Structural basis for the functions of dopamine receptors, neurotransmitter transporters, and sigma 1 receptor

National Institute On Drug Abuse

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Abstract

Fentanyl and its analogs are selective agonists of the micro-opioid receptor (MOR). Among novel synthetic opioids (NSOs), they dominate the recreational drug market and are the main culprits for the opioid crisis, which has been exacerbated by the COVID-19 pandemic. By taking advantage of the crystal structures of the MOR, several groups have investigated the binding mechanism of fentanyl, but have not reached a consensus, in terms of both the binding orientation and the fentanyl conformation. Thus, the binding mechanism of fentanyl at the MOR remains an unsolved and challenging question. Here, we carried out a systematic computational study to investigate the preferred fentanyl conformations, and how these conformations are being accommodated in the MOR binding pocket. We characterized the free energy landscape of fentanyl conformations with metadynamics simulations, and compared and evaluated several possible fentanyl binding conditions in the MOR with long-timescale molecular dynamics simulations. Our results indicate that the most preferred binding pose in the MOR binding pocket corresponds well with the global minimum on the energy landscape of fentanyl in the absence of the receptor, while the energy landscape can be reconfigured by modifying the fentanyl scaffold. The interactions with the receptor may stabilize a slightly unfavored fentanyl conformation in an alternative binding pose. By extending similar investigations to fentanyl analogs, our findings establish a structure-activity relationship of fentanyl binding at the MOR. In addition to providing a structural basis to understand the potential toxicity of the emerging NSOs, such insights will contribute to developing new, safer analgesics. -arrestins bind GPCRs to terminate G protein signaling and to facilitate other downstream signaling pathways. Using single-molecule fluorescence resonance energy transfer imaging, we show that -arrestin is strongly autoinhibited in its basal state. Its engagement with a phosphopeptide mimicking phosphorylated receptor tail efficiently releases the -arrestin tail from its N domain to assume distinct conformations. Unexpectedly, we find that -arrestin binding to phosphorylated receptor, with a phosphorylation barcode identical to the isolated phosphopeptide, is highly inefficient and that agonist-promoted receptor activation is required for -arrestin activation, consistent with the release of a sequestered receptor C tail. These findings, together with focused cellular investigations, reveal that agonism and receptor C-tail release are specific determinants of the rate and efficiency of -arrestin activation by phosphorylated receptor. We infer that receptor phosphorylation patterns, in combination with receptor agonism, synergistically establish the strength and specificity with which diverse, downstream -arrestin-mediated events are directed.