Molecular determinants of neuropeptide-mediated activation mechanisms in tachykinin NK1 and NK2 receptors

Substance P and neurokinin A are closely related neuropeptides belonging to the tachykinin family. Their receptors are neurokinin one receptor (NK1R) and neurokinin two receptor (NK2R), G protein–coupled receptors that transmit Gs and Gq-mediated downstream signaling. We investigate the importance of sequence differences at the bottom of the receptor orthosteric site for activity and selectivity, focusing on residues that closely interact with the C-terminal methionine of the peptide ligands. We identify a conserved serine (NK1R-S2977.45) and the position of the tryptophan residue within the canonical “toggle switch” motif, CWxP of TM6, neighboring a phenylalanine in NK1R (NK1R-F2646.51) and a tyrosine in NK2R (NK2R-Y2666.51), giving rise to distinct microenvironments for the neuropeptide C terminals. Mutating these residues results in dramatic activity changes in both NK1R and NK2R due to a close interaction between the ligand and toggle switch. Structural analysis of active and inactive NKR structures suggests only a minor change in sidechain rotation of toggle switch residues upon activation. However, extensive molecular dynamics simulations of receptor:neuropeptide:G protein complexes indicate that a major, concerted motion happens in the toggle switch tryptophan indole group and the sidechains of the microswitch motif Pro-Ile-Phe (PIF). This rotation establishes a tight hydrogen bond interaction from the tryptophan indole to the conserved serine (NK1R-S2977.45) and a mainchain carbonyl (NK1R-A2947.41) in the kink of TM7. This interaction facilitates communication with the NPxxY microswitch motif of TM7, resulting in stabilization of the G protein–binding region. NK1R-S2977.45 is consequently identified as a central hub for the activation of NKRs.