The orphan nuclear pregnane X receptor (PXR) regulates the expression of numerous genes vital to xenobiotic and bile acid metabolism. Such genes include cytochrome P450-3A4, which metabolizes greater than 50 percent of human drugs, and the drug efflux pump MDR1. In contrast to most nuclear receptors, PXR is promiscuous and responds to a wide variety of structurally- diverse drugs and endogenous compounds. To unravel the structural basis of PXR's promiscuity and function, we determined crystal structures of the ligand binding domain of human PXR (hPXR) by itself and in complex with the cholesterol-lowering drug SR12813. hPXR has a novel nuclear receptor fold and contains a large, elliptical ligand binding pocket. Surprisingly, this pocket allows SR12813 to bind in three orientations at once -- a direct observation of binding promiscuity. These results provide a structural framework for further examination of this receptor. We are now poised to address two fundamental questions regarding PXR. First, how does this single protein respond to such a wide variety of distinct compounds? Second, how does PXR mediate the signal to activate transcription? The goals of these investigations are to elucidate PXR's role in molecular endocrinology and drug metabolism, and to facilitate the development of novel methods for drug screening involving PXR. Four hypotheses will be tested: - The positioning of PXR surface residues and the binding of the co-activator SRC-1 to PXR mediate transcriptional activation by stabilizing a particular conformation of the receptor. - PXR uses shuffled polar interactions to allow small ligands to bind in multiple orientations. - PXR changes in structure to accommodate large ligands. - Polar residues in the ligand binding pocket of PXR dictate the """"""""directed promiscuity"""""""" exhibited by PXRs across species.
Six specific aims will be pursued using the tools of x-ray crystallography, molecular biology and biochemistry: 1. Determine crystal structures of hPXR in complex with fragments of the transcriptional co-activator SRC-1. 2. Examine structures of hPXR variants containing surface changes known to affect its ability to activate transcription. 3. Unravel PXR's promiscuity by examining mutations designed to impact the binding of SR12813. 4. Determine structures of hPXR in complexes with small drugs and endogenous compounds. 5. Elucidate structures of hPXR in complexes with large drugs like rifampicin and taxol. 6. Examine structures of mouse and rabbit PXR to determine why different species respond to distinct xenobiotics.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Endocrinology Study Section (END)
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Margolis, Ronald N
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University of North Carolina Chapel Hill
Schools of Arts and Sciences
Chapel Hill
United States
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