Neuropeptide S Receptor in nanodiscs: SPR-based screening of a peptide library for ligand binding

Neuropeptide S Receptor (NPSR) is a G protein-coupled receptor (GPCR) activated by Neuropeptide S, a ligand recently characterized. NPSR is involved in various physiological processes, such as arousal, anxiety, locomotion, and food intake. Studying the binding site and affinity between NPSR and NPS is crucial for gathering a deeper understanding about the downstream signaling mechanisms and the NPSR therapeutic potential, even to develop new peptide drugs. To investigate the receptor-ligand interactions, Surface Plasmon Resonance (SPR) analysis were performed: NPSR extracellular domain, membrane fragments and nanodiscs containing NPSR were immobilized on specific sensor chips. Affinity constants of a small library of ligand peptides (NPS wild-type, fragment, or modified variants) were measured via SPR. Nanodisc technology is fundamental in this study. It exploits a membrane scaffold protein which, upon acquiring its native conformation, embeds phospholipids and one NPSR molecule, thus creating a nanodisc. This approach allows the NPSR to be studied in a near-physiological environment, with a controlled stoichiometry (one nanodisc per one NPSR). Our findings reveal, for the first time, that NPS peptides specifically bind to the extracellular domain of NPSR. Furthermore, we demonstrate that SPR technology is a reliable and effective method to study these receptor-peptide interactions.