Researchers have examined the molecular details of how neuropeptide FF receptor 1 (NPFFR1) recognizes and responds to different ligands, using cryo-electron microscopy (cryo-EM) to reveal atomic structures. NPFFR1 is a Gi/o-coupled receptor that interacts with endogenous RF-amide peptides such as RFRP-3 and NPFF. This receptor is involved in regulating opioid function, pain response, and energy balance.
The study resolved the structures of two NPFFR1-Gi complexes: one bound to RFRP-3 and another to NPFF. Functional assays confirmed differences in ligand potency, while mutagenesis and molecular dynamics simulations validated important interactions at the binding site.
The research identified a "message-address" mechanism for ligand binding. The conserved C-terminal PQRF-NH₂ motif acts as a "message," inserting into NPFFR1’s orthosteric pocket to activate the receptor through specific π-π stacking, hydrogen bonds, and salt bridges. The variable N-terminal region ("address") of each ligand determines subtype selectivity.
It was found that RFRP-3 has about 20 times higher potency than NPFF due to its N-terminal contacts with extracellular loop 2 (ECL2) and parts of the receptor that stabilize its active conformation. In contrast, NPFF's flexible N-terminus forms fewer stabilizing interactions.
A single amino acid residue at position 45.51 influences subtype selectivity between related receptors. Changing this residue can increase or decrease responsiveness to certain ligands, which was supported by simulation data.
Conserved residues across the RF-amide receptor family are important for ligand binding, while unique negatively charged pockets in NPFFR1/2 enable them to recognize a range of positively charged RF-amide motifs.
According to the researchers, these findings provide guidance for designing new selective ligands targeting NPFFR1. Such compounds could be developed by modifying the N-terminal region or adding polar groups or conformational constraints. These new ligands may improve opioid-based pain therapies by increasing analgesic effectiveness and reducing tolerance and dependence risks. The study suggests this approach could lead to new advances in clinical pain management.
