Computational protocol: The “Phantom Effect” of the Rexinoid LG100754: Structural and Functional Insights

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[…] The crystals were mounted in fiber loops and flash-cooled in liquid nitrogen after cryoprotection with the reservoir solution plus 5% ethylene glycol. Of 100 crystals tested, only one diffracted to 2.75 Å. Data collection from the frozen crystal was performed at 100 K on the beamline ID23-1 at the ESRF (Grenoble, France). The crystal belongs to the tetragonal space group P43212, with one heterodimer per asymmetric unit. The data were integrated and scaled using HKL2000 (statistics in ). The structure was solved by molecular replacement using the program AMoRe . The structure of the antagonist-bound heterodimer (PDB ID: 1DKF) was used as a starting model. Refinement involved iterative cycles of manual building and refinement calculations. The programs CNS , REFMAC , phenix.refine , O and COOT were used throughout structure determination and refinement. Several terminal residues and 13 residues between H1 and H3 of RXR (251-263) are not modelled as the electron density map was poor in the corresponding regions. The TIF-2 peptide and the ligand molecules were only included at the last stage of the refinement. The omit map from the refined atomic model of the heterodimer was used to fit the peptide and ligands to their electron densities (). Anisotropic scaling, a bulk solvent correction and TLS restraints were used for the refinement. Seven TLS groups for each LBD and one group for the peptide were generated by using the program TLSMD . Individual atomic B factors were refined isotropically. Solvent molecules were then placed according to unassigned peaks in the electron density map. In the RARα-atRA/RXRα-LG100754 LBDs and TIF-2 peptide complex, refined at 2.75 Å with no σ cutoff, the final model contains 239 residues (177–415) for hRARα LBD, 218 residues (230–250, 265–461) for mRXRα LBD, 11 residues for TIF-2 peptide, 2 ligand molecules, and 115 water molecules. According to PROCHECK , 91.5% of the peptide lies in the core regions, 8.2% in the allowed regions and 0.2% in the generous region. The peptide classified in the generous region of the Ramachandran plot is around Asp449 of RXR. Since this peptide is located at the loop between H11 and H12, such unfavourable conformation is adapted due to intermolecular interaction imposed on H12 by crystal packing (described below). Data are summarized in . The volumes of the ligand-binding pockets and ligands were calculated by using the program VOIDOO and GRASP , respectively. Structural figures were generated by using the Pymol program and CCP4MG . [...] To quantify the electrostatic and van der Waals contributions to dimer association, a free energy decomposition analysis was performed on the present structure, as well as on the fully agonists and fully antagonists conformations –. Starting from the crystal structures hydrogen atoms were added using the HBUILD module of the CHARMM (Chemistry at HARvard Macromolecular Mechanics) program . The structures were energy minimized and subsequently used in a MM/PBSA decomposition protocol. Details of the protocol are described in reference . Docking of the ligand LG100754 in the binding pocket of hRARα LBD in agonist conformation was performed with Autodock 4.0 using standard input parameters. [...] The synchrotron radiation X-ray scattering data were collected at the storage ring DORIS III of the Deutsches Elektronen Synchrotron (DESY) . The scattering patterns were recorded in the range of momentum transfer 0.15

Pipeline specifications

Software tools CHARMM, AutoDock
Application Protein interaction analysis
Diseases Hyperlipoproteinemia Type II
Chemicals Tretinoin