Structural basis and specificity of acetylated transcription factor GATA1 recognition by BET-family bromodomain protein Brd3ed]

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MATERIALS AND METHODS Preparation of proteins.A construct encoding the first bromodomain (BD1) of Brd3 (residues 25 to 147) was codon optimized by GENEART (Regensburg, Germany) for expression in Escherichia coli and cloned into pGEX-6P (GE Healthcare). Brd3 BD2 (residues 307 to 419) and BD1 mutants were all cloned into the same expression vector. All constructs were overexpressed as fusions with glutathione (GSH) S-transferase (GST) at 37°C upon induction with IPTG (isopropyl-β-D-thiogalactopyranoside) under standard conditions; isotopically labeled BD1 and BD2 constructs were overexpressed using the protocol described in reference 7. Proteins were purified using GSH affinity chromatography and subjected to PreScission protease cleavage and gel filtration (Superdex-75 in NMR buffer [20 mM Tris, 100 mM NaCl, 1 mM dithiothreitol, pH 7.0]). Protein concentrations were verified by determining absorbances at 215, 225, and 280 nm. The correct folding of BD1 point mutants was confirmed by one-dimensional (1D) 1H NMR spectroscopy. Preparation of peptides.All acetylated and nonacetylated GATA1 peptides were synthesized by Peptide 2.0 Inc. (Chantilly, VA). Peptides were subjected to high-pressure liquid chromatography (HPLC) purification, resulting in a purity of at least 90%. They were dissolved in NMR buffer prior to usage, and their concentrations were verified by determining absorbances at 215 and 225 nm (60). The sequence used for the monoacetylated peptides [K(Ac)312, K(Ac)314, K(Ac)315, and K(Ac)316], diacetylated peptides [K(Ac)312/314, K(Ac)314/315, and K(Ac)312/315], and a triacetylated peptide [K(Ac)312/314/315] was CRKASGKGKKKRGSNL, that for the tetra-acetylated (4Ac) peptide was KASGKGKKKRGSN, that for the peptide used in the NMR structure determination [K(Ac)312/315] was KASGKGKKKRGSN, that for competition experiments [K(Ac)308/312/315] was RNRKASGKGKKKRGS, and that for sequence specificity experiments [K(Ac)312/315 with a flanking K] was KASKKKKKKRGSN. All sequences are based on the mouse GATA1 sequence and were acetylated at the positions indicated in their corresponding names (see above; acetylated lysines are in italics). Peptide affinity assays.Peptides were synthesized by Rockefeller University and Peptide 2.0 Inc. (Chantilly, VA). An N-terminal cysteine was added to allow coupling to Sulfo-link resin (Pierce). Peptides were coupled according to the manufacturer's instructions. Peptide affinity assays were performed by incubating 25 to 50 ng immobilized peptide with nuclear extracts prepared from 8 million to 10 million G1E-ER4 cells stably expressing hemagglutinin (HA)-Brd3. Nuclear extracts were prepared as described previously (3) and diluted to 150 mM NaCl. Following stringent NaCl washes, resin was boiled in sodium dodecyl sulfate (SDS) sample buffer, separated on a 10% SDS-PAGE gel, transferred to a nitrocellulose membrane, and assayed by anti-HA Western blotting. Affinity assays were performed in the presence of 10 mM sodium butyrate, 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitor cocktail (Sigma), which was added according to the manufacturer's recommendation. For the GST-pulldown assays, 1 μg of purified GST Brd3 BD1 protein was incubated with 25 to 50 ng immobilized peptide overnight in the presence of 10 mM sodium butyrate, 1 mM PMSF, and protease inhibitor cocktail (Sigma). Resin was washed five times with buffer containing 450 mM NaCl, 50 mM Tris, pH 7.5, and 0.5% Igepal and eluted by boiling it in SDS sample buffer. Western blotting was performed using anti-GST antibodies (sc-138; Santa Cruz). SPR measurements.Kinetic analysis was carried out on a Biacore 3000 surface plasmon resonance (SPR) instrument (Biacore AB, Uppsala, Sweden). Biotinylation of tetra-acetylated GATA1 peptides was performed by chemical synthesis as follows. The biotin labeling reagent EZ-Link maleimide-polyethylene glycol 2 (PEG2)-biotin (Thermo Scientific, Rockford, IL), dissolved in NMR buffer to a final concentration of 20 μM, was added to the peptides in a 20-fold excess. The reaction mixture was then left at 4°C for 5 h. Labeled peptide was then separated from excess biotin using a Superdex peptide HR 10/30 column operating on a BioLogic fast protein liquid chromatography (FPLC) system by monitoring the absorbance at 215 nm. Biotinylated 4Ac GATA1 peptides were immobilized on a streptavidin-coated SA sensor chip (Biacore AB, Uppsala, Sweden). The buffer used for all experiments was NMR buffer with 0.005% P20 detergent. The chip was pretreated according to the manufacturer's instructions with conditioning solution (three 1-min injections at 20 μl/min with 50 mM NaOH, 1 M NaCl). The biotinylated GATA1 peptide was diluted to 100 nM and injected onto one of the sensor chip channels (Fc-2 or Fc-4) at a flow rate of 20 μl/min for 2 min, resulting in an immobilization level of approximately 50 to 100 response units (RU). The sensor chip was then washed with running buffer. Upstream, unmodified channel surfaces were used for reference subtraction. Kinetic measurements with Brd3 BD1 protein concentrations across the range of 1 μM to 200 μM (40 μl) were performed at 25°C with a KINJECT protocol and a flow rate of 20 μl/min. Wild-type and mutant protein samples were sampled alternately, zero-concentration samples were included for double-referencing, and 3 to 5 cycles were performed. Data analysis was initially performed with the BIAevaluation software (Biacore). However, no reliable off rates could be obtained due to the fast off-kinetics (measurement was limited by the data collection rate); therefore, a steady-state analysis (48) using Origin 7.0 (OriginLab Corp., Northampton, MA) was performed. Interactions for which an injection of 50 μM BD1 did not result in an SPR signal of >10 RU were considered not detectable (ND). For the Biacore competition experiments, the published methods (50, 52) for the estimation of the binding constant were used. NMR spectroscopy.NMR samples contained 0.5 to 1.5 mM purified 15N and 13C-labeled, 15N-labeled, and unlabeled Brd3 BD1 in NMR buffer (1 μl 10 μM 2,2-dimethyl-2-silapentane-5-sulfonic acid [DSS]) as a chemical shift reference and either 5 to 10% (vol/vol) D2O or 100% D2O. Samples of [15N]Brd3 BD2 and BD1 mutants were prepared similarly. BD1-GATA1 complex samples were prepared by adding 1.0 to 1.1 molar equivalents of GATA1 K(Ac)312/315 peptide to Brd3 BD1 at 0.5 to 1.5 mM for structure determination and at 300 μM for all HSQC titrations. Spectra were recorded at 298 K on Bruker 600-MHz and 800-MHz spectrometers equipped with cryoprobes. All homonuclear 2D data were collected and analyzed as described previously (37). Mixing times were 60 and 150 ms for all total correlation spectroscopy (TOCSY) and nuclear Overhauser enhancement spectroscopy (NOESY) spectra, respectively. 15N and 13C chemical shift assignments were made from the standard suite of triple-resonance experiments as described previously (11). NOE-derived distance restraints were obtained from 3D 13C-separated NOESY and 3D 15N-separated NOESY spectra. Intermolecular NOEs were also obtained from 3D 13C-separated 15N13C-filtered (in F1 or F3) NOESY experiments (5, 27, 65). GATA1 peptide assignments were made based on a series of 2D 15N13C-filtered (in F1 and F2) NOESY and TOCSY experiments recorded in the absence and presence of increasing amounts of BD1. All NMR data were processed using TOPSPIN (Bruker, Karlsruhe, Germany) and analyzed with SPARKY 3 (21). The weighted chemical shift changes were calculated according to the protocol of Ayed et al. Structure calculations.Initial structures of Brd3 BD1 without the GATA1 peptide were calculated in CYANA (23) from manually assigned unambiguous NOEs from 15N and 13C NOESY spectra. Φ and Ψ restraints for BD1 were included on the basis of an analysis of backbone chemical shifts in the program TALOS (10). GATA1 residues 308 to 310 as well as 317 to 320 were disordered in solution and therefore not included in the structure calculations. Final calculations of the BD1-GATA1 complex structure were then carried out using ARIA 1.2 (38) implementing CNS 1.1 (6), using the standard protocols provided, with the experimentally determined tautomeric state of the histidine side chains fixed (51) and the preassigned intermolecular NOEs present. Final assignments made by ARIA 1.2 were checked manually and corrected where necessary. In the final set of calculations, the 20 lowest-energy structures were refined in a 9-Å shell of water using standard ARIA 1.2 water refinement modules (minimization and dynamics steps; for details, see references 29 and 39). The 20 conformers with the lowest value of total energy were analyzed and visualized using MOLMOL (31), PYMOL (Schrödinger, NY), and PROCHECK-NMR (34) (for structure calculation statistics, see Table 1). Abstract: Recent data demonstrate that small synthetic compounds specifically targeting bromodomain proteins can modulate the expression of cancer-related or inflammatory genes. Although these studies have focused on the ability of bromodomains to recognize acetylated histones, it is increasingly becoming clear that histone-like modifications exist on other important proteins, such as transcription factors. However, our understanding of the molecular mechanisms through which these modifications modulate protein function is far from complete. The transcription factor GATA1 can be acetylated at lysine residues adjacent to the zinc finger domains, and this acetylation is essential for the normal chromatin occupancy of GATA1. We have recently identified the bromodomain-containing protein Brd3 as a cofactor that interacts with acetylated GATA1 and shown that this interaction is essential for the targeting of GATA1 to chromatin. Here we describe the structural basis for this interaction. Our data reveal for the first time the molecular details of an interaction between a transcription factor bearing multiple acetylation modifications and its cognate recognition module. We also show that this interaction can be inhibited by an acetyllysine mimic, highlighting the importance of further increasing the specificity of compounds that target bromodomain and extraterminal (BET) bromodomains in order to fully realize their therapeutic potential. This work was supported in part by a program grant from the National Health and Medical Research Council of Australia to J.P.M., by an NIH grant (RO1 DK054937) to G.A.B., and by an NIH predoctoral training grant (T32 HL007971-07) to J.M.L.