The C-terminal activation function-2 (AF-2) helix plays a crucial role in retinoid X receptor alpha (RXR alpha)-mediated gene expression. Here, we report a nuclear magnetic resonance (NMR) study of the RXR alpha ligand-binding domain complexed with 9-cis-retinoic acid and a Rlucocorticoid receptor-interacting protein 1 peptide. The AF-2 helix and most of the C-terminal residues were undetectable due to a severe line-broadening effect. Due to its outstanding signal-to-noise ratio, the C-terminus residue, threonine 462 (T462) exhibited two distinct crosspeaks during peptide titration, suggesting that peptide binding was in a slow exchange regime on the chemical shift time-scale. Consistently, the K-d derived from T462 intensity decay agreed with that derived from isothermal titration calorimetry. Furthermore, the exchange contribution to the N-15 transverse relaxation rate was measurable in either T462 or the bound peptide. These results suggest that T462 is a sensor for coactivator binding and is a potential probe for AF-2 helix mobility. Published by Elsevier Inc.