Computational protocol: Mimicking of Estradiol Binding by Flame Retardants and Their Metabolites: A Crystallographic Analysis

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[…] Chemicals. TBBPA (analytical grade; ), E2 (), and PAP, all ≥ 97% purity, were purchased from Sigma-Aldrich (St. Louis, MO); and 3-OH-BDE-47 (97% purity; ) was purchased from AccuStandard (New Haven, CT).Protein expression, purification, and crystallization. In these experiments, we used the SULT1E1 mutant V269E [expressed and purified as described previously ()] because it facilitates crystallization. This mutation lies on the surface of the protein remote from the active site at the dimer interface and favors monomer formation in solution, yet it still forms the expected physiological dimer in the crystal, as seen in other sulfotransferase structures (; ). Crystal structures of the complex of SULT1E1–PAP with TBBPA and with E2 were obtained by co-crystallization studies. The protein used for crystallization was concentrated to 13.6 mg/​mL in a solution at pH 7.5 and containing 1.5 mM sodium phosphate dibasic, 0.15 mM monopotassium phosphate, 40 mM sodium chloride, 1 mM dithiothreitol, and 4 mM PAP. TBBPA or E2 dissolved in 100% dimethyl sulfoxide (DMSO) was added to the protein stock for a final concentration of 8 mM. Protein–TBBPA solution or protein–E2 solution was mixed in equal volume with 0.1 M HEPES, pH 7.5, and 18–24% (wt/vol) polyethylene glycol 8000. For crystal growth, we used sitting drop vapor diffusion at 293K. Crystals were transferred to a cryoprotectant solution containing 0.1 M HEPES, pH 7.5, 22% polyethylene glycol 8000, 4 mM PAP, 15% ethylene glycol, and 8 mM TBBPA or E2 and flash frozen in liquid nitrogen.To obtain the crystal structure of SULT1E1 in complex with 3-OH-BDE-47 and PAP, protein stock was mixed in equal volume with 0.1 M 2-[N-morpholino]​ethane sulfonic acid, pH 6.0, and 17–22% (wt/vol) polyethylene glycol 8000, then placed at 293K, where crystals were grown using hanging drop vapor diffusion. SULT1E1–PAP crystals were transferred in three steps into a cryoprotectant solution consisting of 3-OH-BDE-47 suspended at a concentration of 5 mM in 0.1 M 2-[N-morpholino]ethane sulfonic acid, pH 6.0, 20% (wt/vol) polyethylene glycol 8000, 4 mM PAP, and 15% (vol/vol) ethylene glycol. Crystals were soaked in the cryoprotectant solution for 5 days before flash freezing in liquid nitrogen for data collection.Competitive crystallization experiment. Crystal structure of the complex of SULT1E1–​PAP–E2–TBBPA was obtained by co-crystallization studies. TBBPA and E2 dissolved in 100% DMSO were added to the protein stock for final concentrations of 8 mM each. Protein–E2–TBBPA solution was mixed in equal volume with 0.1 M HEPES, pH 7.5, and 18–24% (wt/vol) polyethylene glycol 8000. Crystal were grown using sitting drop vapor diffusion at 293K. For data collections, crystals were then transferred to a cryoprotectant solution containing 0.1 M HEPES, pH 7.5, 22% (wt/vol) polyethylene glycol 8000, 4 mM PAP, 15% (vol/vol) ethylene glycol, 8 mM TBBPA, and 8 mM E2 and flash frozen in liquid nitrogen.Crystallographic data collection, processing, and structure refinement. Data were collected for all the crystals using a Saturn 92 X-ray detector with a Micromax-007 HF X-ray generator (Rigaku, The Woodlands, TX). The crystallographic data statistics are presented in Supplemental Material, Table S1. All data were indexed and scaled using HKL-2000 data processing software (). The structures were solved using the structure of SULT1E1 [Protein Data Bank (PDB; http://www.rcsb.org/pdb/home/home.do) ID 1G3M ()] as a starting model. Reference Rfree reflections were maintained in all three structures. PHENIX, version 1.8 () and Coot, version 0.6.1 () were used to obtain the structures by iterative cycles of refinement and model building. We assessed model quality using MolProbity, version 1.5.0.2 (). All structural figures were prepared using PyMOL (http://www.pymol.org/).Atomic coordinates and structure factors for the reported crystal structures have been deposited with the Protein Data Bank under PDB IDs 4JVM for the SULT1E1–PAP–TBBPA structure, 4JVN for the SULT1E1–PAP–3-OH-BDE-47 structure, and 4JVL for the SULT1E1–PAP–E2 structure. […]

Pipeline specifications

Software tools HKL-2000, PHENIX, Coot, MolProbity, PyMOL
Application Protein structure analysis
Organisms Dipturus trachyderma, Homo sapiens
Diseases Neurotoxicity Syndromes, Drug-Related Side Effects and Adverse Reactions
Chemicals Estradiol, Estrogens