title: Two Na+ Sites Control Conformational Change in a Neurotransmitter Transporter Homolog.
abstract: In LeuT, a prokaryotic homolog of neurotransmitter transporters, Na(+) stabilizes outward-open conformational states. We examined how each of the two LeuT Na(+) binding sites contributes to Na(+)-dependent closure of the cytoplasmic pathway using biochemical and biophysical assays of conformation. Mutating either of two residues that contribute to the Na2 site completely prevented cytoplasmic closure in response to Na(+), suggesting that Na2 is essential for this conformational change, whereas Na1 mutants retained Na(+) responsiveness. However, mutation of Na1 residues also influenced the Na(+)-dependent conformational change in ways that varied depending on the position mutated. Computational analyses suggest those mutants influence the ability of Na1 binding to hydrate the substrate pathway and perturb an interaction network leading to the extracellular gate. Overall, the results demonstrate that occupation of Na2 stabilizes outward-facing conformations presumably through a direct interaction between Na(+) and transmembrane helices 1 and 8, whereas Na(+) binding at Na1 influences conformational change through a network of intermediary interactions. The results also provide evidence that N-terminal release and helix motions represent distinct steps in cytoplasmic pathway opening.
authors.affiliation: From the Departments of Pharmacology and.
authors.forename: Sotiria
authors.initials: S
authors.lastname: Tavoulari
authors.affiliation: From the Departments of Pharmacology and.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Margheritis
authors.affiliation: the Computational Structural Biology Section, NINDS, National Institutes of Health, Rockville, Maryland 20852.
authors.forename: Anu
authors.initials: A
authors.lastname: Nagarajan
authors.affiliation: Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520 and.
authors.forename: David C
authors.initials: DC
authors.lastname: DeWitt
authors.affiliation: From the Departments of Pharmacology and.
authors.forename: Yuan-Wei
authors.initials: YW
authors.lastname: Zhang
authors.affiliation: From the Departments of Pharmacology and.
authors.forename: Edwin
authors.initials: E
authors.lastname: Rosado
authors.affiliation: From the Departments of Pharmacology and.
authors.forename: Silvia
authors.initials: S
authors.lastname: Ravera
authors.affiliation: Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520 and.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Rhoades
authors.affiliation: the Computational Structural Biology Section, NINDS, National Institutes of Health, Rockville, Maryland 20852.
authors.forename: Lucy R
authors.identifier: http://orcid.org/0000-0003-1855-7985
authors.initials: LR
authors.lastname: Forrest
authors.affiliation: From the Departments of Pharmacology and gary.rudnick@yale.edu.
authors.forename: Gary
authors.identifier: http://orcid.org/0000-0002-7622-4110
authors.initials: G
authors.lastname: Rudnick
mesh_terms: D019943:Amino Acid Substitution; D026905:Amino Acid Transport Systems / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D059001:Aquatic Organisms / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D006090:Gram-Negative Bacteria / Q000378:metabolism*; D008024:Ligands; D008081:Liposomes; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D050480:Plasma Membrane Neurotransmitter Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D055550:Protein Stability; D011510:Proteolipids; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*
chemical_list: D026905:Amino Acid Transport Systems; D001426:Bacterial Proteins; D008024:Ligands; D008081:Liposomes; D050480:Plasma Membrane Neurotransmitter Transport Proteins; D011510:Proteolipids; D011994:Recombinant Proteins; C020485:proteoliposomes; D012964:Sodium; D003545:Cysteine
keywords: alternating access; conformational change; coupling; membrane transport; molecular dynamics; neurotransmitter; single-molecule biophysics; sodium; transport