title: Identification of a chloride ion binding site in Na+/Cl -dependent transporters.
abstract: The recent determination of the crystal structure of the leucine transporter from Aquifex aeolicus (aaLeuT) has provided significant insights into the function of neurotransmitter:sodium symporters. Transport by aaLeuT is Cl(-) independent, whereas many neurotransmitter:sodium symporters from higher organisms depend on Cl(-) ions. However, the only Cl(-) ion identified in the aaLeuT structure interacts with nonconserved residues in extracellular loops, and thus the relevance of this binding site is unclear. Here, we use calculations of pK(A)s and homology modeling to predict the location of a functionally important Cl(-) binding site in serotonin transporter and other Cl(-)-dependent transporters. We validate our model through the site-directed mutagenesis of residues predicted to coordinate the Cl(-) ion and through the observation of sequence conservation patterns in other Cl(-)-dependent transporters. The proposed site is located midway across the membrane and is formed by residues from transmembrane helices 2, 6, and 7. It is close to the Na1 sodium binding site, thus providing an explanation for the coupling of Cl(-) and Na(+) ions during transport. Other implications of the model are also discussed.
authors.affiliation: Howard Hughes Medical Institute, Center for Computational Biology and Bioinformatics, and Department of Biochemistry and Molecular Biophysics, Columbia University, 1130 St. Nicholas Avenue, Room 815, New York, NY 10032, USA.
authors.forename: Lucy R
authors.initials: LR
authors.lastname: Forrest
authors.forename: Sotiria
authors.initials: S
authors.lastname: Tavoulari
authors.forename: Yuan-Wei
authors.initials: YW
authors.lastname: Zhang
authors.forename: Gary
authors.initials: G
authors.lastname: Rudnick
authors.forename: Barry
authors.initials: B
authors.lastname: Honig
mesh_terms: D000595:Amino Acid Sequence; D026923:Amino Acid Transport Systems, Basic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002712:Chlorides / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007477:Ions / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D050480:Plasma Membrane Neurotransmitter Transport Proteins / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011522:Protons
chemical_list: D026923:Amino Acid Transport Systems, Basic; D001426:Bacterial Proteins; D002712:Chlorides; D007477:Ions; D026901:Membrane Transport Proteins; D050480:Plasma Membrane Neurotransmitter Transport Proteins; D011522:Protons; C065171:cif nucleoside transporter