| Bile acids are the catabolic products of hepatic cholesterol, and play a key role in eliminating excess cholesterol from the body. Moreover, bile acids act as signaling molecules to transcriptionally regulate the homeostasis of cholesterol and bile acid through bile acid receptor-FXR. As a member of the Class II nuclear receptor superfamily, FXR forms a heterodimer with RXR to regulate gene transcription via additional molecular interaction with coregulator proteins. This dissertation presents our work on the biochemical and structural analysis of FXR and its binding interactions. To make the necessary proteins for our studies, we constructed standard protein expression systems for FXR and related receptors, and also a bicistronic expression system for making heterodimers with RXR. In order to study the protein-ligand interaction, we synthesized a fluoresein labeled bile acid molecule. This ligand has provided a powerful tool for measuring direct ligand interactions with FXR, FXR mutants and the FXR-RXR heterodimer. Moreover, we demonstrate the usefulness of this molecule in a high-throughput screening assays which look for new FXR ligands. By using purified proteins and a separate fluorescence assay, we also studied how FXR interacts with coactivators and corepressors and to what extend various FXR ligands alter these interactions. Although both CDCA and GW4064 are agonists of FXR, we found that these ligands regulate the activation of FXR in different ways. On the basis of our biochemical findings, we crystallized the FXR ligand binding domain complex with bile acid homologs and coactivator peptides. Bile acids are amphipathic molecules with a distinctive A/B Juncture. These properties are utilized by FXR in the ligand discrimination. A π-cation interaction between a His-Trp pair was identified to be required for the state in which FXR is able to bind to coactivators. Furthermore, we found that two coactivator peptides could cooperatively bind to one FXR LBD at the same time. The implication of these discoveries was further explored by detailed stereochemical comparisons of our FXR structures with other steroid-receptor complexes. We found that the ligand binding pocket is quite adaptable and accommodating for ligand of various size and chemical structure. This comparison also allowed us to identify for the first time an arginine/hydrophilic residue pair in all steroid receptors that appears to confer ligand specificity through discrimination of the end-to-end distances in steroids. |
