Characterization of the ligand-binding domain of the ecdysteroid receptor from Drosophila melanogaster.
Mutants created by site-directed mutagenesis were used to elucidate the function of amino acids involved in ligand binding to ecdysteroid receptor (EcR) and heterodimer formation with ultraspiracle (USP). The results demonstrate the importance of the C-terminal part of the D-domain and helix 12 of EcR for hormone binding. Some amino acids are involved either in ligand binding to EcR (E476, M504, D572, I617, N626) or ligand-dependent heterodimerization as determined by gel mobility shift assays (A612, L615, T619), while others are involved in both functions (K497, E648). Some amino acids are suboptimal for ligand binding (L615, T619), but mediate ligand-dependent dimerization. We conclude that the enhanced regulatory potential by ligand-dependent modulation of dimerization in the wild type is achieved at the expense of optimal ligand binding. Mutation of amino acids (K497, E648) involved in the salt bridge between helix 4 and 12 impair ligand binding to EcR more severely than hormone binding to the heterodimer, indicating that to some extent heterodimerization compensates for the deleterious effect of certain mutations. Different effects of the same point mutations on ligand binding to EcR and EcR/USP (R511, A612, L615, I617, T619, N626) indicate that the ligand-binding pocket is modified by heterodimerization.
Grebe
Marco
M
Abteilung für Allgemeine Zoologie und Endokrinologie, Universität Ulm, Albert-Einstein-Allee 11, D-89081 Ulm, Germany.
Przibilla
Sabina
S
Henrich
Vincent C
VC
Spindler-Barth
Margarethe
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D015153:Blotting, Western; D004268:DNA-Binding Proteins / Q000378:metabolism; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D024202:Electrophoretic Mobility Shift Assay; D008024:Ligands; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation / Q000502:physiology; D011487:Protein Conformation; D011987:Receptors, Steroid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
D000596:Amino Acids; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D008024:Ligands; D011987:Receptors, Steroid; D011994:Recombinant Proteins; D014157:Transcription Factors; C066030:USP protein, Drosophila; C034808:ecdysteroid receptor