title: Properties of an inward-facing state of LeuT: conformational stability and substrate release.
abstract: The leucine transporter (LeuT) is a bacterial homolog of the human monoamine transporters, which are important pharmaceutical targets. There are no high-resolution structures of the human transporters available; however, LeuT has been crystallized in several different conformational states. Recently, an inward-facing conformation of LeuT was solved revealing an unexpectedly large movement of transmembrane helix 1a (TM1a). We have performed molecular dynamics simulations of the mutated and wild-type transporter, with and without the cocrystallized Fab antibody fragment, to investigate the properties of this inward-facing conformation in relation to transport by LeuT within the membrane environment. In all of the simulations, local conformational changes with respect to the crystal structure are consistently observed, especially in TM1a. Umbrella sampling revealed a soft potential for TM1a tilting. Furthermore, simulations of inward-facing LeuT with Na(+) ions and substrate bound suggest that one of the Na(+) ion binding sites is fully disrupted. Release of alanine and the second Na(+) ion is also observed, giving insight into the final stage of the translocation process in atomistic detail.
authors: Center for Insoluble Protein Structures and Interdisciplinary Nanoscience Center, Department of Chemistry, Aarhus University, Aarhus, Denmark.
authors: Julie
authors: J
authors: Grouleff
authors: Center for Insoluble Protein Structures and Interdisciplinary Nanoscience Center, Department of Chemistry, Aarhus University, Aarhus, Denmark.
authors: Siri
authors: S
authors: Søndergaard
authors: Center for Insoluble Protein Structures and Interdisciplinary Nanoscience Center, Department of Chemistry, Aarhus University, Aarhus, Denmark.
authors: Heidi
authors: H
authors: Koldsø
authors: Center for Insoluble Protein Structures and Interdisciplinary Nanoscience Center, Department of Chemistry, Aarhus University, Aarhus, Denmark. Electronic address: birgit@chem.au.dk.
authors: Birgit
authors: B
authors: Schiøtt
mesh_terms: D000409:Alanine / Q000737:chemistry; D026905:Amino Acid Transport Systems / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D007477:Ions / Q000737:chemistry; D056004:Molecular Dynamics Simulation; D009154:Mutation; D050480:Plasma Membrane Neurotransmitter Transport Proteins / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D055550:Protein Stability; D012964:Sodium / Q000737:chemistry
chemical_list: D026905:Amino Acid Transport Systems; D001426:Bacterial Proteins; D007477:Ions; D050480:Plasma Membrane Neurotransmitter Transport Proteins; D012964:Sodium; D000409:Alanine