Mapping the binding site pocket of the serotonin 5-Hydroxytryptamine2A receptor. Ser3.36(159) provides a second interaction site for the protonated amine of serotonin but not of lysergic acid diethylamide or bufotenin.
Like other amine neurotransmitters that activate G-protein-coupled receptors, 5-hydroxytryptamine (5-HT) binds to the 5-HT2A receptor through the interaction of its cationic primary amino group with the conserved Asp3.32(155) in transmembrane helix 3. Computational experiments with a 5-HT2A receptor model suggest that the same functional group of 5-hydroxytryptamine also forms a hydrogen bond with the side chain of Ser3.36(159), which is adjacent in space to Asp3.32(155). However, other 5-HT2A receptor ligands like lysergic acid diethylamide (LSD), in which the amine nitrogen is embedded in a heterocycle, or N,N-dimethyl 5-HT, in which the side chain is a tertiary amine, are found in the computational simulations to interact with the aspartate but not with the serine, due mainly to steric hindrance. The predicted difference in the interaction of various ligands in the same receptor binding pocket was tested with site-directed mutagenesis of Ser3.36(159) --> Ala and Ser3.36(159) --> Cys. The alanine substitution led to an 18-fold reduction in 5-HT affinity and the cysteine substitution to an intermediate 5-fold decrease. LSD affinity, in contrast, was unaffected by either mutation. N,N-Dimethyl 5-HT affinity was unaffected by the cysteine mutation and had a comparatively small 3-fold decrease in affinity for the alanine mutant. These findings identify a mode of ligand-receptor complexation that involves two receptor side chains interacting with the same functional group of specific serotonergic ligands. This interaction serves to orient the ligands in the binding pocket and may influence the degree of receptor activation.
Almaula
N
N
Fishberg Research Center in Neurobiology, Mount Sinai School of Medicine, New York, New York 10029, USA.
Ebersole
B J
BJ
Zhang
D
D
Weinstein
H
H
Sealfon
S C
SC
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002027:Bufotenin / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003198:Computer Simulation; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010716:Phosphatidylinositols / Q000378:metabolism; D017433:Protein Structure, Secondary; D044402:Receptor, Serotonin, 5-HT2A; D011985:Receptors, Serotonin / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine; D012701:Serotonin / Q000378:metabolism* / Q000494:pharmacology; D014162:Transfection
D010716:Phosphatidylinositols; D044402:Receptor, Serotonin, 5-HT2A; D011985:Receptors, Serotonin; D011994:Recombinant Proteins; D002027:Bufotenin; D012701:Serotonin; D012694:Serine