Structure and mechanism of human vitamin D receptor (hVDR) with its inhibitors
Mentor 1
Nicholas Silvaggi
Location
Union Wisconsin Room
Start Date
24-4-2015 2:30 PM
End Date
24-4-2015 3:45 PM
Description
The human vitamin D receptor (hVDR) is a ligand-dependent transcriptional regulator that controls calcium metabolism, cell growth and differentiation, homeostasis, development, and several physiological processes. The antiproliferative effect of VDR on cancer cell growth has been demonstrated. Known inhibitors with high binding affinity exist, but induce hypercalcemia and hypercalcinuria, which can cause psychosis, bone pain, calcification of soft tissue, and coronary artery disease. A compound developed in the Arnold laboratory, PS121912, inhibits the binding of hVDR with its coregulator, SRC-2, and induces apoptosis in cancer cells without causing hypercalcemia. We are working to determine how PS121912 inhibits the interaction between hVDR and SRC-2 using X-ray crystallography. Crystallization requires large amounts of protein, so significant work has been done to optimize the heterologous expression of hVDR in E. coli, as well as the purification method. Here we describe our optimized expression and purification methods, as well as efforts to grow diffraction-quality crystals of hVDR. Crystals have been soaked in solutions containing 1 mM PS121912 or other hVDR inhibitors, and diffraction data were collected at the Advanced Photon Source, Argonne National Laboratory. PS121912 and its analogs are thought to bind covalently to Cys, Ser, or Tyr residues due to the presence of a Michael acceptor group in the inhibitor. This group would also react with the reducing agent dithithreitol (DTT), which is present in the crystallization solution. Since DTT may affect the ligand, we tested different DTT concentrations to find the minimum amount that would still give good quality crystals. It was found that DTT is essential for hVDR-LBD to crystallize, and that the concentration must be greater than 1 mM.
Structure and mechanism of human vitamin D receptor (hVDR) with its inhibitors
Union Wisconsin Room
The human vitamin D receptor (hVDR) is a ligand-dependent transcriptional regulator that controls calcium metabolism, cell growth and differentiation, homeostasis, development, and several physiological processes. The antiproliferative effect of VDR on cancer cell growth has been demonstrated. Known inhibitors with high binding affinity exist, but induce hypercalcemia and hypercalcinuria, which can cause psychosis, bone pain, calcification of soft tissue, and coronary artery disease. A compound developed in the Arnold laboratory, PS121912, inhibits the binding of hVDR with its coregulator, SRC-2, and induces apoptosis in cancer cells without causing hypercalcemia. We are working to determine how PS121912 inhibits the interaction between hVDR and SRC-2 using X-ray crystallography. Crystallization requires large amounts of protein, so significant work has been done to optimize the heterologous expression of hVDR in E. coli, as well as the purification method. Here we describe our optimized expression and purification methods, as well as efforts to grow diffraction-quality crystals of hVDR. Crystals have been soaked in solutions containing 1 mM PS121912 or other hVDR inhibitors, and diffraction data were collected at the Advanced Photon Source, Argonne National Laboratory. PS121912 and its analogs are thought to bind covalently to Cys, Ser, or Tyr residues due to the presence of a Michael acceptor group in the inhibitor. This group would also react with the reducing agent dithithreitol (DTT), which is present in the crystallization solution. Since DTT may affect the ligand, we tested different DTT concentrations to find the minimum amount that would still give good quality crystals. It was found that DTT is essential for hVDR-LBD to crystallize, and that the concentration must be greater than 1 mM.
