title: Conformational dynamics of a neurotransmitter:sodium symporter in a lipid bilayer.
abstract: Neurotransmitter:sodium symporters (NSSs) are integral membrane proteins responsible for the sodium-dependent reuptake of small-molecule neurotransmitters from the synaptic cleft. The symporters for the biogenic amines serotonin (SERT), dopamine (DAT), and norepinephrine (NET) are targets of multiple psychoactive agents, and their dysfunction has been implicated in numerous neuropsychiatric ailments. LeuT, a thermostable eubacterial NSS homolog, has been exploited as a model protein for NSS members to canvass the conformational mechanism of transport with a combination of X-ray crystallography, cysteine accessibility, and solution spectroscopy. Despite yielding remarkable insights, these studies have primarily been conducted with protein in the detergent-solubilized state rather than embedded in a membrane mimic. In addition, solution spectroscopy has required site-specific labeling of nonnative cysteines, a labor-intensive process occasionally resulting in diminished transport and/or binding activity. Here, we overcome these limitations by reconstituting unlabeled LeuT in phospholipid bilayer nanodiscs, subjecting them to hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS), and facilitating interpretation of the data with molecular dynamics simulations. The data point to changes of accessibility and dynamics of structural elements previously implicated in the transport mechanism, in particular transmembrane helices (TMs) 1a and 7 as well as extracellular loops (ELs) 2 and 4. The results therefore illuminate the value of this strategy for interrogating the conformational mechanism of the more clinically significant mammalian membrane proteins including SERT and DAT, neither of which tolerates complete removal of endogenous cysteines, and whose activity is heavily influenced by neighboring lipids.
authors.affiliation: Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520.
authors.forename: Suraj
authors.initials: S
authors.lastname: Adhikary
authors.affiliation: Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Deredge
authors.affiliation: Computational Structural Biology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.
authors.forename: Anu
authors.initials: A
authors.lastname: Nagarajan
authors.affiliation: Computational Structural Biology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.
authors.forename: Lucy R
authors.identifier: 0000-0003-1855-7985
authors.initials: LR
authors.lastname: Forrest
authors.affiliation: Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201.
authors.forename: Patrick L
authors.initials: PL
authors.lastname: Wintrode
authors.affiliation: Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520; satinder.k.singh@yale.edu.
authors.forename: Satinder K
authors.initials: SK
authors.lastname: Singh
mesh_terms: D001679:Biogenic Amines / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D004298:Dopamine / Q000737:chemistry* / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D056004:Molecular Dynamics Simulation; D018377:Neurotransmitter Agents / Q000737:chemistry* / Q000378:metabolism; D009638:Norepinephrine / Q000737:chemistry / Q000378:metabolism; D012701:Serotonin / Q000737:chemistry* / Q000378:metabolism; D050608:Sodium-Phosphate Cotransporter Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001679:Biogenic Amines; D008051:Lipid Bilayers; D008565:Membrane Proteins; D018377:Neurotransmitter Agents; D050608:Sodium-Phosphate Cotransporter Proteins; D012701:Serotonin; D003545:Cysteine; D004298:Dopamine; D009638:Norepinephrine
keywords: conformational dynamics; hydrogen–deuterium exchange mass spectrometry; molecular dynamics simulations; nanodisc; neurotransmitter symporter