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 G(s) and G(q)-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-S297(7.45)) and the position of the tryptophan residue within the canonical "toggle switch" motif, CWxP of TM6, neighboring a phenylalanine in NK1R (NK1R-F264(6.51)) and a tyrosine in NK2R (NK2R-Y266(6.51)), 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-S297(7.45)) and a mainchain carbonyl (NK1R-A294(7.41)) 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-S297(7.45) is consequently identified as a central hub for the activation of NKRs.