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.

Transmembrane Domain 6 of the Human Serotonin Transporter Contributes to an Aqueously Accessible Binding Pocket for Serotonin and the Psychostimulant 3,4-Methylene Dioxymethamphetamine

“…In LeuT Aa , TMs 1, 3, 6, and 8 form the leucine binding pocket. Biochemical analyses and homology modeling of SERT proteins predict a similar binding pocket for 5-HT (32)(33)(34)(35)(36). Consistent with these models, pre-structure studies identified residues in hSERT TMs 1 and 3 that confer high affinity interactions and ligand selectivity to substrates and antagonists.…”

“…Impact of Na ϩ on MTSET Inactivation-The LeuT Aa crystal structure and hSERT homology models derived from it predict that TM6 is involved in the coordination of a sodium ion (27,(32)(33)(34)36). Interestingly, replacement of sodium with NMDG in the MTS incubation buffer resulted in protection of three TM6 Cys mutants and exposure of a single mutant (Fig.…”

Binding of the Amphetamine-like 1-Phenyl-piperazine to Monoamine Transporters

“…Since threonine is smaller than isoleucine the loss of affinity cannot be ascribed to a simple steric repulsion; rather it seems to be an effect of changing the hydrophobic nature of isoleucine to the more hydrophilic threonine. From our previous study, 16 it is known that mutation of Ala173 and Thr439 can affect the environment in a hydrophilic pocket housing the hydroxyl group of the cognate substrate, 5-HT. The K i values of 2-OEt-PP in the Ala173Ser, Ala173Met, Thr439Ser, and Thr439Ala mutants suggest that the hydrophobic moiety of the OEt-group can reach into this pocket.…”

Synthesis and serotonin transporter activity of 1,3-bis(aryl)-2-nitro-1-propenes as a new class of anticancer agents

“…Currently, there is no existing crystal structure of SERT; however, a number of homology models of hSERT are available which were constructed using the bacterial leucine transporter (LeuT) as a template 32,33 . For this study the homology model of hSERT constructed by Jørgensen et al (2007) was used 33 .…”

“…5-HT, the natural substrate of SERT, and amphetamines such as 4-MTA interact with residues Ala96, Asp98 and Phe335 via binding of the protonated amine (Fig. 3A), while the tricyclic antidepressant imipramine is also thought to interact strongly with the Asp98 residue 13,32,37 . A study by Koldsø et al (2010) discovered that the two enantiomers of the SSRI citalopram bind to a central binding site of the hSERT homology model with reversed orientations 38 .…”