Biology subjects

Brust, R.

Publications and source records attributed to Brust, R..

5 recordsLinked to original sources

bioRxiv2022

Structural basis of interdomain communication in PPARγ

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR{gamma}) regulates transcription via two activation function (AF) regulatory domains: a ligand-dependent AF-2 coregulator interaction surface within the C-terminal ligand-binding domain (LBD), and an N-terminal disordered AF-1 domain (NTD or A/B region) that functions through poorly understood structural mechanisms. Here, we show the PPAR{gamma} AF-1 contains an evolutionary conserved Trp-Pro motif that undergoes cis/trans isomerization, populating two long-lived conformations that participate in intradomain AF-1 and interdomain interactions including two surfaces in the C-terminal LBD ({beta}-sheet and the AF-2 surface), which are predicted in AlphaFold 3 models but not AlphaFold 2. NMR and chemical crosslinking mass spectrometry show that interdomain interactions occur for soluble isolated AF-1 and LBD proteins, as well as in full-length PPAR{gamma} in a phase separated state. Mutation of the region containing the Trp-Pro motif, which abrogates cis/trans isomerization of this region, impacts LBD interaction and reduces basal PPAR{gamma}-mediated transcription and agonist-dependent activation of PPAR{gamma}. Our findings provide structural insight into published in vitro and cellular studies that reported interdomain functional communication between the PPAR{gamma} AF-1 and LBD suggesting some of these effects may be mediated via AF-1/LBD interactions.

biochemistry↗

bioRxiv2019

Quantitative Structural Assessment of Graded Receptor Agonism

Ligand-receptor interactions, which are ubiquitous in physiology, are described by theoretical models of receptor pharmacology. Structural evidence for graded-efficacy receptor conformations predicted by receptor theory has been limited, but is critical to fully validate theoretical models. We applied quantitative structure-function approaches to characterize the effects of structurally similar and structurally diverse agonists on the conformational ensemble of nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR{gamma}). For all ligands, agonist efficacy is correlated to a shift in the conformational ensemble equilibrium from a ground state towards an active state, which is detected by NMR spectroscopy but not observed in crystal structures. For the structurally similar ligands, ligand potency is also correlated to efficacy and conformation, indicating ligand residence times among related analogs can influence receptor conformation and function. Our results derived from quantitative graded activity-conformation correlations provide new experimental evidence and a platform with which to extend and test theoretical models of receptor pharmacology to more accurately describe and predict ligand-dependent receptor activity.

biophysics↗

bioRxiv2018

Structure-function analysis of 1-hydroxypioglitazone, a major in vivo metabolite of the PPARγ agonist pioglitazone

The thiazolidinedione (TZD) pioglitazone (Pio) is an FDA-approved drug for type 2 diabetes mellitus that binds and activates the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR{gamma}). Although TZDs have potent antidiabetic effects, they also display harmful side effects that have necessitated a better understanding of their mechanisms of action. In particular, little is known about the effect of in vivo TZD metabolites on the structure and function of PPAR{gamma}. Here, we present a structure-function comparison of Pio and a major in vivo metabolite, 1-hydroxypioglitazone (PioOH). PioOH displayed a lower binding affinity and reduced potency in coregulator recruitment assays compared to Pio. To determine the structural basis of these findings, we solved an X-ray crystal structure of PioOH bound to PPAR{gamma} ligand-binding domain (LBD) and compared it to a published Pio-bound crystal structure. PioOH exhibited an altered hydrogen bonding network that could underlie its reduced affinity and potency compared to Pio. Solution-state structural analysis using NMR spectroscopy and hydrogen/deuterium exchange mass spectrometry (HDX-MS) analysis revealed that PioOH stabilizes the PPAR{gamma} activation function-2 (AF-2) coactivator binding surface better than Pio. In support of AF-2 stabilization, PioOH displayed stabilized coactivator binding in biochemical assays and better transcriptional efficacy (maximal transactivation response) in a cell-based assay that reports on the activity of the PPAR{gamma} LBD. These results, which indicate that Pio hydroxylation affects both its potency and efficacy as a PPAR{gamma} agonist, contribute to our understanding of PPAR{gamma}-binding drug metabolite interactions and may assist in future PPAR{gamma} drug design efforts.

biochemistry↗

bioRxiv2018

Coincident binding of synthetic and natural ligands to the nuclear receptor PPARγ

Crystal structures of peroxisome proliferator-activated receptor gamma (PPAR{gamma}) have revealed overlapping binding modes for synthetic and natural/endogenous ligands, indicating competition for the orthosteric pocket. Here we show that cobinding of a synthetic ligand to the orthosteric pocket can push natural and endogenous PPAR{gamma} ligands (fatty acids) out of the orthosteric pocket towards an alternate ligand-binding site near the functionally important omega ({Omega}) loop. X-ray crystallography, NMR spectroscopy, all-atom molecular dynamics simulations, and mutagenesis coupled to quantitative functional assays reveal that synthetic ligand and fatty acid cobinding can form a \"ligand link\" to the {Omega} loop and synergistically affect the structure and function of PPAR{gamma}. These findings contribute to a growing body of evidence indicating ligand binding to nuclear receptors can be more complex than the classical one-for-one orthosteric exchange of a natural or endogenous ligand with a synthetic ligand.

biochemistry↗

bioRxiv2018

A structural mechanism for directing inverse agonism of PPARγ

Small chemical modifications can have significant effects on ligand efficacy and receptor activity, but the underlying structural mechanisms can be difficult to predict from static crystal structures alone. Here we show how a simple phenyl-to-pyridyl substitution between two common covalent orthosteric ligands targeting peroxisome proliferator-activated receptor gamma (PPAR{gamma}) converts a transcriptionally neutral antagonist (GW9662) into an inverse agonist (T0070907). X-ray crystallography, molecular dynamics simulations, and mutagenesis coupled to activity assays reveal a water-mediated hydrogen bond network linking the T0070907 pyridyl group to Arg288 that is essential for inverse agonism. NMR spectroscopy reveals that PPAR{gamma} exchanges between two long-lived conformations when bound to T0070907 but not GW9662, including a conformation that prepopulates a corepressor-bound state, priming PPAR{gamma} for high affinity corepressor binding. Our findings demonstrate that ligand engagement of Arg288 may provide new routes for developing PPAR{gamma} inverse agonist.

biochemistry↗