The conformational analysis and biophysical studies of retinoic acid interactions with nuclear receptor homodimers and heterodimers

Vitamin A metabolites are compounds that perform critical functions for mammalian life. The retinoic acid metabolites of vitamin A regulate genetic transcription. Retinoic acids function by binding to type II nuclear receptor proteins (NRs), which modulates interactions between nuclear receptors and transcription initiation machinery (such as coactivator proteins). It is the focus of this dissertation to expand our understanding of retinoic acids in their interactions with the nuclear receptor protein Retinoid X Receptor alpha (RXRalpha).;Retinoic acids are composed of a trimethylcyclohexenyl ring, polyene chain, and terminal carboxylic acid. The first chapter investigates the energetics of the ring-chain conformation using computational methods. The lowest energy conformer of retinoic acids was found to be a distorted geometry. The steric interactions that favor the distorted conformer over the planar conformer were determined using model molecules. We also explored how ring inversion, polyene chain isomerization, and nature of polar end groups affected the relative energies of the low-energy conformers.;RXRalpha forms heterodimers partnered with other NRs (such as Peroxisome Proliferator-Activated Receptor gamma, PPARgamma), and the heterodimer is responsible for modulating activity of target genes. The 9-cis retinoic acid (9cRA) binds to RXRalpha and regulates its ability to recruit coactivator proteins. The second chapter in this dissertation compares the binding mode of 9cRA to RXRalpha homodimers with 9cRA binding to RXRalpha/PPARgamma ligand binding domain heterodimers. The results indicate that the 9cRA binding affinity and conformation is similar in RXRalpha homodimers and RXRalpha/PPARgamma heterodimers, but the surrounding protein environment may differ. We also compare the binding mode of the PPAR agonist Rosiglitazone to homodimers and heterodimers.;The third chapter investigates how the presence of 9cRA affects the binding of coactivator protein models (coactivator peptides that model the LxxLL motif) to RXRalpha homodimers and RXRalpha/PPARgamma heterodimers by isothermal titration calorimetry. Two coactivator peptides were studied and found to bind differently to RXRalpha and PPARgamma These studies found that the presence of 9cRA increased the stoichiometry of dimer -- peptide binding for both RXRalpha homodimers and RXRalpha/PPARgamma heterodimers. Also, the capability of PPARgamma to bind coactivator peptides depended largely on the peptide sequence about the LxxLL motif.