Ligand binding is without effect on complex formation of the ligand binding domain of the ecdysone receptor (EcR).
The ligand-binding domain (LBD) encompassing the C-terminal parts of the D- and the complete E-domains of the ecdysteroid receptor (EcR) fused to Gal4(AD) is present in two high molecular weight complexes (600 and 150 kDa) in yeast extracts according to size exclusion chromatography (Superdex 200 HR 10/30). Hormone binding is mainly associated with 150-kDa complexes. Complex formation is not influenced by hormone, but the ligand stabilizes the complexes at elevated salt concentrations. Mutational analysis of Gal4(AD)-EcR(LBD) revealed that formation of 600-kDa, but not 150-kDa, complexes depends on dimerization mediated by the EcR(LBD). Deletion of helix 12 is without effect. Mutation of K497 in helix 4, known to be essential for comodulator binding, abolishes 600-KDa complexes, but does not interfere with the formation of 150-kDa complexes. In contrast, the DE-domains of USP fused to Gal4(DBD) elute as monomer after elimination of the dimerization capacity of the ligand-binding domains by mutation of P463 in helix 10. The data presented here reveal that the complex formation of ligand-binding domains EcR and USP ligand is different.
Greb-Markiewicz
B
B
Department of General Zoology and Endocrinology, University of Ulm, Albert-Einstein-Allee 11, D-89081 Ulm, Germany.
Fauth
T
T
Spindler-Barth
M
M
D015153:Blotting, Western; D002850:Chromatography, Gel; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins; D019281:Dimerization; D008024:Ligands; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011987:Receptors, Steroid / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; D008024:Ligands; D046912:Multiprotein Complexes; D011987:Receptors, Steroid; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C055469:ecdysone receptor