Two Na+ Sites Control Conformational Change in a Neurotransmitter Transporter Homolog.
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.
['Tavoulari|Sotiria|S|', 'Margheritis|Eleonora|E|', 'Nagarajan|Anu|A|', 'DeWitt|David C|DC|', 'Zhang|Yuan-Wei|YW|', 'Rosado|Edwin|E|', 'Ravera|Silvia|S|', 'Rhoades|Elizabeth|E|', 'Forrest|Lucy R|LR|http://orcid.org/0000-0003-1855-7985', 'Rudnick|Gary|G|http://orcid.org/0000-0002-7622-4110']
D019943:Amino Acid Substitution D026905:Amino Acid Transport Systems D059001:Aquatic Organisms D001426:Bacterial Proteins D001665:Binding Sites D003545:Cysteine D006090:Gram-Negative Bacteria D008024:Ligands D008081:Liposomes D008958:Models, Molecular D056004:Molecular Dynamics Simulation D016297:Mutagenesis, Site-Directed D009154:Mutation D050480:Plasma Membrane Neurotransmitter Transport Proteins D011487:Protein Conformation D017510:Protein Folding D055550:Protein Stability D011510:Proteolipids D011994:Recombinant Proteins D012964:Sodium
conformational change neurotransmitter transport alternating access coupling membrane transport molecular dynamics single-molecule biophysics sodium