Year of Award

2018

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Biochemistry & Biophysics

Department or School/College

Department of Chemistry and Biochemistry

Committee Chair

Bruce Bowler

Commitee Members

Stephen Sprang, Kent Sugden, Xi Chu, Travis Hughes (advisor)

Keywords

Conformational ensembles, Ligand Dependent, Nuclear receptors, PPAR, Transcription Factors

Abstract

The nuclear receptor PPARγ is a lipid-dependent transcription factor which regulates many pathways in lipid metabolism. Due to functions in adipogenesis, glucose uptake and insulin sensitivity it has been a target of type II diabetes mellitus therapeutics since the 1990s. Due to a poorly understood anti-inflammatory pathway it is also an emerging target in neurodegenerative disease. Despite extensive research the majority of the structural mechanism underlying the regulation of this proteins’ function is poorly understood. It is often thought that nuclear receptors function in a simple two-state model with defined on and off states. This model only considers one region of the protein, the coregulator interaction surface. In this mechanism ligands function only to modulate the equilibrium population of the active and inactive states. In this work we examine the conformational ensemble of PPARγ in response to the binding of a wide variety of structurally and functionally diverse ligands and find clear evidence that this model is only correct for the most efficacious activating ligand. In all others the ligand induced conformational ensemble is highly diverse and contains multiple populations. Additionally, the mechanisms of inverse agonism and agonism are examined. Through this a novel mechanism of inverse agonism is determined. As well we demonstrate that the mechanistic underpinnings of agonism are not strictly through the coregulator interaction surface. The most efficacious ligands also make important interactions in other regions of the protein which are often disregarded. Among the most interesting findings is the identification of a highly conserved motif on helix 4 of the ligand binding domain of nuclear receptors. We demonstrate that this motif is responsible for a large degree of the specificity observed in coactivator interactions. This provides a substantial amount of insight into the mechanism by which these proteins function as it had previously been observed that coactivator recruitment was exceptionally specific, but no mechanistic reason was known. Through this work we have gleaned a deeper understanding of the mechanism of specific ligand-dependent conformations in this important protein. This fundamental understanding allows for more efficient and intelligent design of drugs which target PPARγ and other nuclear receptors.

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© Copyright 2018 Ian Michael Chrisman