Molecular Mechanisms Of Activation Of Human Musk Odorant Receptors By Musk-smelling Compounds

Musk compounds have been widely used in the perfumery and in traditional medicine with a long history for more than 2000 years.Molecular mechanisms of activation of human musk odorant receptors(ORs)have received less attention in the past.Understanding olfaction at the molecular level is challenging due to the lack of crystallographic models of odorant receptors.Our study combined the biological experiments with computational models to synthetically analyze the mechanisms of activation of human musk odorant receptors by musk-smelling compounds.By ligand-based screening for ORs through the OR heterologous expression system,here we identified two human musk odorant receptors,OR5AN1 and OR1A1.We examined the response profiles by testing OR5AN1 and OR1A1 against all three classes of synthetic musks including macrocyclic musks,nitromusks and polycyclic musks,as well as selected structurally-related compounds.To further explore the ligand specificity and selectivity,we also performed a ligand screening for OR5AN1.We found that,OR5AN1 is a highly specific musk odorant receptor with highly responsive to nitromusks over macrocyclic musks and responds at nanomolar concentrations to musk ketone,and robustly to macrocyclic sulfoxides and fluorine-substituted macrocyclic ketones.Nevertheless,OR1A1 responds only to nitromusks.By examining the responses of OR5AN1 against a series of fluorinated muscone structural analogs and analyzing the X-ray crystal structures of these fluorinated derivatives,we explored preferred agonist conformations of flexible rings of muscone for OR5AN1.Structural models of OR5AN1 and OR1A1 based on homology modeling,quantum mechanics/molecular mechanics(QM/MM)and molecular dynamics were validated through direct comparisons with activation profiles from site-directed mutagenesis experiments and analysis of binding energies for the musk-related odorants.Structural models show that OR5AN1 stabilizes odorants by hydrogen bonding to Y260 of transmembrane ?-helix 6(TM6)and hydrophobic interactions with surrounding hydrophobic residues F105,F194 and F207.The binding of OR1A1 to nitromusks is stabilized by hydrogen bonding to Y258 along with hydrophobic interactions with surrounding aromatic residues Y251 and F206.Hydrophobic/non-polar and hydrogen bonding interactions contribute,respectively,77% and 13% to the odorant binding affinities,as shown by an atom-based quantitative structure activity relationship(QSAR)model.