title: Binding of serotonin to the human serotonin transporter. Molecular modeling and experimental validation.
abstract: Molecular modeling and structure-activity relationship studies were performed to propose a model for binding of the neurotransmitter serotonin (5-HT) to the human serotonin transporter (hSERT). Homology models were constructed using the crystal structure of a bacterial homologue, the leucine transporter from Aquifex aeolicus, as the template and three slightly different sequence alignments. Induced fit docking of 5-HT into hSERT homology models resulted in two different binding modes. Both show a salt bridge between Asp98 and the charged primary amine of 5-HT, and both have the 5-HT C6 position of the indole ring pointing toward Ala173. The difference between the two orientations of 5-HT is an enantiofacial discrimination of the indole ring, resulting in the 5-hydroxyl group of 5-HT being vicinal to either Ser438/Thr439 or Ala169/Ile172/Ala173. To assess the binding experimentally, binding affinities for 5-HT and 17 analogues toward wild type and 13 single point mutants of hSERT were measured using an approach termed paired mutant-ligand analogue complementation (PaMLAC). The proposed ligand-protein interaction was systematically examined by disrupting it through site-directed mutagenesis and re-establishing another interaction via a ligand analogue matching the mutated residue, thereby minimizing the risk of identifying indirect effects. The interactions between Asp98 and the primary amine of 5-HT and the interaction between the C6-position of 5-HT and hSERT position 173 was confirmed using PaMLAC. The measured binding affinities of various mutants and 5-HT analogues allowed for a distinction between the two proposed binding modes of 5-HT and biochemically support the model for 5-HT binding in hSERT where the 5-hydroxyl group is in close proximity to Thr439.
authors.affiliation: The iNANO and inSPIN Centers, the Department of Chemistry, University of Aarhus, Aarhus, Denmark.
authors.forename: Leyla
authors.initials: L
authors.lastname: Celik
authors.forename: Steffen
authors.initials: S
authors.lastname: Sinning
authors.forename: Kasper
authors.initials: K
authors.lastname: Severinsen
authors.forename: Carsten G
authors.initials: CG
authors.lastname: Hansen
authors.forename: Maria S
authors.initials: MS
authors.lastname: Møller
authors.forename: Mikael
authors.initials: M
authors.lastname: Bols
authors.forename: Ove
authors.initials: O
authors.lastname: Wiborg
authors.forename: Birgit
authors.initials: B
authors.lastname: Schiøtt
mesh_terms: D001665:Binding Sites; D002460:Cell Line; D002478:Cells, Cultured; D003198:Computer Simulation; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D015394:Molecular Structure; D011485:Protein Binding; D012701:Serotonin / Q000031:analogs & derivatives / Q000737:chemistry* / Q000493:pharmacokinetics; D050486:Serotonin Plasma Membrane Transport Proteins / Q000737:chemistry*; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D014018:Tissue Distribution
chemical_list: D008024:Ligands; D050486:Serotonin Plasma Membrane Transport Proteins; D012701:Serotonin