Crystal structure of ZNF217 bound to DNA, P6522 crystal form

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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.