title: Direct assessment of substrate binding to the Neurotransmitter:Sodium Symporter LeuT by solid state NMR.
abstract: The Neurotransmitter:Sodium Symporters (NSSs) represent an important class of proteins mediating sodium-dependent uptake of neurotransmitters from the extracellular space. The substrate binding stoichiometry of the bacterial NSS protein, LeuT, and thus the principal transport mechanism, has been heavily debated. Here we used solid state NMR to specifically characterize the bound leucine ligand and probe the number of binding sites in LeuT. We were able to produce high-quality NMR spectra of substrate bound to microcrystalline LeuT samples and identify one set of sodium-dependent substrate-specific chemical shifts. Furthermore, our data show that the binding site mutants F253A and L400S, which probe the major S1 binding site and the proposed S2 binding site, respectively, retain sodium-dependent substrate binding in the S1 site similar to the wild-type protein. We conclude that under our experimental conditions there is only one detectable leucine molecule bound to LeuT.
authors.affiliation: Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, Copenhagen, Denmark.|Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Copenhagen, Denmark.|Molecular Neuropharmacology Laboratory, Department of Neuroscience and Pharmacology, University of Copenhagen, Copenhagen, Denmark.|Lundbeck Foundation Center for Biomembranes in Nanomedicine, University of Copenhagen, Copenhagen, Denmark.|Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Simon
authors.identifier: 0000-0002-6378-870X
authors.initials: S
authors.lastname: Erlendsson
authors.affiliation: Molecular Neuropharmacology Laboratory, Department of Neuroscience and Pharmacology, University of Copenhagen, Copenhagen, Denmark.|Lundbeck Foundation Center for Biomembranes in Nanomedicine, University of Copenhagen, Copenhagen, Denmark.|Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Kamil
authors.initials: K
authors.lastname: Gotfryd
authors.affiliation: Quality and Technology, Department of Food Science, Faculty of Life Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Flemming Hofmann
authors.initials: FH
authors.lastname: Larsen
authors.affiliation: Molecular Neuropharmacology Laboratory, Department of Neuroscience and Pharmacology, University of Copenhagen, Copenhagen, Denmark.|Lundbeck Foundation Center for Biomembranes in Nanomedicine, University of Copenhagen, Copenhagen, Denmark.|Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Jonas Sigurd
authors.identifier: 0000-0002-6743-276X
authors.initials: JS
authors.lastname: Mortensen
authors.affiliation: Leibniz-Institut für Molekulare Pharmakologie FMP, Berlin, Germany.
authors.forename: Michel-Andreas
authors.initials: MA
authors.lastname: Geiger
authors.affiliation: Leibniz-Institut für Molekulare Pharmakologie FMP, Berlin, Germany.
authors.forename: Barth-Jan
authors.initials: BJ
authors.lastname: van Rossum
authors.affiliation: Leibniz-Institut für Molekulare Pharmakologie FMP, Berlin, Germany.
authors.forename: Hartmut
authors.initials: H
authors.lastname: Oschkinat
authors.affiliation: Molecular Neuropharmacology Laboratory, Department of Neuroscience and Pharmacology, University of Copenhagen, Copenhagen, Denmark.|Lundbeck Foundation Center for Biomembranes in Nanomedicine, University of Copenhagen, Copenhagen, Denmark.|Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Ulrik
authors.initials: U
authors.lastname: Gether
authors.affiliation: Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, Copenhagen, Denmark.|Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Kaare
authors.initials: K
authors.lastname: Teilum
authors.affiliation: Molecular Neuropharmacology Laboratory, Department of Neuroscience and Pharmacology, University of Copenhagen, Copenhagen, Denmark.|Lundbeck Foundation Center for Biomembranes in Nanomedicine, University of Copenhagen, Copenhagen, Denmark.|Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Claus J
authors.identifier: 0000-0002-1773-1446
authors.initials: CJ
authors.lastname: Loland
mesh_terms: D001419:Bacteria / Q000201:enzymology; D001665:Binding Sites; D007930:Leucine / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D050480:Plasma Membrane Neurotransmitter Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D050480:Plasma Membrane Neurotransmitter Transport Proteins; D007930:Leucine
keywords: LeuT; aquifex aeolicus; biophysics; neuroscience; neurotransmitter transporters; solid state NMR; structural biology; substrate binding site