title: Structure and regulatory interactions of the cytoplasmic terminal domains of serotonin transporter.
abstract: Uptake of neurotransmitters by sodium-coupled monoamine transporters of the NSS family is required for termination of synaptic transmission. Transport is tightly regulated by protein-protein interactions involving the small cytoplasmic segments at the amino- and carboxy-terminal ends of the transporter. Although structures of homologues provide information about the transmembrane regions of these transporters, the structural arrangement of the terminal domains remains largely unknown. Here, we combined molecular modeling, biochemical, and biophysical approaches in an iterative manner to investigate the structure of the 82-residue N-terminal and 30-residue C-terminal domains of human serotonin transporter (SERT). Several secondary structures were predicted in these domains, and structural models were built using the Rosetta fragment-based methodology. One-dimensional (1)H nuclear magnetic resonance and circular dichroism spectroscopy supported the presence of helical elements in the isolated SERT N-terminal domain. Moreover, introducing helix-breaking residues within those elements altered the fluorescence resonance energy transfer signal between terminal cyan fluorescent protein and yellow fluorescent protein tags attached to full-length SERT, consistent with the notion that the fold of the terminal domains is relatively well-defined. Full-length models of SERT that are consistent with these and published experimental data were generated. The resultant models predict confined loci for the terminal domains and predict that they move apart during the transport-related conformational cycle, as predicted by structures of homologues and by the "rocking bundle" hypothesis, which is consistent with spectroscopic measurements. The models also suggest the nature of binding to regulatory interaction partners. This study provides a structural context for functional and regulatory mechanisms involving SERT terminal domains.
authors.affiliation: Computational Structural Biology Group, Max Planck Institute of Biophysics , 60438 Frankfurt am Main, Germany.
authors.forename: Cristina
authors.initials: C
authors.lastname: Fenollar-Ferrer
authors.forename: Thomas
authors.initials: T
authors.lastname: Stockner
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Schwarz
authors.forename: Aritra
authors.initials: A
authors.lastname: Pal
authors.forename: Jelena
authors.initials: J
authors.lastname: Gotovina
authors.forename: Tina
authors.initials: T
authors.lastname: Hofmaier
authors.forename: Kumaresan
authors.initials: K
authors.lastname: Jayaraman
authors.forename: Suraj
authors.initials: S
authors.lastname: Adhikary
authors.forename: Oliver
authors.initials: O
authors.lastname: Kudlacek
authors.forename: Ahmad Reza
authors.initials: AR
authors.lastname: Mehdipour
authors.forename: Sotiria
authors.initials: S
authors.lastname: Tavoulari
authors.forename: Gary
authors.initials: G
authors.lastname: Rudnick
authors.forename: Satinder K
authors.initials: SK
authors.lastname: Singh
authors.forename: Robert
authors.initials: R
authors.lastname: Konrat
authors.forename: Harald H
authors.initials: HH
authors.lastname: Sitte
authors.forename: Lucy R
authors.initials: LR
authors.lastname: Forrest
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003593:Cytoplasm / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D050486:Serotonin Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins
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