Neo plate reader

Fluorescence anisotropy titrations were performed in triplicate at 25°C using a BioTek Neo plate reader with Hop2–Mnd1 and 5′-fluorescein-labeled 40 bp dsDNA or 40-mer ssDNA (50 nM) in a buffer containing 20 mM Tris-HCl (pH 7.5), 100 mM NaCl and 5 mM MgCl₂. The dissociation constant was derived from the equations;

where A_t is the total anisotropy and

[Hop2]_t and [DNA]_t are the total concentrations of Hop2–Mnd1 and DNA. [Hop2.DNA] is the concentration of the Hop2–Mnd1:DNA complex. Q is the fluorescence intensity of Hop2–Mnd1:DNA relative to DNA.

Assays utilized a fluorescein-labelled probe (6 N-FAM) that is displaced by unlabelled competitors³⁹ . Competition assays were conducted in opaque, black 384 assay plates using a combination of 10 nM 6 N-FAM with 5 nM LRH-1 ligand binding domain (LBD) or 30 nM 6 N-FAM with 25 nM SF-1 LBD (these concentrations were determined through optimization steps in our previous work)³⁹ . Competitor ligand concentrations ranged from 2^–11 to 2^–4 M. A constant amount of DMSO was present in each well (6.7% v/v). Assays were conducted three times in quadruplicate. FP was measured on a BioTek Neo plate reader. GraphPad Prism (v.8) was used to analyze the data using a one-site, fit K_i curve, which fits the K_i of the unlabelled competing ligand using the EC₅₀ of the competition curve, the concentration of the fluorescent probe, and the K_d of the probe for each receptor, as follows:
Equation 1: Calculating the EC₅₀ of the competing ligand.
$$\mathrm{Y}=\mathrm{Bottom}+(\mathrm{Top}-\mathrm{Bottom})/(1+{10}^{\wedge }(X-{\mathrm{LogEC}}_{50}))$$
Equation 2: Calculating K_i from EC₅₀, probe concentration, and probe K_d.
$${\text{logEC}}_{50}=\log ({10}^{\wedge }\log {\text{K}}_{\text{i}}\ast (1+[\mathrm{Probe},\mathrm{nM}]/\mathrm{Probe}{\mathrm{K}}_{\mathrm{d}},\mathrm{nM})).$$
Significance of differences in K_i values from parallel experiments was determined using
two-tailed, paired t-tests, with p < 0.05 considered statistically significant.

The same human SF-1 LBD (2CS) construct used for crystallization was complexed with dioleoyl PIP3 and subjected to anion exchange chromatography to separate PIP3-bound species from bacterial phospholipid-bound species. PIP3-bound SF-1 elutes during anion exchange chromatography at higher ionic strength than bacterial phospholipid-bound SF-1 because of the negatively charged phosphates on the PIP3 headgroup. The PIP3-bound and bacterial phospholipid–bound fractions were pooled and concentrated as above, then serially diluted into assay buffer (20 mM Hepes [7.5], 150 mM NaCl) in black-walled 384-well nonprotein binding plates, with 50 μM SF-1 LBD as the highest concentration and a constant 25 μL volume in each well. Reproducibility in the FP assay critically relies on using nonprotein binding plates. To each well, an equal volume of N-terminal 5Fam-labeled PGC1α co-activator peptide (5Fam-EEPSLLKKLLLAPA-OH) was added to a final concentration of 100 nM fluorescently labeled peptide (purchased from New England Peptide). After a 1-h incubation nutating at room temperature in the dark, fluorescence polarization was measured on the Biotek Neo Plate reader at the Vanderbilt High-throughput Screening Center. Data were fit to a standard one-site binding model in GraphPad Prism, and all data represent at least three independent replicates.