The crystal structures of vitamin D analogues in complex with mutant Vitamin D Receptor (Moras VDR) are carried out in collaboration with Prof. Dino Moras and Natacha Rochel at the IGBMC (Ilkirtch-France). Protein purification and crystallization. zVDR LBD (residues 156-453) are cloned as an N-terminal hexahistidine-tagged fusion protein in pET28b expression vector and overproduced in Escherechia coli BL21 DE3 strain. The protein is purified by affinity chromatography using a cobalt-chelating resin. The protein is incubated with the ligand in presence of a peptide. Crystals of the complex ligand-protein are obtained by vapor-diffusion in hanging drops. X-ray data collection. Data collection from a single frozen crystal is performed by Grenoble (Beamline ID23-European Synchrotron Radiation Facility, ESRF). Structure refinement. A rigid body refinement is used with the structure of the 1,25D-zVDR(LBD) complex as starting model. Refinement involves iterative cycles of manual building and refinement calculations. The programs CNS-SOLVE and O are used for the structure determination and refinement.
The figures below depict the superimposition of the crystal structures of a few vitamin D analogues synthesized in our laboratory in complex with the wild-type VDR(LBD).
Superimposition of the crystal structure of 1 with the hormone 1,25D in the binding pocket (Arch. Biochem. Biophys. 2007, 460(2), 172-176).
Superimposition of the crystal structures of superagonist AMCR277A and AMCR277B in complex with wild VDR(LBD) (Chem. Biol. 2008, 15, 383-392).
Superimposition of the crystal structures of the corresponding 2Me-analogues 2Me-AMCR277A and 2Me-AMCR277B in complex with wild VDR(LBD) (J. Med. Chem. 2010, 53, 1159-1171).
Superimposition of the crystal structures of the new metabolite 3-Epi-1,25-D and 1,25D in complex with mutant VDR(LBD) (PLoS ONE. 2011, 66, e18124).