PMC:2728203 / 22168-34190 JSONTXT

{"project":"2_test","denotations":[{"id":"18190927-11403570-62517993","span":{"begin":771,"end":773},"obj":"11403570"},{"id":"18190927-11403570-62517994","span":{"begin":884,"end":886},"obj":"11403570"},{"id":"18190927-15208322-62517995","span":{"begin":3970,"end":3972},"obj":"15208322"},{"id":"18190927-8918934-62517996","span":{"begin":4051,"end":4053},"obj":"8918934"},{"id":"18190927-8589602-62517997","span":{"begin":7524,"end":7526},"obj":"8589602"},{"id":"18190927-1522591-62517998","span":{"begin":8109,"end":8111},"obj":"1522591"},{"id":"18190927-16552146-62517999","span":{"begin":8264,"end":8266},"obj":"16552146"},{"id":"18190927-8272427-62517999","span":{"begin":8264,"end":8266},"obj":"8272427"},{"id":"18190927-8736557-62518000","span":{"begin":8468,"end":8470},"obj":"8736557"},{"id":"18190927-15312772-62518000","span":{"begin":8468,"end":8470},"obj":"15312772"},{"id":"18190927-1303746-62518001","span":{"begin":9283,"end":9285},"obj":"1303746"},{"id":"18190927-15299312-62518001","span":{"begin":9283,"end":9285},"obj":"15299312"},{"id":"18190927-1303746-62518002","span":{"begin":9850,"end":9852},"obj":"1303746"},{"id":"18190927-15299374-62518003","span":{"begin":9890,"end":9892},"obj":"15299374"},{"id":"18190927-15299374-62518004","span":{"begin":9942,"end":9944},"obj":"15299374"},{"id":"18190927-15299374-62518005","span":{"begin":10052,"end":10054},"obj":"15299374"},{"id":"18190927-15299374-62518006","span":{"begin":10134,"end":10136},"obj":"15299374"}],"text":"Materials and Methods\n\nStrains, plasmids, and chemicals\nAll DNA manipulations were performed according to standard methods,35 and all media were prepared by standard procedures.36 All yeast strains and plasmids used are described in Table 3. Chemicals were obtained from Ambion (Austin, TX), Sigma Chemical Co. (St. Louis, MO), US Biological (Swampscott, MA), or Fisher Scientific (Pittsburgh, PA) unless otherwise noted. Site-directed mutagenesis was performed using the Stratagene QuikChange mutagenesis kit. All mutants were sequenced to ensure that the intended change was introduced and no additional changes to the sequence were present.\n\nYeast two-hybrid analysis\nThe yeast two-hybrid reporter strain (EGY48) containing the DNA-binding domain (DBD) vector (pEG202)29 or the appropriate DBD fusion protein plasmids was transformed with the activation domain (AD) vector (pJG4–5)29 or the indicated AD fusion protein plasmids. Briefly, transformants were tested for a positive yeast two-hybrid interaction by expression of lacZ and galactose-dependent growth on media lacking leucine. Cells were grown at 30 °C on glucose or galactose minimal media, minimal media lacking leucine, or minimal media containing 200 μM 5-bromo-4-chloro-3-indolyl-β-d-galactosidase. The expression of the appropriately sized fusion proteins was confirmed by immunoblotting using either LexA antibody (1:1000 dilution, Covance) or HA antibody (1:1000 dilution, Santa Cruz).\n\nIn vitro binding assays\nFor in vitro assays, purified recombinant Nab2-N (amino acids 1–97) and CT-Mlp1 (amino acids 1490–1779) were employed. Nab2-N was expressed as a tobacco etch virus protease-cleavable GST fusion protein to assess direct binding.37 GST-Nab2-N (pAC2058) was expressed in E. coli DE3 cells. Cells were collected and lysed in phosphate-buffered saline (PBS) (137 mM NaCl, 10 mM phosphate, and 2.7 mM KCl, pH 7.4) supplemented with protease inhibitor mixture (1 mM PMSF, 3 ng/ml pepstatin A, leupeptin, aprotinin, and chymostatin) by incubation with lysozyme (0.1 mg/ml) for 30 min on ice followed by sonication to purify the GST fusion proteins. Lysates were clarified by centrifugation and incubated with glutathione Sepharose (Amersham) in buffer A [20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM β-mercaptoethanol, 1 mM PMSF, and 0.1% Igepal] for 2 h at 4 °C with mixing. The beads were then washed with PBS and 0.5% Triton X-100.\nHis-CT-Mlp1 (pAC1486) was expressed in E. coli DE3 cells. Cells were collected and lysed in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole, pH 7.0) supplemented with protease inhibitor mixture by incubation with lysozyme and sonication. Lysates were clarified by centrifugation and incubated with Ni–NTA agarose (Qiagen) in lysis buffer for 2 h at 4 °C with mixing. The beads were then washed with wash buffer (50 mM NaH2PO4, 300 mM NaCl, and 20 mM imidazole). His-Nab2 and His-CT-Mlp1 were eluted from agarose with 250 mM imidazole. Sepharose-bound GST or GST-CT-Mlp1 (6 μg) was incubated with 2 μg of purified His-Nab2 at 4 °C in PBS for 90 min. Sepharose-bound GST-NT-Nab2 WT, F72D, or F73D (6 μg) was incubated with purified His-CT-Mlp1 (2 μg). Unbound fractions were collected and the beads were washed three times with PBS. Bound fractions were eluted with sample buffer (50 mM Tris–HCl, pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 12.5 mM EDTA, and 0.02% bromophenol blue) and analyzed by SDS-PAGE followed by Coomassie Blue staining.\nEach GST-Nab2-N fusion protein or the GST control protein was added to yeast lysate to examine the interaction between the N-terminal domain of Nab2 and full-length Mlp1. GST fusion proteins were then purified on glutathione beads, and copurifying proteins were visualized either by Coomassie Blue staining or by immunoblotting.\nPurified recombinant His-tagged Gfd1 was prepared as previously described to examine the interaction between Nab2-N and Gfd1.27 The Gfd1 protein was attached to CNBr Sepharose beads as previously described.38 Briefly, CNBr Sepharose beads (Amersham Pharmacia Biotech) were swollen and washed in 1 mM HCl. Beads were transferred to coupling buffer (100 mM NaHCO3, pH 8.3, and 500 mM NaCl) and added to 2–5 mg of Gfd1 in coupling buffer. Coupling was carried out at 4 °C overnight. Residual active groups were blocked with 1 M Tris–HCl, pH 8.0, for 2 h at room temperature. Beads were then washed successively and extensively four times in coupling buffer and acid wash buffer (0.1 M sodium acetate, pH 4.0, and 500 mM NaCl). For binding assays, 10 μg of Nab2-N was incubated with 50 μl of Gfd1 beads for 2 h at 4 °C. Beads were then washed twice in PBS, and bound proteins were eluted with 100 μl of sample buffer. Samples were resolved by PAGE, and bound proteins were detected by Coomassie Blue staining.\n\nExpression and purification of the N-terminal domain of Nab2 for structural studies\nResidues 1–105 of Nab2 were cloned into pET28a and pET30a vectors (Novagen) via NdeI and BamHI sites to produce a His-tagged and untagged construct, respectively. Proteins were expressed in E. coli BL21 (DE3) CodonPlus RIL cells by overnight induction with 1 mM IPTG at 25 °C. His-tagged Nab2 1–105 was purified by using Ni–NTA agarose resin according to manufacturer's instructions (Qiagen Inc.) followed by gel filtration on a Superdex 75 HiLoad 26/60 prep grade column (GE Healthcare) with 20 mM Tris–HCl, pH 8.0, 1 mM DTT, 1 mM EDTA, and 50 mM NaCl as buffer. Untagged protein was purified by anion-exchange chromatography (Q-Sepharose HiLoad 16/10 column, GE Healthcare) applying in 20 mM Bis-Tris–HCl, pH 6.0, 1 mM DTT, and 1 mM EDTA and eluting with a linear gradient of NaCl from 0 to 500 mM in the same buffer followed by gel filtration chromatography as above. Typical yields were 40 mg/l of culture. The resultant proteins were \u003e 95% pure as assessed by SDS-PAGE. By mass spectrometry, the untagged material had an Mr of 11,324.6, consistent with Met1 having been removed (theoretical Mr = 11,455.2 for full length, with Mr = 11,324.0 corresponding to loss of the N-terminal Met), whereas the His-tagged construct had an Mr of 13,489 (theoretical Mr = 13487.2, corresponding to loss of the N-terminal Met). Nab2 dual labeled with 15N and 13C was purified from cultures harboring pET30a:Nab2 (1–105) grown in M9 minimal medium supplemented with 13C-labeled glucose and 15N-labeled NH4Cl.\n\nSolution structure\nNMR spectra were acquired at 27 °C on Bruker Avance 800 and DRX500 spectrometers, equipped with triple-resonance (1H/15N/13C) cryoprobes. All data were acquired using a sample containing 2 mM 15N/13C-labeled protein, 25 mM sodium phosphate, pH 6.0, 10 mM sodium chloride, and 10% 2H2O and comprised the following: 2D: [15N–1H] heteronuclear single quantum coherence (HSQC), [13C–1H] HSQC covering full 13C spectral width, constant-time [13C–1H] HSQC covering only aliphatic 13C region, [1H–1H] NOE spectroscopy (NOESY) experiments (τm = 50, 100, and 150 ms), [1H–1H] NOESY experiments filtered to remove 15N-coupled signals in F2 (τm = 50 and 150 ms); 3D data sets: CBCANH, CBCACONH, HBHANH, HBHACONH, [1H–13C–1H] HCCH–total correlated spectroscopy, [1H–13C–1H] HCCH–correlated spectroscopy, 15N NOESY–HSQC (τm = 50 and 150 ms), and 13C NOESY–HSQC (τm = 50 and 150 ms), with separate data sets acquired for 13C aliphatic and aromatic spectral regions. 1H, 15N, and 13C chemical shifts were calibrated using sodium 3,3,3-trimethylsilylpropionate as external 1H reference.39\nNOE distance restraints were derived from analysis of all of the data from NOE-based experiments. Cross-peak intensities were measured using the program SPARKY40 and grouped into four categories. The strongest dαN (i, i + 1) were used to set the upper limit for the category “very strong” (0–2.3 Å), strong dNN (i, i + 1) connectivities in α-helices defined the category “strong” (0–2.8 Å), dαN (i, i + 3) cross peaks in helices defined the category “medium” (0–3.5 Å), and all remaining peaks were classified as “weak” (0–5 Å). Lower bounds for all NOE restraints were set to zero,41 and no multiplicity corrections were required since r− 6 summation was used for restraints involving groups of equivalent or nonstereoassigned spins.33,42\nStructures were calculated from polypeptide chains with randomized ϕ and ψ torsion angles using a two-stage simulated annealing protocol within the program XPLOR, essentially as described elsewhere,43,44 but employing larger numbers of cycles as follows: first-stage calculations comprised Powell energy minimization (500 steps), dynamics at 1000 K (25,000 steps), increase of the van der Waals force constant and tilting of the NOE potential function asymptote (4000 steps), switching to a square-well NOE function then cooling to 300 K in 2000 step cycles, and final Powell minimization (1000 steps). Second-stage calculations used Powell minimization (500 steps), increasing dihedral force constant during 4000 step cycles of dynamics at 1000 K (with a strong van der Waals force constant and square-well NOE potential function), cooling to 300 K in 1000 step cycles, and 2000 steps of final Powell minimization.\nThe program CLUSTERPOSE was used to calculate the mean rmsd of ensembles to their mean structure.45,46\n\nCrystallography\nCrystals were obtained by vapor diffusion after 18 months from a 60-mg/ml solution of His-tagged Nab2 (residues 1–105) with a well buffer containing 0.2 M MgCl2, 0.1 M Tris–HCl, pH 8.5, and 20% (w/v) polyethylene glycol 4000. Crystals were cryoprotected in well buffer containing 20% glycerol and vitrified in a stream of anhydrous nitrogen at 100 K. A 1.8-Å-resolution data set (Table 3) was collected at 100 K in-house using a Rigaku X-ray generator equipped with Osmic mirrors and a MARdtb image plate detector. Data were processed using MOSFLM45 and reduced using SCALA and TRUNCATE.48 A molecular replacement was obtained using Phaser48 using the average NMR structure for residues 6–82 as a structural model. After solvent flattening using DM,48 the best molecular replacement solution was used as a starting model in ArpWarp48 that successfully built residues 6–94. After iterative cycles of refinement with REFMAC5 and model building using O49 and the addition of 59 water molecules, a final structure with an R-factor of 20.7% (Rfree = 26.4%) and excellent geometry was produced (Table 2). Modeling TLS rigid-body motion based on helices H1–H4 and helix H5 reduced the R-factor to 19.2% (Rfree = 25.0%).\n\nGel shift RNA binding assay\nSynthetic 25-nt poly(N) RNAs (Dharmacon) were 5′-end labeled with [γ-32P]ATP (GE Healthcare Life Sciences) using T4 polynucleotide kinase (Promega). The reaction was stopped, and unincorporated nucleotides were removed using Qiagen's Nucleotide Removal Kit. GST, GST-Nab2-N, and, as a positive control, GST-Srm160-N35 were expressed in E. coli and purified using glutathione beads as described above for in vitro binding assays. RNA electrophoretic mobility shift assays were performed by incubating the increasing amounts (0.5–5 μM) of recombinant GST, GST-Nab2-N, or GST-Srm160-N with approximately 30 pM radioactively labeled poly(N) RNA oligonucleotide (a 25-mer RNA oligonucleotide with each position randomized) in binding buffer for 30 min at 20 °C. Binding reactions were loaded onto a 5% native polyacrylamide gel and electrophoresed at 30 mA in 0.3× TBE buffer for 30 min to separate free oligonucleotide from protein–RNA complexes. Gels were dried and exposed overnight using a phosphorimager (Amersham).\n\nData deposition\n1H, 13C, and 15N NMR resonance assignments for the Nab2 N-terminal domain have been deposited at the BioMagResBank under accession code 15263, and the coordinates of the 45 accepted structures have been deposited under the Protein Data Bank (PDB) accession code 2JPS. Coordinates and structure factors for the crystal structure of the Nab2 N-terminal domain have been deposited with the PDB under accession codes 2V75 and R2V75SF, respectively."}