CAREER: The Nature of Interdomain Coupling in the Farnesoid X Receptor

Many hormones and other signaling molecules exert their potent effects by regulating which genes are turned “on” by a process known as transcription. A large family of so-called transcription factors with a similar architecture that includes separate domains for ligand (the hormone/signaling molecule) and DNA binding are known as nuclear hormone receptors. When these proteins bind their ligands, they interact with a specific DNA sequence and allow the machinery necessary to “read” the gene to assemble and proceed. The transcription factors are flexible and the relative positions of the domains can vary dramatically. The PI proposes to understand how the individual domains communicate with each other, so that the DNA-recognition segment can sense that the ligand binding domain is occupied, or how the DNA binding domain might impact the ligand binding domain. Their highly flexible nature is thought to allow for precise biological control of transcription, given that a single member of this family can have distinct responses to very similar ligands. These studies will reveal the key molecular details that drive crucial physiological processes. Additionally, this work will achieve broader impacts by providing educational materials and research training. Curricular materials will be generated to promote teaching and learning of protein dynamics in K-12 and undergraduate courses, while research training will be provided for high school teachers, undergraduate and graduate students. This proposal aims to generate a molecular understanding of how the ligand binding (LBD) and DNA binding (DBD) domains of the farnesoid X receptor (FXR) interact to regulate transcription. The mechanisms of crosstalk between these two domains are uncharacterized in FXR and other nuclear receptors (NR). Thus novel, creative approaches are required to understand how interdomain communication influences transcription. This work will combine molecular experiments with biophysical approaches and computational modeling. This integrative approach will allow molecular-level perturbations of FXR to be linked to structure and dynamic motions, subsequently revealing how FXR function is uniquely regulated and modulated by ligand and/or DNA binding. Molecular dynamics (MD) simulations to model interdomain interactions in various ligand- and DNA-bound states will be followed by hydrogen-deuterium exchange mass spectrometry to experimentally reveal interaction surfaces between the two domains. Small angle X-ray scattering will be used to generate low resolution models of the quaternary structure of FXR. Luciferase reporters of transactivation and nuclear magnetic resonance spectroscopy (NMR) will be used to determine how perturbations of the LBD modulate function in the DBD. Finally, NMR, MD simulations and binding assays will be performed to characterize bidirectional allostery in FXR. By carefully determining how the functions of the NR domains are coupled, this work will provide new insights into how ligands dictate transcriptional outcomes. Importantly, because of the highly conserved functional mechanisms that govern NRs, these studies with FXR will be informative with respect to allosteric mechanisms common to the NR family. This research is funded by the Molecular Biophysics program in the Division of Molecular and Cellular Biosciences in the Directorate of Biological Sciences. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.