PMID- 16460010 OWN - NLM STAT- MEDLINE DCOM- 20060504 LR - 20181203 IS - 0006-2960 (Print) IS - 0006-2960 (Linking) VI - 45 IP - 6 DP - 2006 Feb 14 TI - Analysis of ligand binding and protein dynamics of human retinoid X receptor alpha ligand-binding domain by nuclear magnetic resonance. PG - 1629-39 AB - Retinoid X receptors (RXRs) are nuclear receptors that can activate transcription as homodimers or as obligate heterodimeric partners of other nuclear receptors. While the crystal structures of the RXR ligand-binding domains (LBD) have been previously determined, the dynamics of activation is less well characterized at an atomic level. To probe the effect of ligand binding on RXR LBD dynamics, we initiated nuclear magnetic resonance studies of recombinant human RXRalpha LBD (T223-T462) with and without bound 9-cis-retinoic acid (9cRA). The 1HN, 15N, 13C(alpha), 13CO, and 13C(beta) resonance assignments were established for 164 of 240 residues in apo-RXRalpha LBD. Resonances corresponding to an additional 47 residues emerged upon 9cRA binding. These additional residues included those located in the vicinity of the ligand-binding pocket (helices H3, H5, and strands S1, S2), as well as residues located at the dimerization interface (helices H9 and H10). Thus 9cRA binding stabilized the ligand-binding pocket and had allosteric effects on the dimerization interface. Ligand-induced chemical shift perturbations outside the binding cavity were mapped to helix H3 and the AF-2 helix H12, indicating conformational changes in these regions. However, helix H11, a component of the tetramerization interface, and a large part of helix H10, a component of the dimerization interface, remained undetectable even after 9cRA binding. Although apo- and holo-hRXRalpha LBD existed predominantly as homodimers in solution, exchange between monomeric, dimeric, and tetrameric forms of the protein could have contributed to line broadening of cross-peaks corresponding to helices H10 and H11. 15N T1, T2, and steady-state 1H-15N NOE data collected at 500 and 700 MHz static magnetic fields showed that the internal motions for the residues in the H1-H3 loop (K245-D263) were much less restricted than those in the protein core for both apo- and holo-forms. Significant exchange R(ex) contributions to the transverse relaxation rate were detected for most of the residues measured in both apo- and holo-RXRalpha LBDs by transverse relaxation optimized spectroscopy-Carr-Purcell-Meiboom-Gill (CPMG) experiments at two B1 field strengths. Taken together these results suggest that the RXRalpha LBD exists as a dynamic ensemble of conformations, even after binding its cognate ligand. Such dynamic characteristics may allow RXRalpha to partner with multiple nuclear receptors. FAU - Lu, Jianyun AU - Lu J AD - Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA. FAU - Cistola, David P AU - Cistola DP FAU - Li, Ellen AU - Li E LA - eng GR - DK59501/DK/NIDDK NIH HHS/United States GR - P30 DK056341/DK/NIDDK NIH HHS/United States GR - RR015715/RR/NCRR NIH HHS/United States GR - P30 DK056341-06/DK/NIDDK NIH HHS/United States GR - P30 DK056341-05S2/DK/NIDDK NIH HHS/United States GR - DK 52574/DK/NIDDK NIH HHS/United States GR - DK56341/DK/NIDDK NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PL - United States TA - Biochemistry JT - Biochemistry JID - 0370623 RN - 0 (Ligands) RN - 0 (Retinoid X Receptor alpha) RN - 1UA8E65KDZ (Alitretinoin) RN - 5688UTC01R (Tretinoin) SB - IM MH - Alitretinoin MH - Allosteric Regulation MH - Dimerization MH - Humans MH - Ligands MH - Magnetic Resonance Spectroscopy/methods MH - Models, Molecular MH - Protein Binding MH - Protein Conformation MH - Protein Structure, Secondary MH - Retinoid X Receptor alpha/*chemistry/metabolism MH - Stereoisomerism MH - Tretinoin/*chemistry/metabolism EDAT- 2006/02/08 09:00 MHDA- 2006/05/05 09:00 CRDT- 2006/02/08 09:00 PHST- 2006/02/08 09:00 [pubmed] PHST- 2006/05/05 09:00 [medline] PHST- 2006/02/08 09:00 [entrez] AID - 10.1021/bi051474j [doi] PST - ppublish SO - Biochemistry. 2006 Feb 14;45(6):1629-39. doi: 10.1021/bi051474j.