Molecular Recognition at the Opioid-modulating Neuropeptide FF Receptor 1

The neuropeptide FF receptor 1 (NPFFR1) regulates opioid-induced analgesia via signaling pathways activated by RF-amide peptides such as RFRP-3 and NPFF. It also modulates opioid-induced adverse effects, including analgesic tolerance and hyperalgesia. As a key anti-opioid modulator, NPFFR1's activation mechanism and ligand selectivity remain poorly understood, hindering drug development. This study utilizes cryo-electron microscopy (cryo-EM) to elucidate the structure of NPFFR1-Gi complexes bound with the peptides RFRP-3 and NPFF, revealing crucial insights into ligand recognition and receptor activation. The ligands RFRP-3 and NPFF bind to the receptor in a manner consistent with the 'message-address' model observed in opioid receptor systems, albeit in reverse orientation. We identified the position 45.51 within the receptor's 'address' binding site that may play a key role in mediating subtype selectivity. Mutagenesis and molecular dynamics analysis further validated the structural findings. These insights could aid in future drug design for opioid modulation.