Crystal structure of ZNF217 bound to DNA, P6522 crystal form

EXPERIMENTAL PROCEDURES Expression and Purification of Recombinant ZNF217_F67 A construct encoding F67 of human ZNF217 (amino acids 467–523) was cloned into pMALC2 and pGEX2T vectors to allow the expression of MBP and GST fusion proteins, respectively. The MBP construct was expressed in Escherichia coli Rosetta2 cells overnight at 25 °C following the addition of 0.7 mM isopropyl-1-thio-β-D-galactopyranoside and 1 μM ZnSO4 to the log phase culture. Expression of the GST fusion construct was induced overnight at 22 °C by the addition of 0.4 mM isopropyl-1-thio-β-D-galactopyranoside to E. coli BL21 cells supplemented with 1 μM ZnSO4. Cells were lysed in a buffer containing 50 mM Tris-HCl (pH 8), 1 M NaCl, 1 mM DTT, and 1 mM PMSF. MBP and GST fusion proteins were recovered from the soluble fraction and purified by affinity chromatography. The fusion tags were cleaved using thrombin (3 h at room temperature) in 50 mM Tris (pH 8), 1 M NaCl, 10 mM CaCl2, and 1 mM DTT. F67 was then dialyzed into 50 mM Tris (pH 7), 1 mM DTT and further purified by cation-exchange chromatography (UnoS1, Bio-Rad). The construct identity and correct folding of F67 were confirmed by DNA sequencing and one-dimensional 1H NMR spectroscopy, respectively. 15N-labeled ZNF217_F67 was prepared following the procedure of Cai et al. (35) and purified as described above. Design and Preparation of the Oligonucleotides Used in the Crystallization Trials Three different double-stranded oligonucleotides, containing either one or two copies of the 8-bp consensus sequence TGCAGAAT, were used in efforts to crystallize a ZNF217_F67-DNA complex. All oligonucleotides were 20 residues in length with two complementary overhang nucleotides at the 5′ extremities of each strand. The first set of oligonucleotides contains two binding sites running in the same direction (forward, 5′-TTTGCAGAATCGTGCAGAAT-3′; reverse, 5′-ACGTCTTAGCACGTCTTAAA-3′). The second contains two binding sites running in opposite directions (forward, 5′-TTTGCAGAATCGATTCTGCA-3′; reverse, 5′-ACGTCTTAGCTAAGACGTAA-3′). The last contains a single binding site (forward, 5′-TTTCCATTGCAGAATTGTGG-3′; reverse, 3′-AGGTAACGTCTTAACACCAA-5′). ssDNA oligonucleotides were purchased from Sigma and heated at 95 °C for 15 min in a 50 mM Tris-HCl (pH 7.4) buffer containing 150 mM NaCl. Oligonucleotides were then annealed at room temperature overnight and purified by size exclusion chromatography (Sephadex-75, GE Healthcare). Crystallization and Data Collection Purified ZNF217_F67 and the different DNA duplexes were dialyzed in 20 mM Tris (pH 7), 50 mM NaCl. and 1 mM DTT before being mixed together (ZNF217_F67-DNA, 1:0.6 with the DNA duplexes that carried two binding sites and 1:1.2 with the oligonucleotide that contained a single site). The final protein concentration was 10 mg/ml. Initial crystallization trials were set up at 298 K as vapor diffusion hanging drops using a Mosquito robot (Molecular Dimensions) by mixing 400 nl of sample solution and 400 nl of reservoir solution and placing the resultant drop over 80 μl of reservoir solution in flat-bottom 96-well PS microplates (Greiner Bio-One). JSGC+ and PACT (Qiagen) screens were trialed. Large crystals in two different space groups were obtained with the oligonucleotide containing two binding sites running in opposite directions. Crystals in space group P6522 grew in the presence of 200 mM sodium acetate (pH 7), and 20% (w/v) polyethylene glycol (PEG) 3350 precipitant solution. Crystals in space group C2 grew in 100 mM MES (pH 6), 10 mM zinc chloride, and 20% (v/v) PEG 6000. Diffraction data were recorded on a mar345 image plate detector (Marresearch) using x-rays produced by a Rigaku RU200H rotating-anode generator (CuKα) focused with Osmic mirrors (MSC Rigaku). The diffraction data were integrated and scaled with HKL-2000 (36). Solution and Refinement of the Crystal Structures Phases for the P6522 crystal form were determined using the SIRAS technique with a lead derivative. Crystals were soaked for 2 h in crystallization buffer containing 10 mM trimethyl lead, and a 3.0-Å data set was collected. SIRAS phasing was realized using AutoSol (37), which identified two lead atoms and resulted in a mean figure of merit after density modification of 0.69. The resulting electron density was of sufficient quality to allow building of the oligonucleotide and peptide backbone. Successive rounds of model building were carried out using Coot (38), and refinement utilized REFMAC5 (39). Combined TLS (translation/libration/screw) and individual atomic displacement parameter refinement were also carried out in the final stages. The C2 crystal form phase was solved by molecular replacement with the P6522 model using PHASER (40). Model building and refinement were performed similarly to that described for the P6522 form. Four additional zinc ions were identified in the asymmetric unit. Due to their absence in the P6522 crystal form and their location on the surface of the protein-nucleic acid complex, we attribute these atoms to the presence of 10 mM ZnCl2 in the crystallization solution. Fluorescence Anisotropy Titrations Cleaved or GST-tagged ZNF217_F67 and 5′-fluorescein-labeled dsDNA oligonucleotides (WT sequence forward, 5′-Fl-TCCATTGCAGAATTGTGG-3′; mutated sequence, forward, 5′-Fl-TCCATCTGGAGTATGTGG-3′; poly(A), forward, 5′-Fl-(A)18-3′; the bold sequences correspond to the 8 bp consensus sequence and its mutated version recognized by ZNF217) were dialyzed into a 10 mM phosphate buffer, pH 7, containing 50 mM NaCl and 1 mM DTT. Fluorescence anisotropy titrations were performed at 25 °C on a Cary Eclipse fluorescence spectrophotometer with a slit width of 10 nm, and data were averaged over 15 s. The excitation and detection wavelengths were 495 and 520 nm, respectively. In each titration, the fluorescence anisotropy of a solution of 50 nM fluorescein-tagged dsDNA was measured as a function of the added protein concentration. Binding data were fitted to a simple 1:1 binding model by nonlinear least squares regression. Each titration was performed three times, and the final affinity was taken as the mean of these measurements. Isothermal Titration Calorimetry (ITC) ZNF217_F67 and the two DNA duplexes (see above) were dialyzed overnight against the same reservoir of buffer containing 10 mM Tris buffer, pH 7.0, 50 mM NaCl, and 1 mM tris(2-carboxyethyl)phosphine. Titrations were also carried out at 150 mM NaCl. ZNF217_F67 (200 μM) was titrated into DNA (20 μM). Titrations were carried out on a MicroCal i200 ITC microcalorimeter (GE Healthcare) at 25 °C. For each titration, an initial injection of 0.2 μl (data from which were discarded) and 20 injections of 2 μl of titrant were made at 120-s intervals. Data were corrected for heats of dilution from control experiments of the protein into buffer and analyzed using Origin7.0 (MicroCal Software, Northampton, MA). The two-binding-event titration curve observed for the ZNF217 binding to the specific DNA sequence could not be fitted with confidence using a two-site model because the error associated with this fit was above 100%. We therefore made the assumption that the second, low affinity binding event was identical to the single binding event observed during the titration of the mutated sequence. Using this assumption, we subtracted from the first titration the data points observed for the latter titration and could then fit the remaining data to a single binding event with an associated error under 20%. The derived dissociation constant for the tight interaction was indistinguishable from that obtained with the two-site model, except that the uncertainty in the fit was substantially lower for the single-site fit. NMR Spectroscopy For 15N HSQC chemical shift perturbation experiments, purified 15N-labeled ZNF217_F67 and the different dsDNA oligonucleotides were extensively dialyzed into a buffer comprising 10 mM Na2HPO4 (pH 7.0), 50 mM NaCl, and 1 mM DTT and were concentrated to ∼300 μM. All NMR samples contained 5–10% D2O and 10 μM 2,2-dimethyl-2-silapentane-5-sulfonic acid as a chemical shift reference. All experiments were run at 298 K on either a 600-MHz or an 800-MHz Bruker AvanceIII spectrometer equipped with a cryoprobe. 15N HSQC spectra were recorded for the ZNF217_F67 alone and following the addition of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1, 1.2, and 1.5 molar eq of either wild type (WT) or nonspecific DNA. The interaction between ZNF217_F67 and nonspecific DNA was in fast exchange, allowing straightforward resonance assignment from the titration data. NOESY spectra were also recorded to confirm these assignments. NMR data were processed using Topspin (Bruker, Karlsruhe, Germany) and analyzed with SPARKY. Background: Classical zinc finger proteins are extremely abundant and interact with DNA using a well defined recognition code. Results: We solved the structure of ZNF217 bound to its cognate DNA. Conclusion: ZNF217 presents a unique DNA interaction pattern including a new type of protein-DNA contact. Significance: This study deepens our understanding of DNA recognition by classical zinc fingers. Abstract: Classical zinc fingers (ZFs) are one of the most abundant and best characterized DNA-binding domains. Typically, tandem arrays of three or more ZFs bind DNA target sequences with high affinity and specificity, and the mode of DNA recognition is sufficiently well understood that tailor-made ZF-based DNA-binding proteins can be engineered. We have shown previously that a two-zinc finger unit found in the transcriptional coregulator ZNF217 recognizes DNA but with an affinity and specificity that is lower than other ZF arrays. To investigate the basis for these differences, we determined the structure of a ZNF217-DNA complex. We show that although the overall position of the ZFs on the DNA closely resembles that observed for other ZFs, the side-chain interaction pattern differs substantially from the canonical model. The structure also reveals the presence of two methyl-π interactions, each featuring a tyrosine contacting a thymine methyl group. To our knowledge, interactions of this type have not previously been described in classical ZF-DNA complexes. Finally, we investigated the sequence specificity of this two-ZF unit and discuss how ZNF217 might discriminate its target DNA sites in the cell.

实验方法
## 重组ZNF217_F67的表达与纯化
将编码人类ZNF217的F67片段（氨基酸467–523）的构建体克隆至pMALC2和pGEX2T载体，分别以实现麦芽糖结合蛋白（MBP，Maltose-Binding Protein）和谷胱甘肽S-转移酶（GST，Glutathione S-Transferase）融合蛋白的表达。将该MBP构建体转化至大肠杆菌（Escherichia coli）Rosetta2菌株中，待培养液生长至对数生长期后，加入0.7 mM异丙基-1-硫代-β-D-半乳糖吡喃糖苷（IPTG，isopropyl-1-thio-β-D-galactopyranoside）与1 μM硫酸锌（ZnSO4），于25 ℃下过夜诱导表达。将GST融合构建体转化至大肠杆菌BL21菌株中，补加1 μM硫酸锌后，加入0.4 mM IPTG，于22 ℃下过夜诱导表达。
收集菌体后，使用含50 mM Tris-HCl（pH 8）、1 M氯化钠（NaCl）、1 mM二硫苏糖醇（DTT）与1 mM苯甲基磺酰氟（PMSF）的缓冲液裂解细胞。通过可溶性组分回收MBP与GST融合蛋白，并经亲和色谱进行纯化。使用凝血酶（thrombin）于含50 mM Tris（pH 8）、1 M NaCl、10 mM氯化钙（CaCl2）与1 mM DTT的缓冲液中室温切割3小时，以移除融合标签。随后将F67透析至含50 mM Tris（pH 7）与1 mM DTT的缓冲液中，并通过阳离子交换色谱（UnoS1，伯乐（Bio-Rad）公司）进一步纯化。通过DNA测序确认构建体序列正确性，通过一维1H核磁共振波谱（1H NMR spectroscopy）验证F67的正确折叠。参照Cai等人（文献35）的方法制备15N标记的ZNF217_F67，并按上述流程纯化。

## 结晶实验所用寡核苷酸的设计与制备
我们设计了三种不同的双链寡核苷酸，均包含1或2份8 bp共有序列TGCAGAAT，用于尝试结晶ZNF217_F67-DNA复合物。所有寡核苷酸链长均为20个碱基，每条链的5'端均带有两个互补的突出端核苷酸。
第一组寡核苷酸包含两个同向排列的结合位点（正义链：5'-TTTGCAGAATCGTGCAGAAT-3'；反义链：5'-ACGTCTTAGCACGTCTTAAA-3'）。第二组包含两个反向排列的结合位点（正义链：5'-TTTGCAGAATCGATTCTGCA-3'；反义链：5'-ACGTCTTAGCTAAGACGTAA-3'）。第三组仅包含单个结合位点（正义链：5'-TTTCCATTGCAGAATTGTGG-3'；反义链：3'-AGGTAACGTCTTAACACCAA-5'）。
单链寡核苷酸购自西格玛（Sigma）公司，于含50 mM Tris-HCl（pH 7.4）与150 mM NaCl的缓冲液中95 ℃加热15分钟，随后于室温下退火过夜，并通过尺寸排阻色谱（Sephadex-75，通用电气医疗（GE Healthcare）集团）纯化。

## 结晶与数据收集
将纯化后的ZNF217_F67与不同DNA双链体透析至含20 mM Tris（pH 7）、50 mM NaCl与1 mM DTT的缓冲液中，随后按比例混合：携带两个结合位点的DNA双链体与蛋白的摩尔比为0.6:1，携带单个结合位点的寡核苷酸与蛋白的摩尔比为1.2:1。最终蛋白浓度为10 mg/ml。
初始结晶实验于298 K下通过悬滴气相扩散法开展，使用Mosquito机器人（Molecular Dimensions公司）将400 nl样品溶液与400 nl储液混合，将形成的悬滴置于含80 μl储液的平底96孔PS微孔板（Greiner Bio-One公司）中。我们尝试了Qiagen公司的JSGC+与PACT筛选试剂盒。
使用携带两个反向排列结合位点的寡核苷酸时，获得了两种不同空间群的大晶体。空间群为P6522的晶体在含200 mM乙酸钠（pH 7）与20%（w/v）聚乙二醇3350（PEG 3350）的沉淀剂溶液中生长。空间群为C2的晶体在含100 mM 2-(N-吗啉代)乙磺酸（MES，pH 6）、10 mM氯化锌（ZnCl2）与20%（v/v）聚乙二醇6000（PEG 6000）的溶液中生长。
衍射数据使用mar345图像板探测器（Marresearch公司）收集，X射线由Rigaku RU200H旋转阳极X射线发生器产生（CuKα射线），并通过Osmic聚焦镜（MSC Rigaku）聚焦。衍射数据使用HKL-2000软件（文献36）进行积分与标度。

## 晶体结构解析与精修
使用单同晶置换反常散射（SIRAS，Single Isomorphous Replacement with Anomalous Scattering）技术结合铅衍生物解析空间群P6522晶体的相位信息。将晶体在含10 mM三甲基铅的结晶缓冲液中浸泡2小时，收集分辨率为3.0埃的数据集。使用AutoSol软件（文献37）完成SIRAS相位解析，该软件可识别两个铅原子，经密度修正后的平均优值为0.69。得到的电子云密度质量足够用于构建寡核苷酸与肽骨架模型。后续通过Coot软件（文献38）开展多轮模型构建，使用REFMAC5软件（文献39）进行精修。在精修的最终阶段，同时开展了平动/扭转/螺旋（TLS，translation/libration/screw）与单个原子位移参数精修。
使用P6522晶体的模型作为搜索模型，通过分子置换法解析空间群C2晶体的相位信息（PHASER软件，文献40）。模型构建与精修流程与P6522晶体一致。在该晶体的不对称单元中发现了4个额外的锌离子。由于这些锌离子未在P6522晶体中出现，且位于蛋白-核酸复合物的表面，我们推断其来源于结晶液中添加的10 mM ZnCl2。

## 荧光各向异性滴定
将切割后的或带有GST标签的ZNF217_F67与5'端荧光素标记的双链DNA寡核苷酸（野生型序列正义链：5'-Fl-TCCATTGCAGAATTGTGG-3'；突变序列正义链：5'-Fl-TCCATCTGGAGTATGTGG-3'；聚腺苷酸序列正义链：5'-Fl-(A)18-3'；加粗序列为ZNF217识别的8 bp共有序列及其突变版本）透析至含10 mM磷酸盐缓冲液（pH 7）、50 mM NaCl与1 mM DTT的缓冲液中。
荧光各向异性滴定实验于25 ℃下在Cary Eclipse荧光分光光度计上开展，狭缝宽度为10 nm，数据采集时长为15秒并取平均值。激发波长与检测波长分别为495 nm与520 nm。每次滴定中，我们检测50 nM荧光素标记的双链DNA溶液的荧光各向异性随添加蛋白浓度的变化情况。结合数据通过非线性最小二乘回归拟合至简单的1:1结合模型。每个滴定实验重复三次，最终亲和力取三次测量的平均值。

## 等温滴定量热法（ITC）
将ZNF217_F67与两种DNA双链体（见上文）透析过夜至含10 mM Tris缓冲液（pH 7.0）、50 mM NaCl与1 mM三(2-羧乙基)膦的缓冲液中，同时在150 mM NaCl浓度下开展滴定实验。将200 μM的ZNF217_F67滴定至20 μM的DNA溶液中。滴定实验于25 ℃下在MicroCal i200 ITC微量热计（通用电气医疗集团）上开展。每次滴定先注入0.2 μl滴定液（弃去该部分数据），随后以120秒的间隔注入20次2 μl的滴定液。通过将蛋白滴定至缓冲液中的对照实验校正稀释热，并使用Origin7.0软件（MicroCal软件公司，马萨诸塞州北安普顿）分析数据。
针对ZNF217与特异性DNA序列结合的双结合事件滴定曲线，无法通过双位点模型可靠拟合，因为该拟合的误差超过100%。因此我们假设：第二个低亲和力结合事件与滴定突变序列时观察到的单个结合事件完全一致。基于该假设，我们从首次滴定的数据中减去后者滴定得到的数据点，随后可将剩余数据拟合至单个结合事件模型，拟合误差低于20%。得到的紧密相互作用的解离常数与双位点模型得到的结果无显著差异，但单一位点拟合的不确定性显著降低。

## 核磁共振波谱学
针对15N异核单量子相干谱（15N HSQC）化学位移扰动实验，将纯化后的15N标记的ZNF217_F67与不同双链DNA寡核苷酸充分透析至含10 mM磷酸氢二钠（Na2HPO4，pH 7.0）、50 mM NaCl与1 mM DTT的缓冲液中，并浓缩至约300 μM。所有NMR样品均包含5–10%的重水（D2O）与10 μM 2,2-二甲基-2-硅戊烷-5-磺酸（DSS）作为化学位移参考物。所有实验均于298 K下在配备低温探头的600 MHz或800 MHz布鲁克AvanceIII核磁共振波谱仪上开展。
分别在单独的ZNF217_F67样品中，以及分别添加0.1、0.2、0.3、0.4、0.5、0.6、0.8、1、1.2与1.5摩尔当量的野生型（WT）或非特异性DNA后，采集15N HSQC谱图。ZNF217_F67与非特异性DNA的相互作用处于快速交换状态，可直接通过滴定数据完成共振归属。我们同时采集了核Overhauser效应谱（NOESY）以验证这些归属。NMR数据使用Topspin软件（布鲁克公司，卡尔斯鲁厄，德国）处理，使用SPARKY软件进行分析。

---
## 研究背景
经典锌指蛋白丰度极高，可通过明确的识别代码与DNA结合。
## 研究结果
我们解析了ZNF217与其同源DNA结合的复合物结构。
## 研究结论
ZNF217具有独特的DNA相互作用模式，包含一种新型的蛋白-DNA接触方式。
## 研究意义
本研究加深了我们对经典锌指蛋白DNA识别机制的理解。

---
## 摘要
经典锌指（ZF，zinc finger）结构域是丰度最高、研究最透彻的DNA结合结构域之一。通常，三个及以上锌指的串联阵列可高亲和力、高特异性地结合DNA靶序列，且其DNA识别模式已被充分阐明，因此可用于工程化定制锌指DNA结合蛋白。我们此前的研究表明，转录辅调控因子ZNF217中存在的双锌指单元可识别DNA，但其亲和力与特异性均低于其他锌指串联阵列。为探究这些差异的分子基础，我们解析了ZNF217-DNA复合物的结构。结果显示，尽管锌指在DNA上的整体位置与其他锌指阵列高度相似，但侧链相互作用模式与经典模型存在显著差异。该结构同时揭示了两个甲基-π相互作用的存在，每个相互作用均由一个酪氨酸残基与一个胸腺嘧啶甲基基团形成。据我们所知，此类相互作用此前从未在经典锌指-DNA复合物中被报道。最后，我们探究了该双锌指单元的序列特异性，并讨论了ZNF217如何在细胞中区分其靶DNA位点。