Helix 1/8 interactions influence the activity of nuclear receptor ligand-binding domains.
The ligand-binding domain (LBD) of apo-nuclear receptors in solution is thought to be a very dynamic structure with many possible conformations. Upon ligand binding, the structure is stabilized to a more rigid conformation. The dynamic stabilization assay is a LBD reassembly assay that takes advantage of the high specificity of the intramolecular interactions that comprise the ligand-bound LBD. Here, we demonstrate dynamic stabilization for the nuclear receptors peroxisome proliferator-activated receptor (PPAR)gamma and nerve growth factor inducible (NGFIB)beta and identify residues important for stabilization of the intramolecular interactions induced by PPARgamma ligands. Site-directed mutagenesis studies identified residues in helices 1 and 8 required for LBD reassembly. Further, disrupting the helix 1/8 interaction in the context of the holo-LBD alters the response of the receptor in a compound-specific manner, suggesting that residues far from the ligand-binding pocket can influence the stability of the ligand-bound receptor. Thus, these results support and extend models of the apo-LBD of PPARgamma as a dynamic structure.
Holt
Jason A
JA
GlaxoSmithKline Research and Development, Research Triangle Park, North Carolina 27709-3398, USA.
Consler
Thomas G
TG
Williams
Shawn P
SP
Ayscue
Andrea H
AH
Leesnitzer
Lisa M
LM
Wisely
G Bruce
GB
Billin
Andrew N
AN
D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D009154:Mutation; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D011987:Receptors, Steroid; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D008024:Ligands; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D014157:Transcription Factors