Mechanistic insights into the synergistic activation of the RXR–PXR heterodimer by endocrine disruptor mixtures

Abstract

Humans are chronically exposed to mixtures of xenobiotics referred to as endocrine-disrupting chemicals (EDCs). A vast body of literature links exposure to these chemicals with increased incidences of reproductive, metabolic, or neurological disorders. Moreover, recent data demonstrate that, when used in combination, chemicals have outcomes that cannot be predicted from their individual behavior. In its heterodimeric form with the retinoid X receptor (RXR), the pregnane X receptor (PXR) plays an essential role in controlling the mammalian xenobiotic response and mediates both beneficial and detrimental effects. Our previous work shed light on a mechanism by which a binary mixture of xenobiotics activates PXR in a synergistic fashion. Structural analysis revealed that mutual stabilization of the compounds within the ligand-binding pocket of PXR accounts for the enhancement of their binding affinity. In order to identify and characterize additional active mixtures, we combined a set of cell-based, biophysical, structural, and in vivo approaches. Our study reveals features that confirm the binding promiscuity of this receptor and its ability to accommodate bipartite ligands. We reveal previously unidentified binding mechanisms involving dynamic structural transitions and covalent coupling and report four binary mixtures eliciting graded synergistic activities. Last, we demonstrate that the robust activity obtained with two synergizing PXR ligands can be enhanced further in the presence of RXR environmental ligands. Our study reveals insights as to how low-dose EDC mixtures may alter physiology through interaction with RXR–PXR and potentially several other nuclear receptor heterodimers.

Helix 12 stabilization contributes to basal transcriptional activity of PXR

“…( 27 ) reported that AF2 in the crystal structures of the unliganded PPARγ LBD is also located slightly distant from of its position in ligand-bound PPARγ. In contrast, the conformation of the AF2 domain in the reported crystal structures of the unliganded PXR LBD (PDBIDs; 7AX8, 4J5W, 3CTB, and 1ILG) ( 14 , 28 , 29 , 30 ) was found to be different from other nuclear receptors. In these cases, the AF2 domain was located in the same position as the reported crystal structures of ligand (rifampicin or SR12813)-bound LBDs with a coactivator peptide (PDBIDs; 1SKX, 3HVL, 1NRL, and 4J5X) ( 14 , 29 , 31 , 32 ).…”

A Comparative Study of Human and Zebrafish Pregnane X Receptor Activities of Pesticides and Steroids Using In Vitro Reporter Gene Assays

“…To gain structural insights into zfPXR ligand binding selectivity, we generated a model of zfPXR LBD using the web-based server EDMon (Endocrine Disruptor Monitoring; ) ( 21 , 22 ), and superposed it on the experimental crystal structures of hPXR in complex with SR12813 ( 23 ) and clotrimazole ( 16 ). As previously observed, the ligand-binding pockets (LBP) of hPXR and zfPXR show significant differences in their size and residue composition ( 7 , 8 ).…”

“…In a recent study on the human receptor, we observed that PXR contains an aromatic cage deeply buried at the bottom of the LBP and made up of residues F288, W299 and Y306. This unique aromatic triad (referred to as the π-trap) catches compounds through their most hydrophobic moieties and constitutes the main anchoring point of many compounds, including clotrimazole ( 16 ). Interestingly, residues forming the π-trap are fully conserved in zfPXR ( Figure 5 ).…”

“…Overall, some of the specific structural features of hPXR ( e.g. large LBP size and high plasticity) most likely account for its refractoriness to antagonism ( 16 , 30 ), while in contrast, the smaller and less malleable zfPXR LBP ( 17 ) could explain why the zebrafish receptor is more prone to antagonism. Notably, the substitution of a comfortable methionine residue in helix H12 of hPXR for a bulky and rigid tryptophan residue in zfPXR ( Figure 5 ) may enable easier destabilization of the active conformation of the activation helix by chemicals.…”

“…(10)(11)(12)(13)(14). Besides, we have recently described the capacity of environmental pollutants to bind simultaneously to the hPXR, leading to its synergic activation (15,16). Since X-ray crystallography of the PXR has only been reported for human, little is known regarding the structure of the PXR in other species.…”