Conformational Ensembles from Experimental Data and Computer Simulations

Conformational Ensembles from Experimental Data

and Computer Simulations

Poster Abstracts

104

69-POS

Board 29

Coregulators Select Conformational States from Drug Specific Peroxisome Proliferator-

activated Receptor Gamma (PPARγ) Conformational Ensembles

Michelle Nemetchek

1,2,3

, Ian M. Chrisman

1,2

, Travis S. Hughes

1,2,3

The University of Montana, Missoula, MT, USA,

The University of Montana, Missoula, MT,

USA,

The University of Montana, Missoula, MT, USA.

Nuclear receptors are transcription factors that, when bound to agonists, cause transcription of

regulatory genes. Peroxisome Proliferator-activated receptor gamma (PPARγ) is a nuclear

receptor that is an important target of many FDA-approved type II diabetes drugs. When agonist

bound, PPARγ recruits the coactivator Mediator complex subunit 1 (MED1), which is part of the

mediator complex and triggers transcription of PPARγ-regulated genes. These genes control

adipogenesis and lipid storage, and may enhance insulin sensitivity. When bound to inverse

agonists, PPARγ complexes with Nuclear Receptor Corepressor I (NCoR1) and is blocked from

transcriptional activation. By using Time-Resolved Förster Resonance Energy Transfer (TR-

FRET), PPARγ binding drugs are shown to produce differing affinities for MED1 and NCoR1

peptides which contain the essential nuclear receptor binding motif. We covalently label PPARγ

with a fluorine containing probe on the AF2 surface (the surface where coregulators have been

demonstrated to interact) and use Fluorine-19 (19F) Nuclear Magnetic Resonance to determine

how coregulator binding affects this surface. The AF2 surface conformations are highly similar

for both full agonist bound and full agonist and MED1 co-bound states, similarly inverse

agonists induce conformations that are relatively unchanged by NCoR1 binding. Our data also

suggest that coregulators select conformations from a preexisting ensemble which is highly drug

specific. This work links structure of the LBD to the functional outcome of transcription.