Data

Solution structure of CHD4-PHD2 in complex with H3K9me3

The University of Sydney
Ann Kwan (Aggregated by) Joel Mackay (Associated with, Aggregated by)
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ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Adc&rfr_id=info%3Asid%2FANDS&rft.title=Solution structure of CHD4-PHD2 in complex with H3K9me3&rft.identifier=https://mds.sydney.edu.au/redbox/published/detail/2eecf5b060cccc25cbdaaed43b210085&rft.publisher=The University of Sydney&rft.description=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 (&rft.creator=Ann Kwan&rft.creator=Ann Kwan&rft.creator=Joel Mackay&rft.date=2014&rft.relation=http://dx.doi.org/10.2210/pdb2l75/pdb&rft.relation=http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=21278251&rft.relation=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3064229&rft.relation=http://dx.doi.org/10.1074/jbc.M110.208207&rft_subject=Adenosine Triphosphatases&rft_subject=Plants&rft_subject=Protein Structure&rft_subject=Tertiary &rft_subject=Autoantigens&rft_subject=Chromatin Assembly and Disassembly&rft_subject=Histones&rft_subject=Humans&rft_subject=K562 Cells&rft_subject=Methylation&rft_subject=Mi-2 Nucleosome Remodeling and Deacetylase Complex&rft_subject=Nucleosomes&rft.type=dataset&rft.language=English Access the data

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Please forward data access requests to School of Molecular Biosciences, University of Sydney, NSW 2006, Australia. Tel.: 61-2-9351-3906; E-mail: joel.mackay@sydney.edu.au.

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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 (

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