A Mechanistic Study Of The Metal Effect And Metal Response Of Mammalian Olfactory Receptors

Mammals are extremely sensitive to sulfur-containing compounds,which play important roles in foraging,reproduction and communication.(Methylthio)methanethiol(MTMT)is a highly volatile semiochemical found in the male mice urine.It mediates the attraction of the female towards the male mice.In a previous study,MOR244-3 and MOR256-17 were identified as MTMT receptors.Copper enhanced the response of MOR244-3 significantly.MOR256-17 did not exhibit copper effect when responding to MTMT.Through sequence alignment,homology modeling,QM/MM,combined with site-directed mutagenesis and functional assays,we found three amino acids,H105,C109 and N202,that might be important for copper/MTMT binding.In addition,we identified another MTMT receptor with copper effect,MOR180-1.We note that the three amino acids that are important in MTMT/Cu binding in MOR244-3 are not seen in MOR180-1.We conclude that there might be different copper-mediated mechanisms for the activation of MTMT receptors.Comparing the sequences and functions of homologs with high sequence identities may shed light on the identification of important sites for receptor functions.With this in mind,we cloned 9 MOR244-3,11 MOR180-1,and 11 MOR256-17 mammalian orthologs,as well as 2 MOR244-3 paralogs,MOR244-1 and MOR244-2,and tested the functions of these homologous receptors.We found that the function of the orthologs of the 3 MTMT receptors varied dramatically during evolution and that the copper effect and the response to MTMT always co-evolved.With respect to the paralogs,while MOR244-3 could respond to a panel of sulfur-containing compounds,we have yet to identify the ligands for MOR244-1 and MOR244-2 after a high-throughput screening of 57 thiols and 30 odorant mixtures.MOR244-1 and MOR244-3 did not respond to 11 metals;MOR244-2,however,could respond to copper and silver.Through ancestral reconstruction of the MOR244-3 rodent lineage,we identified one residue,R260,that might be important for response to MTMT.In addition,the corresponding site R261 in MOR180-1 and R263 in MOR244-2 were also proved to be important for MTMT/Cu-and copper-binding,respectively.QM/MM analysis showed that the potential binding sites as C203/M254/R261 in MOR180-1 and C254/R260/K269 in MOR244-3.Whether metal effect and metal response are common among mammalian olfactory receptors are still unknown.We performed screenings for metal effect using known receptor-ligand pairs and found that metal effect are rather restricted phenomena.Next,with screenings targeting odorants with potential metal-coordinating abilities,we identified a human receptor OR2T11 that responded to tert-butyl mercaptan(TBM)with copper and silver effect,and to silver alone.Two copper-binding sites,M115/C238/H241 and M56/M133/R135/C138,and one silver-binding site,R135/C138,were identified.