Ligand-induced stabilization of PPARgamma monitored by NMR spectroscopy: implications for nuclear receptor activation.
Nuclear receptors are ligand-dependent transcription factors that are mediators of the action of lipophilic hormones and other endogenous ligands and are the targets of drugs useful in a variety of therapeutic areas. Peroxisome proliferator-activated receptor (PPAR)gamma is a nuclear receptor that, acting as a heterodimer with RXR, mediates a variety of cellular effects including adipocyte-differentiation. Due to its role in modulating insulin sensitivity, it is the target of therapeutically active anti-diabetic agents such as rosiglitazone. We have assigned the chemical shifts of the backbone atoms of the 32 kDa ligand-binding domain of PPARgamma in the presence of bound rosiglitazone. Three-dimensional HNCO spectra of the apo ligand-binding domain (LBD) have less than half the expected number of cross-peaks. The missing cross-peaks are restored upon binding strong agonists such as rosiglitazone. The NMR results indicate that the apo-LBD of PPARgamma is in a conformationally mobile state, and that agonist binding is associated with a marked stabilization of the conformation. Mapping the missing peaks to the 3D X-ray crystallographic structure indicates the region of mobility is extensive and includes the ligand-binding region and the cofactor-binding site. This leads to the conclusion that activation of this nuclear receptor is a result of a population shift of a dynamic ensemble of conformations, rather than a two-state switch from an inactive to an active conformation. Our results have important implications for the mechanisms by which antagonists, partial agonists, and agonists of nuclear receptor function operate.
Johnson
B A
BA
Department of Endocrinology and Chemical Biology, Merck Research Laboratories, Rahway, NJ 07065, USA. bruce_johnson@merck.com
Wilson
E M
EM
Li
Y
Y
Moller
D E
DE
Smith
R G
RG
Zhou
G
G
D000595:Amino Acid Sequence; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D005786:Gene Expression Regulation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009038:Motion; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000819:agonists / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D018160:Receptors, Cytoplasmic and Nuclear / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014157:Transcription Factors / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*
D001059:Apoproteins; D008024:Ligands; D010446:Peptide Fragments; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors