Intrinsic Disorder and Allostery in Glucocorticoid Receptor
MetadataShow full item record
Intrinsically disordered (ID) regions of proteins, lacking stable tertiary structure, are malleable and sensitive regulators of cell functions. Allostery is transmittance of a perturbation at one region to distant sites of the same molecule, allowing for precise control of macromolecular function. Nature preferentially uses both ID regions and allostery to regulate protein function, as observed in transcription factors. This motivated us to investigate whether and how ID regions can facilitate allostery. The classic allosteric models all feature a static structural and single molecule view of allostery and are not applicable to study allostery mediated by ID regions. To address this limitation, our group has developed the Ensemble Allosteric Model (EAM), which views allostery as an effector binding driven shift in ensemble probabilities. It is a thermodynamic and quantitative model that can be applied to structured proteins, ID proteins and mixed proteins. In this thesis project, I investigated the intra- and inter- domain allostery mediated by the ID regions in human glucocorticoid receptor (GR). Through thermodynamic and functional studies on eight GR translational isoforms, I found that the ID GR N terminal domain (NTD) is composed of two functionally distinct regions, unfavorably coupled with each other, and both of them are favorably coupled to the DNA binding domain (DBD). Based on these experimental constraints, an EAM was built for GR and reasonable thermodynamic parameter combinations were found to describe both the transcriptional activity and binding affinity of different translational isoforms. We found that GR uses these competing energetic couplings, which are modulated in different translational isoforms, to provide tunable responses to environmental cues. In the context of the EAM predictions, mutagenesis was carried out in different regions of GR and the influence on transcriptional activity and binding affinity was assayed to pinpoint the molecular basis of allostery, which paves a way for allosteric drug design. This study suggests a unifying strategy to investigate thermodynamics and the molecular basis of allostery in any complicated system.