Single-Molecule Insights into Allosteric Regulation of the Nuclear Receptor Protein RXRα

Abstract

Nuclear receptors are crucial transcription factors that respond to a broad range of external signaling ligands by binding cognate DNA response elements and switching on transcription. As such, these proteins form tightly regulated, multi-component allosteric systems. RXRα is one of the key players in this protein family for its role as a promiscuous heterodimerization partner. We employ single-molecule fluorescence techniques to investigate the conformational dynamics of RXRα's ligand binding domain (LBD)—the essential regulatory domain. It has been proposed that ligand binding changes the conformation of helix 12 of the LBD and positions it in a conformation favoring coactivator peptide binding. Our single-molecule FRET studies, however, demonstrate that helix 12 undergoes a substantial conformational change only after the coactivator peptide binds. This suggests an unexpected allosteric mechanism of nuclear receptor activation, which we investigate. In particular, we address the questions: on what time scales do the conformational changes of helix 12 take place? and how are its dynamics affected by modulations in other parts of RXRα and in its binding partners? Our studies will pave the way to better understanding of the complex regulatory machinery embodied in nuclear receptor function.