Human Substance P Interactions with G Protein-Coupled Receptor NK1R Observed by NMR in Solution

Neurokinin 1 receptor (NK1R) is a class A G protein-coupled receptor (GPCR) that interacts with the endogenous undecapeptide agonist Substance P (SP) to activate its role in key physiological processes such as nausea, inflammation, and neurological disorders. Structure determinations by cryogenic electron microscopy (cryo-EM) for NK1R with SP bound provided high-quality views of the bulk of these complexes but failed to observe the N-terminal peptide segments of both components. Here, ¹⁹F-NMR spectroscopy was combined with function assays to explore the impact of these chain ends on structure and function. The ¹⁹F-NMR probe 3-trifluoromethyl-l-phenylalanine (<i>mtfF</i>) was introduced into the SP sequence positions 0, 3, 5, 7, and 8 and observed in aqueous solution, bound to lauryl-maltose-neopentyl-glycol (LMNG)/cholesteryl hemisuccinate (CHS) micelles, and bound to micelle-solubilized NK1R. The NMR data showed for NK1R-bound SP that the N-terminal pentapeptide segment is flexible and solvent-exposed, while the C-terminal hexapeptide segment is rigidly anchored within the orthosteric pocket. Cyclic adenosine monophosphate function tests further showed that transient interactions of the side chains of Lys3 and Gln5 in SP with sites on the NK1R impacted the functional properties of SP. Combined with the rates measured for exchange of SP between all of its states in solutions with micelle-solubilized NK1R, these observations identify specific dynamic intermolecular interactions between the N-terminal peptide segments of SP and NK1R that impact the functional potency of SP.