum of the non-randomly 10% ¹³C-labeled protein.

Figure 2

(a) Ribbon diagram of the lowest-energy NMR-derived structure of the GABPα OST domain (“front” view), with the β-sheet, helix, and two distinct loops (S₃/S₄ and S₄/S₅) shown in yellow, red, and cyan, respectively. Superimposed backbone (b) and hydrophobic sidechain (c) atoms from the structural ensemble of GABPα³⁵⁻¹²¹, with selected residues labelled for reference. (d) Two clusters of negatively-charged Asp and Glu residues (red) are observed on the “front” (left) and “back” (right) of the OST domain. In contrast, no extended clusters of (d) positively-charged Arg, His, or Lys residues (blue) or (e) exposed hydrophobic residues (green) are found on its surface (polar residues in grey). Methods: The tertiary structure of OST was calculated with ARIA(v1.2)/CNS utilizing distance, dihedral angle, and orientation restraints (Table 1). Interproton distance restraints were obtained from 3D ¹⁵N- NOESY-HSQC (600 MHz, 150 msec mixing time), simultaneous aliphatic ¹³C- and amide ¹⁵N-NOESY-HSQC (800 MHz, 125 msec), aromatic ¹³C- NOESY-HSQC (500 MHz, 125 msec), and constant time methyl-methyl and amide-methyl-NOESY spectra (600 MHz, 140 msec), followed by the automatic and iterative NOE assignments in ARIA. Mainchain dihedral angles were derived from ¹H, ¹³C, and ¹⁵N chemical shifts using TALOS. The predicted dihedral angles were only used when the Φ values agreed with ³J_NH-Hα coupling constants obtained from a HNHA spectrum. The χ₁ angle restraints for residues with prochiral H^β protons were determined in concert with the stereospecific assignments, and those of the Val, Thr, and Ile residues were determined from ³J_NCα and ³J_C’Cα coupling constants measured using N-Cα and C'-Cαspin echo experiments. For residues not showing ³J couplings indicative of rotamer averaging, the χ₁ angles were set to ± 60° or 180° based on a staggered rotamer model. ¹H^N-¹⁵N RDC orientational restraints were measured from ¹H-¹⁵N IPAP-HSQC spectra using protein aligned with stretched gels, and incorporated at iteration 4 with the SANI routine in ARIA, with default energy constants of 0.2 and 1 kcal mol⁻¹ Hz⁻² for the first and second simulated annealing cooling stages, respectively. Values of the alignment tensor (R = 0.3, D_a = 5.3 Hz) were estimated by the histogram method, followed by a grid search for minimal SANI violations. All His imidazole sidechains were determined to be in the neutral N^ε2 tautomeric state from long-range ¹H-¹⁵N correlation measurements. All Xaa-Pro amides were constrained to the trans conformation based on a chemical shift analysis with POP. In the final set of 10 water-refined structures, there were no violations greater than 0.5 Å and 5° for distance and angle restraints, respectively. The structural ensemble was analyzed using Procheck-NMR and secondary structures determined according to Promotif and Vadar. Structural figures were generated using MOLMOL and PyMOL.

Figure 3

Structure comparisons of the GABPα OST domain and ubiquitin (1UBQ.pdb). (a) The global folds of the two proteins are clearly similar. (b) However, secondary structure topology diagrams show that the direction of β-strand S5 in the OST domain is opposite from that of the corresponding strand in ubiquitin. (c) The sequential orders of secondary structural elements are also different. In addition, ubiquitin has a regular α-helix and 2 short 3₁₀-helices, whereas the OST domain has a distorted α-helix preceded by a 3₁₀-helix.

Figure 4

The GABPα OST domain binds to the mCBP CH1 and CH3 domains. (a,c) Superimposed ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled OST domain before (black, 0.1 mM) and after (red, 0.09 mM) addition of unlabeled CH1 (0.09 mM final). The top quartile of residues with backbone or sidechain amides showing intensity decreases due to CH1 domain binding are mapped in green on to the surface of the OST domain. (b,d) Superimposed ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled CH1 before (black, 0.1 mM) and after (red, 0.05 mM) addition of unlabeled OST domain (0.05 mM final). The top quartile of residues showing intensity decrease due to OST domain binding are mapped in green on to the surface of the CH1 domain (1U2N.pdb). (e) Superimposed ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled PNT domain before (black, 0.1 mM) and after (red, 0.1 mM) addition of unlabeled CH1 (0.1 mM final). The absence of any spectral changes indicates that no measurable binding occurs between the PNT and CH1 domains. These conclusions are supported by pulldown assays, involving 200−300 pmol of the indicated GST-tagged mCBP fragments, pre-bound to glutahionine-sepharose beads (20 μL), and 5 μM of FLAG-HMK-tagged (f) GABPα³⁵⁻¹²¹ (OST domain), (g) GABPα^168−256 (PNT domain), and (h) GABPα¹⁻³²⁰ (OST and PNT domains). Following bead collection and washing, binding was analyzed by Western blotting using an anti-FLAG antibody. Approximate molecular weight marker positions on the 4−15% SDS-PAGE gels are shown. The slower migrating band in the GABPα³⁵⁻¹²¹ sample is attributed to disulfide-linked dimers. (i) Cartoon of the domain structure of CBP. NMR Methods: The gene encoding the His₆-tagged CH1 domain (mCBP^340−439) was PCR cloned from the full length CBP gene (Genbank 70995311) into the pET28b (Novagen) vector for expression in E. coli BL21(λDE3) cells. Cells were grown at 30 °C in ZnSO₄ supplemented (150 μM) LB or ¹⁵N-minimal M9T medium to OD₆₀₀ ∼ 1, followed by induction with 1 mM IPTG and growth for overnight at 16 °C. Purification was as in Figure 1, except that buffer A was modified to 150 μM ZnSO₄ , 5 mM imidazole, 50 mM Tris (pH 7.5), 500 mM NaCl, and 5 % glycerol. The purified CHI (mCBP^340−439), OST (GABPα³⁵⁻¹²¹), and PNT (GABPα^168−256) domains were dialyzed into NMR buffer (20 mM Tris (pH 6.9), 50 mM NaCl, 2 mM DTT, ∼10 % D₂O) and concentrated by ultrafiltration. ¹H-¹⁵N HSQC spectra were recorded at 25 °C. GST-pulldown Methods: A DNA cassette encoding the FLAG tag and heart muscle kinase (HMK) target sequences was inserted into pET28a NdeI site of the GABPα constructs to generate clones for His₆-FLAG-HMK-GABPα³⁵⁻¹²¹, His₆-FLAG-HMK-GABPα^168−256, and His₆-FLAG-HMK-GABPα¹⁻³²⁰. Tagged fragments were purified as described for GABPα³⁵⁻¹²¹ in Figure 1. GST pulldown assays were conducted in 20 mM Tris pH 7.9, 10% glycerol, 180 mM KCl, 0.2 mM EDTA, 10 μM ZnSO₄, 0.5% NP40, 0.5 mM PMSF, 2−10 mM DTT, 0.1 mg/mL BSA, with Glutathione-Sepharose 4B beads (GE Healthcare). After incubation for 16 hr at 4 °C with constant rotation, the beads were collected by centrifugation and washed with binding buffer. The bound proteins were eluted using 3x SDS sample buffer, separated by SDS-PAGE, transferred to PVDF membranes, blotted with a 1:20,000 dilution of FLAG M2 antibody (Sigma), and visualized by ECL Plus (GE Healthcare). Panels (f), (g), and (h) are representative immoblots from experiments repeated four times. An SDS-PAGE gel of the protein samples used in these assays is provided in Supplemental Figure S4.