Defining a conformational ensemble that directs activation of PPAR\u03b3

Ian M Chrisman,Jakob Fuhrmann,Jinsai Shang,Zahra Heidari,Anne-Laure Blayo,Yanan Long,Youseung Shin,Michelle D Nemetchek,Douglas J Kojetin,Thomas E Cheatham,Hermes Reyes-Caballero,Ian Mitchelle S De Vera,Travis S Hughes,Theodore M Kamenecka,Rodrigo Galindo-Murillo,Patrick R Griffin

doi:10.1038/s41467-018-04176-x

Abstract

The nuclear receptor ligand-binding domain (LBD) is a highly dynamic entity. Crystal structures have defined multiple low-energy LBD structural conformations of the activation function-2 (AF-2) co-regulator-binding surface, yet it remains unclear how ligand binding influences the number and population of conformations within the AF-2 structural ensemble. Here, we present a nuclear receptor co-regulator-binding surface structural ensemble in solution, viewed through the lens of fluorine-19 (19F) nuclear magnetic resonance (NMR) and molecular simulations, and the response of this ensemble to ligands, co-regulator peptides and heterodimerization. We correlate the composition of this ensemble with function in peroxisome proliferator-activated receptor-γ (PPARγ) utilizing ligands of diverse efficacy in co-regulator recruitment. While the co-regulator surface of apo PPARγ and partial-agonist-bound PPARγ is characterized by multiple thermodynamically accessible conformations, the full and inverse-agonist-bound PPARγ co-regulator surface is restricted to a few conformations which favor coactivator or corepressor binding, respectively.

Highlights

The nuclear receptor ligand-binding domain (LBD) is a highly dynamic entity
Using 19F nuclear magnetic resonance (NMR) combined with biochemical co-regulator interaction analysis and molecular simulations, we define the ligand-dependent conformational ensemble of the co-regulator interaction surface, including helix 12, which controls the transcriptional activity of peroxisome proliferator-activated receptor-γ (PPARγ)
In a time-resolved fluorescence resonance energy transfer (TR-FRET) co-regulator interaction assay, full agonists such as GW1929 and rosiglitazone that induce robust PPARγ transcription[27, 28] increase binding of a peptide derived from mediator of RNA polymerase II transcription subunit 1 (MED1) coactivator (Fig. 1a), and decrease binding of a peptide derived from the nuclear receptor corepressor 1 (NCoR1) referred to as NCoR (Fig. 1b)

Summary

Introduction

The nuclear receptor ligand-binding domain (LBD) is a highly dynamic entity. Crystal structures have defined multiple low-energy LBD structural conformations of the activation function-2 (AF-2) co-regulator-binding surface, yet it remains unclear how ligand binding influences the number and population of conformations within the AF-2 structural ensemble. Crystal structures, HDX-MS, and protein NMR have provided complementary information revealing a relationship between structure and function for PPARγ (e.g., the presence or absence of critical hydrogen bonds between ligand and helix 1220); a direct observation of the ligand-dependent ensemble implied by these data is lacking. This raises the question: are there multiple long-lived conformations that correlate with functional efficacy (e.g., co-regulator affinity) in nuclear receptors?. These data better elucidate how ligands induce functional effects via nuclear receptors

Methods

Results

Conclusion