The RXRalpha C-terminus T462 is a NMR sensor for coactivator peptide binding.
The C-terminal activation function-2 (AF-2) helix plays a crucial role in retinoid X receptor alpha (RXRalpha)-mediated gene expression. Here, we report a nuclear magnetic resonance (NMR) study of the RXRalpha ligand-binding domain complexed with 9-cis-retinoic acid and a glucocorticoid 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 timescale. Consistently, the K(d) derived from T462 intensity decay agreed with that derived from isothermal titration calorimetry. Furthermore, the exchange contribution to the (15)N 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.
Lu
Jianyun
J
Department of Internal Medicine, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8124, St. Louis, MO 63110, USA.
Chen
Minghe
M
Dekoster
Gregory T
GT
Cistola
David P
DP
Li
Ellen
E
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000077556:Alitretinoin; D009682:Magnetic Resonance Spectroscopy; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D047490:Retinoid X Receptor alpha / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013912:Threonine / Q000737:chemistry*; D013997:Time Factors; D014212:Tretinoin / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D010455:Peptides; D047490:Retinoid X Receptor alpha; D000077556:Alitretinoin; D013912:Threonine; D014212:Tretinoin