Sidues 50-212) (Z)-4-Hydroxytamoxifen site containing the F185K mutation were expressed in E.
Sidues 50-212) containing the F185K mutation were expressed in E. coli and purified as described [37].Crystallization and X-ray crystal structure determinationThe absolute binding free energies between the various forms of IN CCD dimer and BI-D were calculated using the double decoupling method (DDM) [65-68] in explicit solvent (TIP3P water model [69] plus counterions) at 300 K. The protein molecules are modeled by the Amber ff99sb-ILDN force field [70], and the ligand BI-D is described by the Amber GAFF [71] parameters set. The partial charges of the ligands are obtained using the AM1-BCC method [72]. A DDM calculation involves two legs of simulation, in which a restrained ligand is gradually decoupled from the receptor binding pocket or from the aqueous solution. In each leg of the decoupling simulations, the Coulomb interaction is turned off first using 11 lambda windows, and the Lennard-Jones interactions are then turned off in 17 lambda windows. The two decoupling free energies Ggas*complex and Ggaswater associated with the two legs of the DDM cycle were determined using thermodynamic integration (TI). The Hamiltonian derivative U/ at a series of l from 0 to 1 were collected and integrated to obtain the free energy difference. For absolute binding free energy calculations, the MD simulation at each was performed using the GROMACS [73,74] version 4.6.4 for 15 ns; the last 10 ns was used for the calculation of binding free energy.SECRecombinant H171T IN CCD (50-212) containing the F185K solubilizing substitution was prepared to 8 mg/ml and grown at 4 using hanging drop vapor diffusion method. The crystallization buffer contains 8 PEG 8 K, 0.1 M Na cacodylate, pH 6.5, 0.1 M ammonium sulfate and 5 mM DTT. Protein (1 l) was mixed with the equal volume of the crystallization buffer. Within 4 weeks the cubic shape crystals reached 0.1- 0.2 mm in size. The soaking buffer containing 5 mM BI-D was prepared by dissolving the compound in crystallization buffer supplemented with 10 DMSO. The protein crystal was soaked in the buffer for 12 hrs at 4 before it was flash-frozen with liquid nitrogen. Diffraction data from the crystals were collected at 100 F on a Rigaku Raxis 4++ image plate detector in OSU Crystallography Facility. The intensity data integration and reduction were PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28242652 performed with HKL2000 program [61]. Molecular Replacement program Phaser [62] in CCP4 package method was used to solveA Superdex 200 10/300 GL column (GE Healthcare) was used to analyze multimeric forms of recombinant WT and H171T INs in buffer containing 20 mM HEPES (pH 6.8), 750 mM NaCl, 10 mM MgSO4, 0.2 mM EDTA, 5 mM BME, 5 glycerol and 200 M ZnCl2. The following proteins were used to calibrate the column: bovine thyroglobulin (670,000 Da), bovine gamma-globulin (158,000 Da), chicken ovalbumin (44,000 Da), horse myoglobin (17,000 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26080418 Da) and vitamin B12 (1,350 Da). Proteins were detected by absorbance at 280 nm. All the procedures were performed at 4 .DLSDLS experiments were carried out as previously reported [44]. Briefly, DMSO, 0.12 M BI-D, or 10 M BI-D was incubated with 200 nM WT or H171T IN. DLS signals wereSlaughter et al. Retrovirology 2014, 11:100 http://www.retrovirology.com/content/11/1/Page 12 ofrecorded at room temperature after 15, 20 and 30 minutes using a Malvern Nano series Zetasizer instrument.SPRAuthors’ contributions MK, AE, RML, EP and JRF designed the study. AS, KJ, LF, JJK and RCL carried out the experiments. ND performed binding free energy c.