Deuterium exchange and mass spectrometry reveal the interaction differences of two synthetic modulators of RXRα LBD

Abstract

Protein amide hydrogen/deuterium (H/D) exchange was used to compare the interactions of two antagonists, UVI 2112 and UVI 3003, with that of the agonist, 9‐ cis ‐retinoic acid, upon binding to the human retinoid X receptor alpha ligand‐binding domain (hRXRα LBD) homodimer. Analysis of the H/D content by mass spectrometry showed that in comparison to 9‐ cis ‐retinoic acid, the antagonists provide much greater protection toward deuterium exchange‐in throughout the protein, suggesting that the protein–antagonist complex adopts a more restricted conformation or ensemble of conformations in which solvent accesses to amide protons are reduced. A comparison between the two antagonists shows that UVI 3003 is more protective in the C‐terminal region due to the extra hydrophobic interactions derived from the atoms in the benzene ring of the carboxylic acid chain. It was less protective within regions comprising peptides 271–278 and 326–330 due to differences in conformational orientation, and/or shorter carboxylic acid chain length. Decreased deuterium exchange‐in in the segment 234–239 where the residues do not involve interactions with the ligand was observed with the two antagonists, but not with 9‐ cis ‐RA. The amide protons of helix 12 of the agonist‐ or antagonist‐occupied protein in solution have the same deuterium exchange rates as the unliganded protein, supporting a suggestion made previously that helix 12 can cover the occupied binding cavity only with the cofactor present to adjust its location.