Interplay of Flexibility and Stability in the Control of Estrogen Receptor Activity

• Published in: Biochemistry (Easton) Vol. 44; no. 2; pp. 790 - 798
• Main Authors: Bouter, A, Le Tilly, V, Sire, O
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 18-01-2005
• Subjects: Animals; Base Pair Mismatch; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Estrogen Receptor alpha - chemistry; Estrogen Receptor alpha - genetics; Estrogen Receptor alpha - metabolism; Fluorescence Polarization; Humans; Kinetics; Ligands; Macromolecular Substances - metabolism; Oncorhynchus mykiss - genetics; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Protein Binding - genetics; Protein Structure, Tertiary; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Response Elements - genetics; Salts - chemistry; Thermodynamics; Transcriptional Activation; Tryptophan - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0483716

Previously, we have identified an imperfect estrogen response element (rtERE) in the promoter of the rainbow trout vitellogenin gene. Although this ERE leads to a lower transcriptional activation, a better estradiol stimulation in vivo as compared to consensus ERE (EREcs) was observed. Here we examine the ability of recombinant human estrogen receptor α (rhERα) to bind DNA containing the EREcs or the natural imperfect rtERE, which contains three mismatches. At low salt concentration, whatever the ERE sequence, dissociation equilibrium constants of the specific rhERα−ERE complexes are similar (K D = 2 nM) with the same stoichiometry. As salt concentration increases from 80 to 200 mM KCl, the affinity of the rhERα−rtERE complex largely diminishes whereas that of rhERα−EREcs seems less affected. Hence the nature of the interactions stabilizing these complexes is different:  more ionic in rhERα−rtERE as compared to rhERα−EREcs. Moreover, kinetic measurements showed that specific rhERα−ERE complexes exhibit shorter half-lives (few seconds) and that the rhERα−EREcs complex is more stable (33 s) than the complex that formed with rtERE (19.8 s), in accordance with equilibrium binding results. Finally, dynamic studies of rhERα have shown that the protein fluctuations are damped when the salt concentration increases or when bound to ERE and all the more with rtERE. The interplay of affinity, complex half-lives, and protein dynamics in the transcriptional regulation of estrogen receptor is discussed.