Substrate-induced Unlocking of the Inner Gate Determines the Catalytic Efficiency of a Neurotransmitter:Sodium Symporter

Billesbølle, Christian B.; Krüger, Mie B.; Shi, Lei; Quick, Matthias; Li, Zheng; Stolzenberg, Sebastian; Kniazeff, Julie; Gotfryd, Kamil; Mortensen, Jonas S.; Javitch, Jonathan A.; Weinstein, Harel; Løland, Claus J.; Gether, Ulrik

doi:10.1074/jbc.m115.677658

Cited by 35 publications

(56 citation statements)

References 58 publications

(64 reference statements)

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“…Thus donor and acceptor were in closer proximity in the presence of Na + . This is consistent with the results from previous studies [10–12, 15], which applied single molecule FRET to detergent solubilized LeuT. In the micelle environment, the distances were 6.26 ± 0.21 nm and 5.16 ± 0.22 nm in the absence and presence of 200 mM Na + , respectively.…”

Section: Resultssupporting

confidence: 92%

“…Substrate and two sodium ions bind to the substrate binding site (S1) located in the center of the membrane [3]. The structural dynamics of LeuT have been directly measured by fluorescence resonance energy transfer microscopy (FRET) [10–13], electron paramagnetic resonance (EPR) [14–17], and investigated by molecular dynamics (MD) simulations [12, 17–37]. These studies revealed that substrate transport by LeuT involves multiple conformations and that the structure of LeuT is highly dynamic.…”

Section: Introductionmentioning

confidence: 99%

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The Environment Shapes the Inner Vestibule of LeuT

et al. 2016

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Human neurotransmitter transporters are found in the nervous system terminating synaptic signals by rapid removal of neurotransmitter molecules from the synaptic cleft. The homologous transporter LeuT, found in Aquifex aeolicus, was crystallized in different conformations. Here, we investigated the inward-open state of LeuT. We compared LeuT in membranes and micelles using molecular dynamics simulations and lanthanide-based resonance energy transfer (LRET). Simulations of micelle-solubilized LeuT revealed a stable and widely open inward-facing conformation. However, this conformation was unstable in a membrane environment. The helix dipole and the charged amino acid of the first transmembrane helix (TM1A) partitioned out of the hydrophobic membrane core. Free energy calculations showed that movement of TM1A by 0.30 nm was driven by a free energy difference of ~15 kJ/mol. Distance measurements by LRET showed TM1A movements, consistent with the simulations, confirming a substantially different inward-open conformation in lipid bilayer from that inferred from the crystal structure.

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Section: Resultssupporting

confidence: 92%

Section: Introductionmentioning

confidence: 99%

The Environment Shapes the Inner Vestibule of LeuT

et al. 2016

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“…For LeuT WT the specific leucine peak was unaffected by lowering the free leucine concentration (Figure 2—figure supplement 1). The F253A mutant has previously been shown to impair binding to the S1 site (Billesbølle et al, 2015; Wang et al, 2012b). Thus, F253A serves as a S1 disturbing mutant at low substrate concentrations.…”

Section: Resultsmentioning

confidence: 99%

“…Expression of LeuT WT from Aquifex aeolicus was performed according to the protocol described previously (Billesbølle et al, 2015). LeuT WT was expressed in E. coli C41(DE3) transformed with pET16b encoding C-terminally 8xHis-tagged transporter (expression plasmid was kindly provided by Dr E. Gouaux, Vollum Institute, Portland, Oregon, USA).…”

Section: Methodsmentioning

confidence: 99%

Direct assessment of substrate binding to the Neurotransmitter:Sodium Symporter LeuT by solid state NMR

Erlendsson

Gotfryd

Larsen

et al. 2017

eLife

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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.DOI: http://dx.doi.org/10.7554/eLife.19314.001

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“…Expression and Purification of LeuT-Expression and purification of LeuT were performed as previously described (66). In short, E. coli strain C41 containing His-tagged LeuT in pET16b expression vector was cultivated in lysogeny broth with 75 g ml Ϫ1 ampicillin at 37°C shaking at 180 r.p.m.…”

Section: Experimental Methodsmentioning

confidence: 99%

Conformational Dynamics on the Extracellular Side of LeuT Controlled by Na+ and K+ Ions and the Protonation State of Glu290

Khelashvili

Schmidt

Shi

et al. 2016

Journal of Biological Chemistry

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Substrate-induced Unlocking of the Inner Gate Determines the Catalytic Efficiency of a Neurotransmitter:Sodium Symporter

Abstract: Background:The mechanism coupling substrate binding to transport in neurotransmitter: sodium symporters (NSSs) is poorly understood. Significance: The data add to our mechanistic understanding of Na ϩ -coupled transport across lipid bilayers.

Cited by 35 publications

References 58 publications

The Environment Shapes the Inner Vestibule of LeuT

The Environment Shapes the Inner Vestibule of LeuT

Direct assessment of substrate binding to the Neurotransmitter:Sodium Symporter LeuT by solid state NMR

Conformational Dynamics on the Extracellular Side of LeuT Controlled by Na+ and K+ Ions and the Protonation State of Glu290

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