The 48 human nuclear receptors (NRs) form a superfamily of transcription factors that regulate major physiological and pathological processes. Emerging evidence suggests that NR crosstalk can fundamentally change our understanding of NR biology, but detailed molecular mechanisms of crosstalk are lacking. Here, we report the molecular basis of crosstalk between the pregnane X receptor (PXR) and constitutive androstane receptor (CAR), where they form a novel heterodimer, resulting in their mutual inhibition. PXR and CAR regulate drug metabolism and energy metabolism. Although they have been broadly perceived as functionally redundant, a growing number of reports suggests a mutual inhibitory relation, but their precise mode of coordinated action remains unknown. Using methods including RNA sequencing, small-angle X-ray scattering and crosslinking mass spectrometry we demonstrate that the mutual inhibition altered gene expression globally and is attributed to the novel PXR–CAR heterodimerization via the same interface used by each receptor to heterodimerize with its functional partner, retinoid X receptor (RXR). These findings establish an unexpected functional relation between PXR, CAR and RXR, change the perceived functional relation between PXR and CAR, open new perspectives on elucidating their role and designing approaches to regulate them, and highlight the importance to comprehensively investigate nuclear receptor crosstalk.

The human cytochrome P450 (CYP) CYP3A4 and CYP3A5 enzymes metabolize more than one-half of marketed drugs. They share high structural and substrate similarity and are often studied together as CYP3A4/5. However, CYP3A5 preferentially metabolizes several clinically prescribed drugs, such as tacrolimus. Genetic polymorphism in CYP3A5 makes race-based dosing adjustment of tacrolimus necessary to minimize acute rejection after organ transplantation. Moreover, the differential tissue distribution and expression levels of CYP3A4 and CYP3A5 can aggravate toxicity during treatment. Therefore, selective inhibitors of CYP3A5 are needed to distinguish the role of CYP3A5 from that of CYP3A4 and serve as starting points for potential therapeutic development. To this end, we report the crystal structure of CYP3A5 in complex with a previously reported selective inhibitor, clobetasol propionate (CBZ). This is the first CYP3A5 structure with a type I inhibitor, which along with the previously reported substrate-free and type II inhibitor-bound structures, constitute the main CYP3A5 structural modalities. Supported by structure-guided mutagenesis analyses, the CYP3A5–CBZ structure showed that a unique conformation of the F–F′ loop in CYP3A5 enables selective binding of CBZ to CYP3A5. Several polar interactions, including hydrogen bonds, stabilize the position of CBZ to interact with this unique F–F′ loop conformation. In addition, functional and biophysical assays using CBZ analogs highlight the importance of heme-adjacent moieties for selective CYP3A5 inhibition. Our findings can be used to guide further development of more potent and selective CYP3A5 inhibitors.

The addition of benzene also enhanced the flexibility and hydrophobic exposure of pockets within the domain III across all simulated systems, suggesting the drug-binding capacity of the envelope protein and the potential to modify or obstruct the interactions between the virus and the host receptors.

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