Conformational Ensembles from Experimental Data and Computer Simulations

Conformational Ensembles from Experimental Data and Computer Simulations

Poster Abstracts

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.

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