Figure 1.
Schematic representation of the structural domains of ER protein.
A) A cartoon representation of the three-dimensional structure of the ligand binding domain is shown, as well as its sequence. Residues belonging to the ligand binding pocket are shown in red in both the structure and sequence. Residues highlighted in yellow belong to the histidine tag, residues in light blue encompass the ligand binding domain. B) Degree of conservation for residues of the ligand binding pocket among the analyzed ER sequences. Full bars correspond to 100% conservation.
Figure 2.
Computational docking models of ligands in the ER binding pocket.
The protein backbone is shown as grey cartoon; ligands and side chain of residue 421 are shown as sticks; hydrogen atoms are not shown for clarity. M421F-ERαLBD mutant bound to bisphenol-A (orange in B), 4-nonylphenol (green in C) and 17β-estradiol (red in D). In contrast to the wt-ERαLBD (shown in A with bisphenol A), the aromatic ring of the F421 mutated ER may increase the affinity for the ligands either by direct stacking interactions or by increasing the aromatic content of the ligand binding pocket; no steric clashes are created by the incorporation of this larger side chain.
Figure 3.
Circular dichroism and SDS-PAGE of recombinant ER proteins.
CD spectra show typical α-helical character and a single band at the expected molecular weight is visible (insets). (A) Far-UV CD and SDS-PAGE of wt-ERαLBD (B) Far-UV CD and SDS-PAGE of M421F-ERαLBD (C) Far-UV CD and SDS-PAGE of M421I-ERαLBD (D) – (F): thermal unfolding data of the three proteins. Intensity of peak at 222 nm plotted as a function of increased temperature. Melting temperature (Tm) reported.
Figure 4.
Chemical structures of tested compounds.
Figure 5.
Competitive binding assay on wt-ERαLBD (blue squares), M421F-ERαLBD (red circles) and M421I-ERαLBD (green triangles) with six different compounds: 17β-estradiol (panel A), 17α-ethinylestradiol (panel B), bisphenol-A (panel C), tamoxifen (panel D), 4-nonylphenol (panel E) and 4-tert-octylphenol (panel F).
Table 1.
IC50 values and standard errors resulting from the competitive binding assay performed in four replicates with wt-ERαLBD (column 2), M421F- ERαLBD (column 3) and M421I- ERαLBD (column 4) and selected compounds (ligands, column 1): 17β-estradiol, 17α-ethinylestradiol, bisphenol-A, tamoxifen, 4-nonylphenol and 4-tert-octylphenol.