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Hydrogen/deuterium-exchange (H/D-Ex) of PPAR LBD in the presence of various modulators

¹ ExSAR Corp., Monmouth Junction, New Jersey 08852, USA
² Sierra Analytics Inc., Modesto, California 95355, USA

(RECEIVED January 19, 2006; FINAL REVISION May 10, 2006; ACCEPTED May 15, 2006)

A nuclear receptor, peroxisome proliferator-activated receptor (PPAR), is a ligand-dependent transcription factor involved in glucose homeostasis and adipocyte differentiation. PPAR is the molecular target of various natural and synthetic molecules, including anti-diabetic agents such as rosiglitazone. Amide hydrogen/deuterium-exchange (H/D-Ex), coupled with proteolysis and mass spectrometry, was applied to study the dynamics of the PPAR ligand binding domain (LBD) with or without molecules that modulate PPAR activity. The H/D-Ex patterns of ligand-free PPAR LBD show that the ligand binding pocket of LBD is significantly more dynamic than the rest of the LBD. Presumably, the binding pocket is intrinsically disordered in order to accommodate different ligands. The presence of two full agonists (rosiglitazone and GW1929), a partial agonist (nTZDpa), and a covalent antagonist (GW9662), changed the dynamics/conformation of PPAR LBD and slowed the H/D exchange rate in various regions of the protein. The full agonists slowed the H/D exchange more globally and to a greater extent than the partial agonist or the antagonist, indicating that the full agonist stabilizes the PPAR LBD more than the partial agonist or the antagonist. One interesting observation is that the two full agonists significantly stabilized helix 12 while the partial agonist and the antagonist did not perturb the H/D exchange of this region. The results showed that the change in protein dynamics induced by ligand binding may be an important factor for the activation of genes and that H/D-Ex is a useful method for analyzing the biological activity of drug leads.

Keywords: hydrogen/deuterium exchange; mass spectrometry; PPAR; protein dynamics; nuclear receptor

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