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
Kamil Gotfryd
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
Flemming Hofmann Larsen
Quality and Technology, Department of Food Science, Faculty of Life Sciences, 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
Michel-Andreas Geiger
Leibniz-Institut für Molekulare Pharmakologie FMP, Berlin, Germany
Barth-Jan van Rossum
Leibniz-Institut für Molekulare Pharmakologie FMP, Berlin, Germany
Hartmut Oschkinat
Leibniz-Institut für Molekulare Pharmakologie FMP, Berlin, Germany
Ulrik Gether
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
Kaare Teilum
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
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.
