Solution structure of CHD4-PHD2 in complex with H3K9me3

EXPERIMENTAL PROCEDURES Sequence Analysis and Molecular Diagrams Sequence analyses and alignments of DNA and proteins were carried out using ClustalW and BioManager 3.0 (no longer available) followed by manual adjustment. Molecular diagrams were produced using MOLMOL (35) or PyMOL. Cloning, Expression, and Purification Constructs of PHD1(365–420), PHD2(446–501), and PHD12(364–506) from human CHD4 were cloned by PCR amplification from a K562 cDNA library and ligated into the pGEX-2P vector (a modified pGEX-2T vector that contains a human rhinovirus 3C protease cleavage site). Each construct was expressed in Escherichia coli BL21(DE3) cells and purified as described previously for PHD2, with minor variations (including purification by size exclusion chromatography in place of anion exchange chromatography). The cleaved, purified proteins contained an additional five amino acids (GPLGS) derived from the human rhinovirus 3C protease cleavage site at the N terminus. The identity of each protein was confirmed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Western Blot Analysis GST fusion CHD4-PHD1 was incubated with C-terminally biotinylated peptides (Upstate Biotechnology, Inc.) corresponding to the unmodified H3 (residues 1–21) and singly modified H3K4me1/3 (residues 1–21), H3K9me1/2/3 (residues 1–21), H3K27me1/2/3 (residues 21–44), and H3K36me1/2/3 (residues 21–44) histone tails in the presence of streptavidin-Sepharose beads (GE Healthcare) in binding buffer containing 50 mm Tris (pH 7.5), 150 mm NaCl, and 0.05% Nonidet P-40. The beads were collected via centrifugation and washed five times with the peptide binding buffer. Bound protein was detected by Western blot using anti-GST horseradish peroxidase (HRP)-conjugate monoclonal antibodies (GE Healthcare). Negative controls using GST fusion proteins in the absence of the peptides were run in parallel to ensure that the proteins did not bind to the streptavidin beads. Combinatorial On-bead Screening Assay A 5000-member PTM-randomized combinatorial peptide library based on the first 10 residues of the histone H3 N terminus was incubated first with the GST-tagged version of CHD4-PHD1; second with a GST-specific primary antibody; third with a biotinylated secondary antibody, and finally with streptavidin-conjugated alkaline phosphatase, catalyzing the turnover of 5-bromo-4-chloroindol-3-yl phosphate, which results in the formation of a turquoise precipitate on beads bearing sequences that bind to the target protein. The bead color intensity is proportional to affinity of the interaction. Peptides from individual beads were cleaved with cyanogen bromide and analyzed by MALDI-TOF mass spectrometry. PTM patterns were determined from the resulting mass ladders. Discrimination factors were obtained by dividing the frequency of each modification observed in the intensely blue beads by the frequency of each corresponding modification from a random group of 100 library members. Discrimination factors represent the likelihood of observing a particular modification in a protein screening experiment relative to random chance. 15N HSQC Titrations All 15N HSQC spectra were recorded at 298 K on a 600 MHz Bruker Avance spectrometer equipped with a TCI cryoprobe. PHD constructs and H3 peptides were dialyzed into buffer containing 10 mm sodium phosphate (pH 7.0), 5 mm NaCl, and 1 mm DTT, using Micro DispoDIALYZERSTM (100-Da molecular mass cutoff, Harvard Apparatus, Holliston, MA) for the H3 peptides. For H3(1–12) peptides containing no aromatic residues, concentrations were determined by absorbance at 215 and 225 nm as described previously. H3(1–12) peptides (2–5 mm; synthesized by the Peptide Core Facility, University of Colorado Denver) were incrementally titrated into solutions containing 15N-labeled PHD constructs (25–50 μm). Association constants were determined from the chemical shift changes of individual resonances by nonlinear least squares regression analysis using a 1:1 binding model as described previously. An additional parameter in the form of a peptide concentration multiplication factor was included to correct for errors in peptide concentration determination. Use of this factor resulted in high convergence and improved χ2 values. NMR Resonance Assignment All spectra were recorded at 298 K on 600 or 800 MHz Bruker Avance spectrometers equipped with TCI cryoprobes. For PHD2-H3K9me3 assignment and structure determination, PHD2 and peptide were prepared as described above. For PHD1 assignment and structure determination, PHD1 was dialyzed into 10 mm sodium phosphate (pH 7.5), 50 mm NaCl, and 1 mm DTT. Protein concentrations were typically 0.3–1.6 mm. 1H, 15N, and 13C assignments of free PHD1 and H3K9me3-bound PHD2 were obtained from HNCA, CBCA(CO)NH, HNCACB, HNHA, HBHA(CO)NH, HNCO, HN(CA)CO, C(C)(CO)NH-TOCSY, (HB)CB(CGCD)HD, (HB)CB(CGCDCE)HE, H(C)CH-TOCSY, and HCC(CO)HN-TOCSY spectra, with the last two listed spectra recorded on samples in D2O buffer. NMR data were processed using Topspin (Bruker) and analyzed with Sparky (41). 1H assignments of unlabeled PHD2-bound H3K9me3 peptide (ARTKQTARKme3STG synthesized by the Peptide Core Facility, University of Colorado Denver, or ARTKQTARKme3STGGY purchased from Peptide 2.0, Chantilly, VA) were obtained from 15N/13C double half-filtered-NOESY, two-dimensional COSY and two-dimensional TOCSY spectra as well as a 13C/13C double half-filtered-NOESY acquired on a sample in D2O buffer. Data Analysis for Structure Determination PHI (ϕ) angle restraints were obtained by analysis of HNHA spectra, and CHI1 (χ1) angle restraints for PHD1 were obtained by analysis of an HNHB and short mixing time (50 ms) TOCSY and NOESY spectra. Additional dihedral angles were calculated using TALOS and TALOS+, and only angles predicted to be reliable by both programs were used as restraints. For the H3K9me3 peptide, additional negative ϕ angle restraints were included for residues for which the intraresidue Hα-HN NOE was clearly weaker than the NOE between Hα and the HN of the following residue. For PHD1 structure calculations, all distance restraints were derived from integration of a two-dimensional NOESY acquired on an unlabeled PHD1 sample. For calculation of the PHD2-H3K9me3 structure, distance restraints were obtained from two-dimensional NOESY, 15N NOESY, and 15N/13C double half-filtered NOESY spectra, as well as 13C NOESY and 13C/13C double half-filtered NOESY spectra acquired on samples in D2O buffer. For NOESY experiments, PHD2 and H3K9me3 were present at a 1:1 molar ratio, or up to a 5% molar excess of H3K9me3 (as judged by inspection of 15N HSQC titration data). Structure Calculations The molecular-viewing programs MOLMOL and PyMOL were used to analyze calculated structures throughout the structure determination process. Initial structure refinement was carried out using CYANA 2.1, and final calculations were performed using ARIA 1.2 for PHD1. For PHD2-H3K9me3, final calculations were performed using ARIA 2.2 with upper distance limits for intermolecular NOEs calibrated using the CALIBA module of CYANA 2.1. Trimethylated lysine was added to the library file of CYANA 2.1 and MOLMOL and to numerous defining files of ARIA 2.2 (topallhdg5.3.pro, parallhdg5.3.pro, PseudoAtom.py, atomnames.xml, and iupac.xml). The coordination geometry for each zinc ion in PHD1 and PHD2 was defined to be consistent with high resolution (实验方法 序列分析与分子绘图 DNA与蛋白质的序列分析及比对工作采用ClustalW与BioManager 3.0（已停止维护）完成，随后进行手动校正。分子绘图通过MOLMOL（参考文献35）或PyMOL生成。 克隆、表达与纯化 人源CHD4的PHD1(365–420)、PHD2(446–501)及PHD12(364–506)重组片段通过K562 cDNA文库的PCR扩增获得，并连接至pGEX-2P载体（一种携带来自人鼻病毒3C蛋白酶切割位点的改造型pGEX-2T载体）。各重组片段均在大肠杆菌BL21(DE3)细胞中表达，并参照此前报道的PHD2纯化方案进行纯化，仅做小幅调整（包括以尺寸排阻色谱替代阴离子交换色谱完成纯化步骤）。经切割纯化后的蛋白质N端额外携带一段源自人鼻病毒3C蛋白酶切割位点的5个氨基酸残基序列（GPLGS）。采用基质辅助激光解吸电离飞行时间（MALDI-TOF）质谱验证各蛋白质的身份。 蛋白质免疫印迹分析 将GST融合的CHD4-PHD1与对应于未修饰组蛋白H3（1-21位残基）以及单修饰H3K4me1/3（1-21位残基）、H3K9me1/2/3（1-21位残基）、H3K27me1/2/3（21-44位残基）和H3K36me1/2/3（21-44位残基）的C端生物素化肽段（购自Upstate Biotechnology, Inc.）在链霉亲和素琼脂糖凝胶珠（GE Healthcare）存在的条件下，于含50 mM Tris（pH 7.5）、150 mM NaCl及0.05% Nonidet P-40的结合缓冲液中共同孵育。通过离心收集凝胶珠，并用肽段结合缓冲液洗涤五次。采用偶联辣根过氧化物酶（HRP）的抗GST单克隆抗体（GE Healthcare）进行蛋白质免疫印迹，检测结合的蛋白质。同时设置不含肽段的GST融合蛋白作为阴性对照，以验证蛋白质不会非特异性结合链霉亲和素凝胶珠。 珠上组合筛选实验 以组蛋白H3 N端前10位残基为模板构建的包含5000个成员的翻译后修饰（PTM）随机组合肽库，首先与带有GST标签的CHD4-PHD1孵育，随后依次与GST特异性一抗、生物素标记二抗孵育，最后加入链霉亲和素偶联的碱性磷酸酶；该酶可催化5-溴-4-氯-3-吲哚磷酸的水解，在结合靶蛋白的肽段所在磁珠上形成绿松石色沉淀。磁珠的显色强度与蛋白质-肽段相互作用的亲和力呈正相关。从单个磁珠上用溴化氰裂解肽段，采用MALDI-TOF质谱进行分析。通过所得的质量阶梯峰确定肽段的翻译后修饰模式。区分因子通过将显色较强的磁珠中观测到的每种修饰的出现频率，除以随机选取的100个文库成员中对应修饰的出现频率得到。区分因子代表在蛋白质筛选实验中，相较于随机概率，观测到某一特定修饰的可能性。 15N异核单量子相干滴定实验（15N HSQC） 所有15N HSQC谱图均在298 K下于配备TCI低温探头的600 MHz Bruker Avance核磁共振波谱仪上采集。将PHD重组片段与H3肽段透析至含10 mM磷酸钠（pH 7.0）、5 mM NaCl及1 mM二硫苏糖醇（DTT）的缓冲液中；其中H3肽段的透析采用Micro DispoDIALYZER™（截留分子量100 Da，哈佛仪器公司，霍利斯顿，马萨诸塞州）完成。对于不含芳香族残基的H3(1–12)肽段，参照此前报道的方法，通过215 nm与225 nm处的吸光度测定其浓度。将H3(1–12)肽段（浓度2–5 mM，由科罗拉多大学丹佛分校肽核心实验室合成）逐步滴加入含15N标记PHD重组片段（浓度25–50 μM）的溶液中。参照此前报道的方法，采用1:1结合模型，通过非线性最小二乘回归分析单个共振峰的化学位移变化，计算结合常数。引入一个以肽段浓度校正因子形式存在的额外参数，以修正肽段浓度测定中的误差。引入该校正因子可提升拟合收敛性并优化χ²统计值。 核磁共振共振峰归属 所有谱图均在298 K下于配备TCI低温探头的600 MHz或800 MHz Bruker Avance核磁共振波谱仪上采集。针对PHD2-H3K9me3复合物的共振峰归属与结构解析，PHD2与肽段的制备参照上述方法完成。针对PHD1的共振峰归属与结构解析，将PHD1透析至含10 mM磷酸钠（pH 7.5）、50 mM NaCl及1 mM DTT的缓冲液中。蛋白质浓度通常为0.3–1.6 mM。游离PHD1以及结合H3K9me3的PHD2的1H、15N及13C共振峰归属通过以下谱图完成：HNCA、CBCA(CO)NH、HNCACB、HNHA、HBHA(CO)NH、HNCO、HN(CA)CO、C(C)(CO)NH-TOCSY、(HB)CB(CGCD)HD、(HB)CB(CGCDCE)HE、H(C)CH-TOCSY及HCC(CO)HN-TOCSY；其中最后列出的两个谱图采集于重水（D₂O）缓冲液的样品。核磁共振数据采用Topspin（Bruker）处理，并通过Sparky（参考文献41）进行分析。未标记的结合PHD2的H3K9me3肽段（序列为ARTKQTARKme3STG，由科罗拉多大学丹佛分校肽核心实验室合成；或序列为ARTKQTARKme3STGGY，购自Peptide 2.0公司，尚蒂伊，弗吉尼亚州）的1H共振峰归属通过以下谱图完成：15N/13C双半滤波NOESY、二维COSY、二维TOCSY，以及采集于重水缓冲液样品的13C/13C双半滤波NOESY。 结构解析数据分析 通过分析HNHA谱图获得φ角约束；针对PHD1的χ1角约束则通过分析HNHB谱图以及短混合时间（50 ms）的TOCSY和NOESY谱图获得。采用TALOS与TALOS+计算额外的二面角约束，仅保留两个程序均预测为可靠的角度作为约束条件。对于H3K9me3肽段，若某残基的残基内Hα-HN核Overhauser效应（NOE）显著弱于该残基Hα与后续残基HN之间的NOE，则为其添加额外的负φ角约束。在PHD1的结构计算中，所有距离约束均来自未标记PHD1样品的二维NOESY谱图积分结果。对于PHD2-H3K9me3复合物的结构计算，距离约束来自二维NOESY、15N NOESY、15N/13C双半滤波NOESY谱图，以及采集于重水缓冲液样品的13C NOESY和13C/13C双半滤波NOESY谱图。在NOESY实验中，PHD2与H3K9me3的摩尔比为1:1，或H3K9me3最多过量5%（通过15N HSQC滴定数据的分析确定）。 结构计算 在整个结构解析过程中，采用分子可视化程序MOLMOL与PyMOL对计算得到的结构进行分析。PHD1的结构初始精修采用CYANA 2.1完成，最终计算则通过ARIA 1.2实现。对于PHD2-H3K9me3复合物，最终计算采用ARIA 2.2完成，并通过CYANA 2.1的CALIBA模块校准分子间NOE的距离上限。将三甲基化赖氨酸的参数添加至CYANA 2.1与MOLMOL的库文件，以及ARIA 2.2的多个定义文件中（包括topallhdg5.3.pro、parallhdg5.3.pro、PseudoAtom.py、atomnames.xml及iupac.xml）。PHD1与PHD2中每个锌离子的配位几何构型被定义为与高分辨率（