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 1 receptor (NK1R) and neurokinin 2 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 micro-environments 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 suggest 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 micro-switch motif 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 micro-switch 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.