Peg 400

The dual mTORC1/2 inhibitor, AZD8055 (AstraZeneca, London, UK), was prepared for in vitro assays by dissolution in DMSO to 10 mM (4.65 mg/mL), per manufacturer instructions. The selective mTORC1 inhibitor, sirolimus (Selleckchem, Houston, TX), was prepared for in vitro assays by dissolution in 100% ethanol to 10.9 mM (10 mg/mL). For in vivo assays, AZD8055 was dissolved by sonication in 30% Captisol (CyDex Pharmaceuticals, Lenexa, KS) to a working concentration of 2 mg/ml and pH of 5.0 per manufacturer instructions. For in vivo assays, sirolimus was dissolved in 5% Tween-80 (Sigma Aldrich) and 5% PEG-400 (Hampton Research, Aliso Viejo, CA) to a working concentration of 0.4 mg/ml. Doses of ~ 200 μl drugs were administered to each animal.

The AtERF96 protein and GCC11 double-stranded DNA probe (5′-TAGCCGCCAGC-3′) were incubated in a tube at a 1:2 molar ratio, and concentrated to 6 mg/mL with GF2 buffer for crystallization. Screening for suitable crystallization conditions was performed using the Crystal Phoenix Liquid Handling System robot (Art Robbins Instruments, LLC). The program was set to a sitting-drop method, which dispensed an equal volume of the protein–DNA mixture and screening buffer to a volume of 1 µL to each well of a 96-well plate. The AtERF96–GCC11 complex crystals were observed at a temperature of 295 K at four crystallization conditions: Natrix™ No. 45 (0.05 M Tris-HCl pH 8.5, 0.025 M MgSO₄·H₂O, 1.8 M (NH₄)₂SO₄), Natrix™ 2 No. 6 (0.05 M sodium cacodylate trihydrate pH 6.0, 35% tacsimate pH 6.0), Natrix™ 2 No. 26 (0.05 M 3-(N-morpholino)propanesulfonic acid (MOPS) pH 7.0, 0.02 M MgCl₂·6H₂O, 55% tacsimate pH 7.0), and PEGRx™ 2 No. 6 (0.1 M sodium citrate tribasic dihydrate pH 5.0, 10% (v/v) 2-propanol, 26% (v/v) PEG 400; Hampton Research, Inc.). Crystals grew to a suitable size for X-ray diffraction after six months. All diffraction data were collected at 100 K on beamline 13C1 at the National Synchrotron Radiation Research Center (NSRRC), Hsinchu, Taiwan. Diffraction data were recorded using the ADSC Quantum-315r CCD detector and collected using Blu-Ice software (McPhillips et al. 2002 (link)).

For crystallization, the D417C mutant was put into a deletion construct (ΔNC) lacking N-terminal residues 2–16 and C-terminal residues 461–464 (Lim et al., 2012 (link)). Purified ΔNC-D417C was cross-linked with 100 µM CuP for 1 h, incubated with excess Fab fragment (Dutzler et al., 2003 (link)) for 30 min, then purified by size exclusion chromatography (Superdex 200) into buffer containing 100 mM NaCl, 5 mM DM, 10 mM Tris (Fisher Scientific, Pittsburgh, PA), pH 7.5. The complex was concentrated to 10–12 mg/mL and mixed with 30% PEG 400 (Hampton Research, Aliso Viejo, CA), 0.075 M K/Na-tartrate (Fluka Analytical, Ronkonkoma, NY), 0.1 M Tris HCl (MP Biomedicals, Santa Ana, CA) (pH 9.0). Crystals were grown by the sitting drop method for 2–4 weeks at 20^oC and were directly harvested from the reservoir, flash frozen and stored in liquid N₂. Diffraction data were collected to 0.9795 Å at the BL12-2 beamline (SLAC) and processed using XDS (Kabsch, 2010 (link)). Phases were obtained by molecular replacement with the WT protein in complex with Fab (PDB 1OTS) using the MOLREP program (Vagin and Teplyakov, 2010 (link)). Refinement was done using the refmac program (Murshudov et al., 1997 (link)). Atomic coordinate and structure factors are deposited in the Protein Data Bank under accession code 5HD8.

Rapamycin (LC Laboratories, Woburn, MA) was reconstituted in Dimethyl Sulphoxide Hybri-Max (Sigma-Aldrich, St. Louis, MO), and then diluted in 5% Tween-80 (Sigma-Aldrich) and 5% Polyethylene glycol (PEG) 400 (Hampton Research, Aliso Viejo, CA). Mice received Rapamycin (4 mg kg⁻¹ ip) every other day for 2 weeks32 .

Rapamycin (LC Laboratories) was reconstituted in absolute ethanol at 10 mg/mL and stored at −20°C. Rapamycin was diluted in 5.2% Tween 80 (Sigma) and 5.2% polyethylene glycol (PEG-400; Hampton Research) and injected i.p. (1 mg/kg) every other day for 15 days [20 (link), 68 (link)]. Only vehicle solution was administered as controls.