title: Genetically encoded photocrosslinkers locate the high-affinity binding site of antidepressant drugs in the human serotonin transporter.
abstract: Despite the well-established role of the human serotonin transporter (hSERT) in the treatment of depression, the molecular details of antidepressant drug binding are still not fully understood. Here we utilize amber codon suppression in a membrane-bound transporter protein to encode photocrosslinking unnatural amino acids (UAAs) into 75 different positions in hSERT. UAAs are incorporated with high specificity, and functionally active transporters have similar transport properties and pharmacological profiles compared with wild-type transporters. We employ ultraviolet-induced crosslinking with p-azido-L-phenylalanine (azF) at selected positions in hSERT to map the binding site of imipramine, a prototypical tricyclic antidepressant, and vortioxetine, a novel multimodal antidepressant. We find that the two antidepressants crosslink with azF incorporated at different positions within the central substrate-binding site of hSERT, while no crosslinking is observed at the vestibular-binding site. Taken together, our data provide direct evidence for defining the high-affinity antidepressant binding site in hSERT.
authors.affiliation: Department of Drug Design and Pharmacology, Center for Biopharmaceuticals, University of Copenhagen, Jagtvej 162, DK-2100 Copenhagen, Denmark.|Laboratory of Chemical Biology &Signal Transduction, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA.
authors.forename: Hafsteinn
authors.initials: H
authors.lastname: Rannversson
authors.affiliation: Department of Drug Design and Pharmacology, Center for Biopharmaceuticals, University of Copenhagen, Jagtvej 162, DK-2100 Copenhagen, Denmark.
authors.forename: Jacob
authors.initials: J
authors.lastname: Andersen
authors.affiliation: Department of Drug Design and Pharmacology, Center for Biopharmaceuticals, University of Copenhagen, Jagtvej 162, DK-2100 Copenhagen, Denmark.
authors.forename: Lena
authors.initials: L
authors.lastname: Sørensen
authors.affiliation: Department of Drug Design and Pharmacology, Center for Biopharmaceuticals, University of Copenhagen, Jagtvej 162, DK-2100 Copenhagen, Denmark.|Lundbeck Research Denmark, H. Lundbeck A/S, Ottiliavej 9, DK-2500 Valby, Denmark.
authors.forename: Benny
authors.initials: B
authors.lastname: Bang-Andersen
authors.affiliation: Laboratory of Chemical Biology &Signal Transduction, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA.
authors.forename: Minyoung
authors.initials: M
authors.lastname: Park
authors.affiliation: Laboratory of Chemical Biology &Signal Transduction, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Huber
authors.affiliation: Laboratory of Chemical Biology &Signal Transduction, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA.|Department of Neurobiology, Care Sciences and Society, Center for Alzheimer Research, Karolinska Institutet, Huddinge 141 57, Sweden.
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Sakmar
authors.affiliation: Department of Drug Design and Pharmacology, Center for Biopharmaceuticals, University of Copenhagen, Jagtvej 162, DK-2100 Copenhagen, Denmark.
authors.forename: Kristian
authors.initials: K
authors.lastname: Strømgaard
mesh_terms: D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000928:Antidepressive Agents / Q000378:metabolism* / Q000494:pharmacology* / Q000627:therapeutic use; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D003863:Depression / Q000188:drug therapy / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055668:Photochemical Processes / Q000528:radiation effects; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D050486:Serotonin Plasma Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014466:Ultraviolet Rays
chemical_list: D000596:Amino Acids; D000928:Antidepressive Agents; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; D010649:Phenylalanine