Computational protocol: Allosteric Modulation of Hormone Release from Thyroxine and Corticosteroid-binding Globulins*

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Protocol publication

[…] All the crystallization studies were based on the TBG-Atl variant. The reactive loop cleaved TBG (cTBG) was concentrated in 10 mm Tris-HCl, 50 mm NaCl, 1 mm EDTA, pH 7.4, and crystallized in sitting drops containing 1 μl of protein (15 mg/ml) and 1 μl of precipitant solution (20% PEG 3350 with 0.2 m CaCl2 or Na2SO4, pH 5.4) at 23 °C for ∼1 week. To soak in thyroxine or other analogues, cTBG crystals were transferred into the corresponding precipitant solution containing corresponding ligands and soaked for ∼12 h. The crystals were cryoprotected with 15% glycerol and flash-cooled in liquid nitrogen. The data set for cTBG (1.5 Å) was collected at Daresbury Synchroton Radiation Source Station 14.1 and the data set for its complex with thyroxine (2 Å) was collected in-house. The data sets for cTBG soaked with T4–6-F, T4–5-F, mefenamic acid, and furosemide were collected at Diamond Light Source station I02. Data were processed by Mosflm () and scaled with Scala (). The initial structure of cTBG was solved by molecular replacement with Phaser () using coordinates of cleaved antitrypsin (Protein Data Bank code 1QMB) () as a search model. One copy of cTBG was found for the cTBG and cTBG-thyroxine structures. The models were built in Coot () and refined with TLS (translation, liberation, screw motion) parameters in Refmac5 (). Subsequent structures of cTBG with different ligands were solved similarly using cTBG as the search model. The T4 fluorescein models were built in Sketcher (CCP4 suite) and the coordinates of other thyroxine analogues were from the CCP4 library. T4–5-CF and T4–6-CF were refined with partial occupancy in the structure. Although both T4 fluorescein analogues can be readily modeled and refined in the structures, the electron densities for the fluorescein moiety are weak, indicating greater flexibility in this region of the ligand. All structures were refined with good geometry, and the final refinement statistics are listed in . The atomic coordinates and structure factors have been deposited in the Protein Data Bank (). The secondary structure was assigned by the program DSSP (). The figures were created using the open source program PyMOL using the following coordinates: native human TBG-thyroxine complex (Protein Data Bank code 2CEO), native rat CBG-cortisol complex (Protein Data Bank code 2V6D), and reactive loop cleaved human CBG (Protein Data Bank codes 2VDX and 2VDY). […]

Pipeline specifications

Software tools Coot, REFMAC5, PyMOL
Application Protein structure analysis
Chemicals Adrenal Cortex Hormones, Calcium Gluconate, Furosemide, Mefenamic Acid, Thyroxine