Deuterium exchange and mass spectrometry reveal the interaction differences of two synthetic modulators of RXRalpha LBD.
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 (hRXRalpha 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.
Yan
Xuguang
X
Department of Chemistry, Oregon State University, Corvallis, OR 97331, USA.
Pérez
Efrén
E
Leid
Mark
M
Schimerlik
Michael I
MI
de Lera
Angel R
AR
Deinzer
Max L
ML
D000595:Amino Acid Sequence; D001665:Binding Sites; D003903:Deuterium / Q000737:chemistry; D019281:Dimerization; D006801:Humans; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods*; D008956:Models, Chemical; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D047490:Retinoid X Receptor alpha / Q000737:chemistry*; D013997:Time Factors
D008024:Ligands; D047490:Retinoid X Receptor alpha; D003903:Deuterium