Structural determinants of species‐selective substrate recognition in human and Drosophila serotonin transporters revealed through computational docking studies

Abstract

To identify potential determinants of substrate selectivity in serotonin (5-HT) transporters (SERT), models of human and Drosophila serotonin transporters (hSERT, dSERT) were built based on the leucine transporter (LeuT Aa ) structure reported by Yamashita et al. (Nature 2005;437:215-223), PBDID 2A65. Although the overall amino acid identity between SERTs and the LeuT Aa is only 17%, it increases to above 50% in the first shell of the putative 5-HT binding site, allowing de novo computational docking of tryptamine derivatives in atomic detail. Comparison of hSERT and dSERT complexed with substrates pinpoints likely structural determinants for substrate binding. Forgoing the use of experimental transport and binding data of tryptamine derivatives for construction of these models enables us to cHitically assess and validate their predictive power: A single 5-HT binding mode was identified that retains the amine placement observed in the LeuT Aa structure, matches sitedirected mutagenesis and substituted cysteine accessibility method (SCAM) data, complies with support vector machine derived relations activity relations, and predicts computational binding energies for 5-HT analogs with a significant correlation coefficient (R = 0.72). This binding mode places 5-HT deep in the binding pocket of the SERT with the 5-position near residue hSERT A169/ dSERT D164 in transmembrane helix 3, the indole nitrogen next to residue Y176/Y171, and the ethylamine tail under residues F335/F327 and S336/S328 within 4 Å of residue D98. Our studies identify a number of potential contacts whose contribution to substrate binding and transport was previously unsuspected.★Correspondence to: Jens Meiler,

Transgenic elimination of high-affinity antidepressant and cocaine sensitivity in the presynaptic serotonin transporter

“…We suspect the inability of the SERT M172 substitution to reduce paroxetine potency reflects a distinct orientation of paroxetine compared with other SSRIs in the 5-HT binding pocket, particularly given that high-affinity SSRI recognition at SERT can be strongly influenced by relatively small changes in transporter structure (34). SERT M172 cell lines should be useful in further exploring the physical basis of interactions of different antidepressants in vitro (26), whereas the SERT M172 mice should help define the requirements for SERT and 5-HT signaling in the in vivo actions of antidepressants and cocaine. Although significant evidence supports a primary role for the 5-HT transporter in the reinforcing properties of psychostimulants (35)(36)(37), SERT blockade also appears to contribute to reinforcement (38)(39)(40).…”

“…Based on hydrophobicity assessments and high-resolution crystal structures of the SLC6 family member LeuT Aa , SERT is proposed to contain 12 transmembrane domains (TMs), four of which-TMs 1, 3, 6, and 8-provide the key residues for ion and substrate recognition (24)(25)(26)(27). By using the pharmacological differences displayed by human and Drosophila melanogaster SERTs (hSERT and dSERT), we identified variation at a single residue in TM3 (I172 in human and mouse, M167 in fly) proximal to the proposed binding site for 5-HT (28).…”

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.…”

“…Because hSERT TM6 is predicted to lie adjacent to and share topology with TM1 (27,(32)(33)(34)(35)(36), we explored potential contributions of TM6 to the interactions of 5-HT, MDMA, and cocaine via an assessment of their ability to protect hSERT C109A Cys mutants from inactivation by MTSET and biotinylation with MTSEA-biotin. Before initiation of protection experiments, we examined the sensitivity of each Cys mutant to MTSET at 0.1, 1, and 10 mM to ensure we were using a concentration of MTSET appropriate for detecting either protection or exposure.…”