A Characteristic Back Support Structure in the Bisphenol A-Binding Pocket in the Human Nuclear Receptor ERRγ

<div><p>The endocrine disruptor bisphenol A (BPA) affects various genes and hormones even at merely physiological levels. We recently demonstrated that BPA binds strongly to human nuclear receptor estrogen-related receptor (ERR) γ and that the phenol-A group of BPA is in a receptacle pocket with essential amino acid residues to provide structural support at the backside. This led BPA to bind to ERRγ in an induced-fit-type binding mode, for example, with a rotated motion of Val313 to support the Tyr326-binding site. A similar binding mechanism appears to occur at the binding site of the BPA phenol-B ring. X-ray crystal analysis of the ERRγ-ligand-binding domain/BPA complex suggested that the ERRγ receptor residues Leu342, Leu345, Asn346, and Ile349 function as intrinsic binding sites of the BPA phenol-B, whereas Leu265, Leu268, Ile310, Val313, Leu324, Tyr330, Lys430, Ala431, and His434 work as structural elements to assist these binding sites. In the present study, by evaluating the mutant receptors replaced by a series of amino acids, we demonstrated that a finely assembled structural network indeed exists around the two adjacent Leu³⁴²-Asn³⁴⁶ and Leu³⁴⁵-Ile³⁴⁹ ridges on the same α-helix 7 (H7), constructing a part of the binding pocket structure with back support residues for the BPA phenol-B ring. The results reveal that the double-layer binding sites, namely, the ordinary ligand binding sites and their back support residues, substantiate the strong binding of BPA to ERRγ. When ERRγ-Asn346 was replaced by the corresponding Gly and Tyr in ERRα and ERRβ, respectively, the binding affinity of BPA and even 4-hydroxytamxifen (4-OHT) is much reduced. Asn346 was found to be one of the residues that make ERRγ to be exclusive to BPA.</p></div

Receptor binding potency of BPA, 4-α-cumylphenol, and 4-OHT in the competitive binding assay using [³H]BPA for human nuclear receptor ERRγ and its mutants with site-directed mutagenesis in the BPA binding site amino acid residues.

1)<p>Specifically mutated residues are designated in italics.</p>2)<p>Because there was "no specific binding" in the saturation binding assay, the competitive binding assay could not be carried out.</p

Receptor binding characteristics of [³H]BPA for wild-type ERRγ and its mutants in the saturation binding assay.

1)<p>Specifically mutated residues are designated in italics. All the saturation binding assays to determine the dissociation constant (<i>K</i>_d) and the receptor density (<i>B</i>_max) for [³H]BPA were carried out at least three times.</p>2)<p>NSB means "no specific binding" in the saturation binding assay.</p

Receptor binding potency of BPA, 4-α-cumylphenol, and 4-OHT in the competitive binding assay using [³H]BPA for human nuclear receptor ERRγ and its mutants with site-directed mutagenesis in the back support residues of BPA binding sites.

1)<p>Specifically mutated residues are designated in italics.</p>2)<p>Because there was "no specific binding" in the saturation binding assay, the competitive binding assay could not be carried out.</p>3)<p>The results of ERRγ mutant receptors of Leu268, Ile310, and Val313 for BPA, except for those for 4-OHT and 4-α-cumylphenol, were re-collected from the previous report <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101252#pone.0101252-Liu1" target="_blank">[24]</a> for providing easiness in understanding.</p

Structural interrelationships between the bisphenol A-binding site amino acid residues Leu³⁴²-Asn³⁴⁶/Leu³⁴⁵-Ile³⁴⁹ and their back support residues present in the binding pocket of ERRγ-LBD.

<p>(<b>A</b>) Leu345 and its back support residue Ala431. There is no direct contact between Ala431 and BPA. (<b>B</b>) Ile349 and its back support residue Val313. There is no direct contact between Val313 and BPA. (<b>C</b>) Leu342 and its back support residue Leu268. (<b>D</b>) Leu268 and its back support residues Leu265 and Tyr330. This Leu268 binds directly to the phenol-A and B rings of BPA as a double-hook to connect both tightly. (<b>E</b>) Ile349/Asn346 and their simultaneous back support residue Leu324. (<b>F</b>) Four amino acids, Leu324, Tyr326, Tyr330, and Met332, stand on the pleated β-sheet, which makes a bottom plate of ERRγ-LBP. (<b>G</b>) Leu345 and its back support residue Lys430. (<b>H</b>) Leu342/Leu345 and their simultaneous back support residue His434.</p

The homologous competitive binding assays between [³H]bisphenol A and non-labeled bisphenol A for the wild-type ERRγ-LBD and its mutants.

<p>The receptors used were the wild-type ERRγ and its mutant receptors. (<b>A</b>) Leu342-substituted ERRγ mutant receptors, (<b>B</b>) Leu345-substituted ERRγ mutant receptors, (<b>C</b>) Asn346-substituted ERRγ mutant receptors, and (<b>D</b>) Ile349-substituted ERRγ mutant receptors. The graphs show representative dose-dependent binding curves, which give the IC₅₀ value closest to the mean IC₅₀ from at least three independent experiments.</p