Dynamics and ligand-induced solvent accessibility changes in human retinoid X receptor homodimer determined by hydrogen deuterium exchange and mass spectrometry.

Receptors for retinoic acid act as ligand activated transcription factors. The three-dimensional structure of the retinoid X receptor (RXR) ligand binding domain has been determined, but little information is available concerning the properties of the protein in solution. Hydrogen/deuterium exchange followed by electrospray ionization mass spectrometry was used to probe the solution conformation of the recombinant human RXRalpha homodimer ligand binding domain in the presence and absence of 9-cis-retinoic acid (9-cis-RA). Within the experimental time domain (0.25-180 min), about 20 amide hydrogens showed decreased exchange rates in the presence of saturating concentrations of 9-cis-RA as compared to those found for the homodimer in the absence of ligand. Most of the amides were located in peptides derived from regions of the protein shown by the X-ray structure to interact with the bound ligand: the amino termini of helices 3 and 9, the two beta sheets, helix 8, the H8-H9 loop, and the carboxyl terminus of helix 11. Unexpectedly, protection was also observed in peptides derived from helices 7, 10, 11, and the H7-H8 and H10-H11 loops, regions that are not directly in contact with bound 9-cis-RA. These results suggest that the binding of ligand results in additional effects on the conformation or dynamics of the homodimer in solution as compared to those observed for the X-ray structure. Overall, the change in deuterium exchange induced by the binding of 9-cis-RA correlated reasonably well with changes in hydrogen bonding, residue depth, and/or solvent accessibility predicted from the crystal structure.