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Introduction: Nuclear receptor transcription factors such as peroxisome proliferator-activated receptor gamma (PPARγ) are the molecular targets of drugs for the treatment of a wide range of diseases. Ligand-induced changes in structure/dynamics of the receptor drive dissociation of co-repressors, heterodimer formation, co-activator binding, and subsequent transcriptional activity. Characterization of the dynamics of the receptor is critical to understanding the mechanism of activation.
Methods: Hydrogen/deuterium (H/D) exchange is a technique for measuring protein dynamics. Here we describe a two-part approach to generate a high-throughput (<1 h/compound) H/D method to measure changes in the dynamics of the receptor ligand-binding domain (LBD). First, a comprehensive set of differential H/D data are acquired with a number of well-characterized ligands (~24 h/compound). From these data, a single period of H/D on-exchange is chosen that provides the maximal distance between dynamics of ligand-bound and apo-receptor. Next, the chromatography is optimized for the rapid elution of a subset of peptides that span the LBD. To aid this step, we incorporated 1.9-μm stationary phase HPLC columns into our H/D exchange platform. Single time point H/D exchange experiments are then used to probe the dynamics of the receptor.
Results: Comprehensive H/D exchange experiments were performed with five ligands that induce varying degrees of PPARγ activity (rosiglitazone, MRL-20, MRL-24, nTZDpa, BVT.13). For all regions of the protein, 60 sec on-exchange provided the maximum difference between the apo- and ligand-bound receptor. Fifteen peptides were selected that span the LBD. A chromatography method was developed that allowed for the separation of the peptides of interest in <10 min. We then proceeded to characterize 27 ligands in a 24-h time period.
Conclusions: We have developed an H/D exchange method for rapid characterization of ligand binding to PPARγ LBD. The approach is suited to the analysis of other nuclear receptors.