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Conformational Ensembles from Experimental Data
and Computer Simulations
Poster Abstracts
45
12-POS
Board 12
Examining the Conformational Ensemble in the Peroxisome Proliferation-activating
Receptor Gamma (PPARγ) Ligand Binding Domain
Ian M. Chrisman
1
, Michelle D. Nemetchek
1
, Travis S. Hughes
1,2,3
.
1
University of Montana, Missoula, MT, USA,
2
University of Montana, Missoula, MT, USA,
3
University of Montana, Missoula, MT, USA.
Relatively little is known about the conformational ensemble in the ligand binding domains
(LBD) of nuclear receptors, a type of transcription factor. They are often assumed to exist in
two-state systems where ligand binding causes conformational change to direct transcriptional
activation or repression. In this study we employ 19F NMR and chemical exchange saturation
transfer (CEST) to examine the conformational states of the LBD of the nuclear receptor PPARγ,
in both an apo state as well as bound to a variety of drugs. Fluorine NMR is employed through
the covalent attachment of a fluorine probe to a cysteine introduced in the AF2 region, a region
of the protein important for binding other effector proteins (coregulators). In an apo state the
AF2 region exists in a wide variety of conformations which are in intermediate exchange. These
conformations are the components of two distinct NMR peaks which are in slower exchange.
Molecular simulations suggest the possible structure of the faster exchanging conformations.
With all ligands examined there exist two or more conformations of the LBD as determined by
objective deconvolution of the NMR signal into Lorentzian populations. This is seen even in the
presence of covalent ligands. We observe very slow exchange (<1s-1) between conformations
detected as well separated 19F NMR peaks and differences in the 19F probe solvent exposure in
these distinct conformations. Such results indicate that the conformational ensemble of PPARγ
LBD supports multiple distinct conformations in a single protein state and a two-state protein
conformation model may be unsuitable for studying of this and other nuclear receptors. Finally,
there is a significant correlation between conformation chemical shift and coregulatory peptide
binding, indicating that distinct NMR observed conformations are also functionally distinct.