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

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.

### 材料与方法
#### 蛋白质制备
编码Brd3第一个溴结构域（BD1，残基25-147）的构建体由GENEART（德国雷根斯堡）进行密码子优化以在大肠杆菌（Escherichia coli）中表达，并克隆到pGEX-6P载体（GE Healthcare）中。Brd3 BD2（残基307-419）及BD1突变体均克隆至同一表达载体。所有构建体均以谷胱甘肽S-转移酶（glutathione S-transferase, GST）融合蛋白形式在37℃下经IPTG（异丙基-β-D-硫代半乳糖苷，isopropyl-β-D-thiogalactopyranoside）诱导过表达；同位素标记的BD1和BD2构建体采用参考文献7所述方案过表达。蛋白质通过谷胱甘肽亲和层析纯化，经PreScission蛋白酶切割后进行凝胶过滤（Superdex-75柱，NMR缓冲液：20 mM Tris、100 mM NaCl、1 mM二硫苏糖醇，pH 7.0）。蛋白质浓度通过测定215、225和280 nm处吸光度验证。BD1点突变体的正确折叠通过一维（1D）1H核磁共振（nuclear magnetic resonance, NMR）光谱确认。

#### 肽段制备
所有乙酰化及非乙酰化GATA1肽段由Peptide 2.0 Inc.（美国弗吉尼亚州尚蒂利）合成。肽段经高效液相色谱（high-pressure liquid chromatography, HPLC）纯化，纯度≥90%。使用前溶解于NMR缓冲液，浓度通过测定215和225 nm处吸光度验证（参考文献60）。单乙酰化肽段[K(Ac)312、K(Ac)314、K(Ac)315、K(Ac)316]、双乙酰化肽段[K(Ac)312/314、K(Ac)314/315、K(Ac)312/315]及三乙酰化肽段[K(Ac)312/314/315]的序列为CRKASGKGKKKRGSNL；四乙酰化（4Ac）肽段序列为KASGKGKKKRGSN；用于NMR结构测定的肽段[K(Ac)312/315]序列为KASGKGKKKRGSN；竞争实验用肽段[K(Ac)308/312/315]序列为RNRKASGKGKKKRGS；序列特异性实验用肽段[带侧翼K的K(Ac)312/315]序列为KASKKKKKKRGSN。所有序列基于小鼠GATA1序列，在对应名称所示位置乙酰化（乙酰化赖氨酸为斜体）。

#### 肽段亲和实验
肽段由洛克菲勒大学及Peptide 2.0 Inc.（美国弗吉尼亚州尚蒂利）合成。添加N端半胱氨酸以偶联至Sulfo-link树脂（Pierce）。按制造商说明进行肽段偶联。肽段亲和实验：将25-50 ng固定化肽段与来自800万-1000万稳定表达血凝素（hemagglutinin, HA）标记Brd3的G1E-ER4细胞的核提取物孵育。核提取物按先前描述制备（参考文献3）并稀释至150 mM NaCl。经高盐（NaCl）洗涤后，树脂在十二烷基硫酸钠（sodium dodecyl sulfate, SDS）样品缓冲液中煮沸，通过10% SDS-PAGE凝胶分离，转移至硝酸纤维素膜，并用抗HA抗体进行Western blot分析。亲和实验在含10 mM丁酸钠、1 mM苯甲基磺酰氟（phenylmethylsulfonyl fluoride, PMSF）及蛋白酶抑制剂混合物（Sigma，按制造商推荐添加）的体系中进行。GST下拉实验：1 μg纯化的GST-Brd3 BD1蛋白与25-50 ng固定化肽段在含10 mM丁酸钠、1 mM PMSF及蛋白酶抑制剂混合物（Sigma）的体系中孵育过夜。树脂用含450 mM NaCl、50 mM Tris（pH 7.5）及0.5% Igepal的缓冲液洗涤5次，煮沸于SDS样品缓冲液中洗脱。使用抗GST抗体（sc-138，Santa Cruz）进行Western blot分析。

#### 表面等离子体共振（SPR）测量
动力学分析在Biacore 3000表面等离子体共振（surface plasmon resonance, SPR）仪器（Biacore AB，瑞典乌普萨拉）上进行。四乙酰化GATA1肽段的生物素化通过化学合成实现：将EZ-Link马来酰亚胺-聚乙二醇2（PEG2）-生物素（Thermo Scientific，美国罗克福德）溶解于NMR缓冲液至终浓度20 μM，以20倍过量加入肽段；反应混合物在4℃静置5 h。标记肽段通过Superdex肽HR 10/30柱（BioLogic快速蛋白液相色谱系统，FPLC）分离，监测215 nm吸光度以去除过量生物素。生物素化4Ac GATA1肽段固定于链霉亲和素包被的SA传感器芯片（Biacore AB，瑞典乌普萨拉）。所有实验使用含0.005% P20去污剂的NMR缓冲液。芯片按制造商说明用条件化溶液预处理（3次1分钟注射，20 μl/min，50 mM NaOH+1 M NaCl）。生物素化GATA1肽段稀释至100 nM，以20 μl/min流速注射到传感器芯片通道（Fc-2或Fc-4）2分钟，固定水平约50-100响应单位（RU）。芯片随后用运行缓冲液洗涤。上游未修饰通道表面用于参考扣除。Brd3 BD1蛋白浓度范围为1 μM至200 μM（40 μl），在25℃下采用KINJECT程序以20 μl/min流速进行动力学测量。野生型与突变体蛋白样品交替取样，纳入零浓度样品用于双参考，重复3-5次循环。数据初始用BIAevaluation软件（Biacore）分析，但因解离动力学过快（受数据采集速率限制）无法获得可靠解离速率，故采用Origin 7.0（OriginLab Corp.，美国北安普顿）进行稳态分析（参考文献48）。若50 μM BD1注射未产生>10 RU的SPR信号，则视为无相互作用（ND）。Biacore竞争实验采用已发表方法（参考文献50、52）估算结合常数。

#### NMR光谱
NMR样品含0.5-1.5 mM纯化的¹⁵N/¹³C标记、¹⁵N标记或未标记Brd3 BD1，溶于NMR缓冲液（含1 μl 10 μM 2,2-二甲基-2-硅戊烷-5-磺酸，DSS，作为化学位移参考），并添加5-10%（体积/体积）D₂O或100% D₂O。[¹⁵N]Brd3 BD2及BD1突变体样品制备方法类似。BD1-GATA1复合物样品：结构测定时，向0.5-1.5 mM BD1中加入1.0-1.1摩尔当量的GATA1 K(Ac)312/315肽段；HSQC滴定实验中BD1浓度为300 μM。光谱在298 K下于配备低温探头的Bruker 600 MHz及800 MHz谱仪上采集。所有同核二维数据按先前描述采集与分析（参考文献37）。总相关光谱（TOCSY）和核Overhauser增强光谱（NOESY）的混合时间分别为60 ms和150 ms。¹⁵N和¹³C化学位移归属通过标准三联共振实验完成（参考文献11）。NOE衍生的距离限制来自3D ¹³C分离NOESY和3D ¹⁵N分离NOESY光谱。分子间NOE通过3D ¹³C分离¹⁵N¹³C过滤（F1或F3）NOESY实验获得（参考文献5、27、65）。GATA1肽段归属基于一系列2D ¹⁵N¹³C过滤（F1和F2）NOESY及TOCSY实验（在有无递增浓度BD1的条件下采集）。所有NMR数据用TOPSPIN软件（Bruker，德国卡尔斯鲁厄）处理，用SPARKY 3分析（参考文献21）。加权化学位移变化按Ayed等人的方案计算。

#### 结构计算
无GATA1肽段的Brd3 BD1初始结构在CYANA软件（参考文献23）中计算，基于¹⁵N和¹³C NOESY光谱中手动指认的明确NOE。BD1的Φ和Ψ限制基于TALOS程序对 backbone化学位移的分析（参考文献10）。溶液中GATA1残基308-310及317-320无序，故未纳入结构计算。BD1-GATA1复合物的最终结构计算使用ARIA 1.2软件（参考文献38）结合CNS 1.1（参考文献6），采用标准协议，固定实验确定的组氨酸侧链互变异构状态（参考文献51）并纳入预指认的分子间NOE。ARIA 1.2的最终归属经手动检查并必要时修正。最终计算中，20个最低能量结构在9 Å水壳中用标准ARIA 1.2水 refinement模块（最小化和动力学步骤；细节见参考文献29、39）优化。20个总能量最低的构象用MOLMOL（参考文献31）、PYMOL（Schrödinger，美国纽约）及PROCHECK-NMR（参考文献34）分析与可视化（结构计算统计见表1）。

### 摘要
近期研究数据表明，特异性靶向溴结构域（bromodomain）蛋白的小型合成化合物可调控癌症相关或炎症基因的表达。尽管这些研究聚焦于溴结构域识别乙酰化组蛋白的能力，但越来越多证据显示，组蛋白样修饰也存在于转录因子等其他重要蛋白上。然而，我们对这些修饰调控蛋白功能的分子机制理解仍不完整。转录因子GATA1的锌指结构域邻近赖氨酸残基可发生乙酰化，该修饰对GATA1正常的染色质占据至关重要。我们近期发现含溴结构域的蛋白Brd3是与乙酰化GATA1相互作用的辅因子，且该相互作用对GATA1靶向染色质不可或缺。本文描述了该相互作用的结构基础：首次揭示了携带多重乙酰化修饰的转录因子与其同源识别模块间相互作用的分子细节；同时表明该相互作用可被乙酰赖氨酸模拟物抑制，强调需进一步提高靶向溴结构域与外端（BET）家族溴结构域化合物的特异性，以充分发挥其治疗潜力。

本研究获澳大利亚国家健康与医学研究委员会（NHMRC）授予J.P.M.的项目基金、美国国立卫生研究院（NIH）授予G.A.B.的RO1 DK054937基金及授予J.M.L.的T32 HL007971-07博士前培训基金部分支持。