title: Ligand Binding in the Extracellular Vestibule of the Neurotransmitter Transporter Homologue LeuT.
abstract: The human monoamine transporters (MATs) facilitate the reuptake of monoamine neurotransmitters from the synaptic cleft. MATs are linked to a number of neurological diseases and are the targets of both therapeutic and illicit drugs. Until recently, no high-resolution structures of the human MATs existed, and therefore, studies of this transporter family have relied on investigations of the homologues bacterial transporters such as the leucine transporter LeuT, which has been crystallized in several conformational states. A two-substrate transport mechanism has been suggested for this transporter family, which entails that high-affinity binding of a second substrate in an extracellular site is necessary for the substrate in the central binding site to be transported. Compelling evidence for this mechanism has been presented, however, a number of equally compelling accounts suggest that the transporters function through a mechanism involving only a single substrate and a single high-affinity site. To shed light on this apparent contradiction, we have performed extensive molecular dynamics simulations of LeuT in the outward-occluded conformation with either one or two substrates bound to the transporter. We have also calculated the substrate binding affinity in each of the two proposed binding sites through rigorous free energy simulations. Results show that substrate binding is unstable in the extracellular vestibule and the substrate binding affinity within the suggested extracellular site is very low (0.2 and 3.3 M for the two dominant binding modes) compared to the central substrate binding site (14 nM). This suggests that for LeuT in the outward-occluded conformation only a single high-affinity substrate binding site exists.
authors.affiliation: Center for Insoluble Protein Structures (inSPIN) and Interdisciplinary Nanoscience Center (iNANO), Department of Chemistry, Aarhus University , Langelandsgade 140, 8000 Aarhus C, Denmark.
authors.forename: Julie
authors.initials: J
authors.lastname: Grouleff
authors.affiliation: Center for Insoluble Protein Structures (inSPIN) and Interdisciplinary Nanoscience Center (iNANO), Department of Chemistry, Aarhus University , Langelandsgade 140, 8000 Aarhus C, Denmark.
authors.forename: Heidi
authors.initials: H
authors.lastname: Koldsø
authors.affiliation: Howard Hughes Medical Institute and Department of Pharmacology, University of California at San Diego , La Jolla, California 92093, United States.
authors.forename: Yinglong
authors.identifier: 0000-0003-3714-1395
authors.initials: Y
authors.lastname: Miao
authors.affiliation: Center for Insoluble Protein Structures (inSPIN) and Interdisciplinary Nanoscience Center (iNANO), Department of Chemistry, Aarhus University , Langelandsgade 140, 8000 Aarhus C, Denmark.
authors.forename: Birgit
authors.identifier: 0000-0001-9937-1562
authors.initials: B
authors.lastname: Schiøtt
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D005110:Extracellular Space / Q000378:metabolism; D006801:Humans; D007930:Leucine / Q000737:chemistry*; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D050480:Plasma Membrane Neurotransmitter Transport Proteins / Q000737:chemistry / Q000235:genetics*; D025341:Principal Component Analysis; D011487:Protein Conformation
chemical_list: D050480:Plasma Membrane Neurotransmitter Transport Proteins; D007930:Leucine
keywords: LeuT; free energy calculations; molecular dynamics; monoamine transporters