Cryo-EM reveals NPFFR1 activation and guides novel ligand design

This study investigates the molecular mechanisms underlying ligand recognition, subtype selectivity, and activation of neuropeptide FF receptor 1 (NPFFR1)-a Gi/o-coupled receptor that responds to endogenous RF-amide peptides (RFRP-3 from pro-NPFFB, NPFF from pro-NPFFA) and regulates physiological processes like opioid function, pain, and energy homeostasis. To address gaps in understanding its role in opioid modulation (due to lack of selective ligands), the authors used cryo-electron microscopy (cryo-EM) to resolve atomic structures of two NPFFR1-Gi complexes: RFRP-3-bound and NPFF-bound. GloSensor cAMP assays confirmed ligand potency differences, and mutagenesis/MD simulations validated key interactions.

Key findings from the study include:

1. A "message-address" binding mechanism: The conserved C-terminal PQRF-NH₂ motif ("message") inserts into NPFFR1's orthosteric pocket (TM2/3, TM5/6, TM7) to drive activation via π-π stacking (Phe8-W287^6.52), hydrogen bonds (Phe8 α-amide-T100^2.61/Q123^3.23/H315^7.39), and salt bridges (Arg7-E205^45.52); divergent N-termini ("address") determine selectivity.

2. RFRP-3 has ~20-fold higher potency than NPFF because its N-terminus forms stabilizing contacts with ECL2 (E185ECL2) and TM3/TM4, enhancing receptor conformational stability and Gi coupling, whereas NPFF's N-terminus is flexible with fewer interactions.

3. Residue 45.51 dictates subtype selectivity: mutating W204^45.51 to Arg enhances NPFF-induced Gi activation, while R207W mutation reduces NPFFR2's response (validated by MD simulations).

4. Conserved residues (e.g., T5.39) across RF-amide receptors (QRFPR, KISS1R, PrRPR) mediate ligand binding. NPFFR1/2 have unique negatively charged pockets that complement positive RF-amide motifs, enabling broad ligand recognition.

These insights offer a strategic framework for designing selective NPFFR1 ligands-through approaches such as N-terminal elongation, incorporation of polar substitutions, or imposing conformational constraints-to address opioid-related disorders. The resulting novel ligands hold promise for co-administration with opioid drugs, enhancing analgesic efficacy while effectively mitigating tolerance and dependence, thereby paving the way for innovative breakthroughs in clinical pain management.