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A mechanism for intracellular release of Na+ by neurotransmitter/sodium symporters

Nature Structural & Molecular Biology volume 21, pages 1006–1012 (2014)Cite this article

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Abstract

Neurotransmitter/sodium symporters (NSSs) terminate synaptic signal transmission by Na+-dependent reuptake of released neurotransmitters. Key conformational states have been reported for the bacterial homolog LeuT and an inhibitor-bound Drosophila dopamine transporter. However, a coherent mechanism of Na+-driven transport has not been described. Here, we present two crystal structures of MhsT, an NSS member from Bacillus halodurans, in occluded inward-facing states with bound Na+ ions and L-tryptophan, providing insight into the cytoplasmic release of Na+. The switch from outward- to inward-oriented states is centered on the partial unwinding of transmembrane helix 5, facilitated by a conserved GlyX9Pro motif that opens an intracellular pathway for water to access the Na2 site. We propose a mechanism, based on our structural and functional findings, in which solvation through the TM5 pathway facilitates Na+ release from Na2 and the transition to an inward-open state.

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Figure 1: MhsT uptake and binding.
Figure 2: MhsT structure.
Figure 3: Comparison of MhsT and LeuT in different functional states.
Figure 4: A proposed TM5 spring mechanism.
Figure 5: Conservation of transmembrane helix 5 GlyX9Pro motif and mutational studies.
Figure 6: Transport mechanism of the NSS family.

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Acknowledgements

We thank A.M. Winther for initial work on crystallization of detergent-solubilized MhsT, A.M. Nielsen for technical assistance and J.L. Karlsen for support with scientific computing. We are grateful to L. Shi and H. Weinstein for valuable discussion. We thank S.G. Rasmussen for access to a LCP dispensing robot. We are thankful to D. Flot and S. Russi at the European Synchrotron Radiation Facility ID23-2, U. Müller and M.S. Weiss at the Helmholtz-Zentrum Berlin synchrotron radiation source BESSY II BL 14.1 and 14.3, and R. Owen and D. Axford at the Diamond Light Source I24 for help with X-ray diffraction data collection. Access to synchrotron facilities was supported by the Danscatt program of the Danish Council for Independent Research and the EU-FP7 infrastructure program Biostruct-X (grants 860 and 5624 to P.N.). This work was supported by research grants from the Lundbeck Foundation (to P.N.) and by US National Institutes of Health grants DA17293 and DA022413 (to J.A.J.). L.M. was supported by a Boehringer-Ingelheim Fonds fellowship. L.R. was supported by the Danish Council for Independent Research in Medical Sciences, and J.A.L. was supported by the Danish Council for Independent Research in Natural Sciences.

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Author notes

  1. Linda Reinhard & Hideaki Yano

    Present address: Present addresses: Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden, and Department of Cell and Molecular Biology, Karolinska Institutet, Hamburg, Germany (L.R.), and National Institute on Drug Abuse, Intramural Research Program, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland, USA (H.Y.).,

Authors and Affiliations

  1. Danish Research Institute of Translational Neuroscience (DANDRITE), Nordic EMBL Partnership for Molecular Medicine, Aarhus University, Aarhus, Denmark

    Lina Malinauskaite, Linda Reinhard, Joseph A Lyons & Poul Nissen

  2. Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

    Lina Malinauskaite, Linda Reinhard, Joseph A Lyons & Poul Nissen

  3. Center for Molecular Recognition, Columbia University College of Physicians and Surgeons, New York, New York, USA.,

    Matthias Quick, Hideaki Yano & Jonathan A Javitch

  4. Department of Psychiatry, Columbia University College of Physicians and Surgeons, New York, New York, USA.,

    Matthias Quick & Jonathan A Javitch

  5. Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York, USA.,

    Matthias Quick & Jonathan A Javitch

  6. Department of Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York, USA.,

    Jonathan A Javitch

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  1. Lina Malinauskaite
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Contributions

The functional characterization was performed by M.Q., H.Y. and L.M. HiLiDe crystallization, data collection and structure determination were performed by L.M. with assistance from L.R. LCP crystallization and data collection were performed by L.M. and J.A.L., and structure determination was performed by L.M. The manuscript was written by L.M., M.Q., J.A.J. and P.N., and all authors commented on the manuscript.

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Correspondence to Matthias Quick or Poul Nissen.

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Integrated supplementary information

Supplementary Figure 1 MhsT and LeuT architecture.

a, Cartoon structure representation and topology diagram for MhsT in the occluded inward-facing state and b, LeuT in the occluded outward-facing state (PDB 2A65). Important structural elements for conformational changes between these states are highlighted. Color scheme: scaffold domain (TM 3-4, 8-9) – grey; bundle elements – TM1 red, TM6 yellow, TM7 and EL4 pink and TM2 orange; TM5 – cyan.

Supplementary Figure 2 Alignment of bacterial and eukaryotic members of the NSS family.

The sequence alignment of helices 1, 4, 5 and 8, which are involved in the formation of the Na2 site and the cytoplasmic pathway leading to it, are shown. Bacterial NSS members: MhsT_Bh (multi-hydrophobic amino acid transporter, Bacillus halodurans, NP_241994), LeuT_Aa (leucine transporter, Aquifex aeolicus, O67854), TnaT_St (tryptophan transporter, Symbiobacterium thermophilum, O50649), MetP_Cg (methionine/alanine transporter, Corynebacterium glutamicum, Q8NRL8), MJ1319_Mj (hypothetical Na+-dependent transporter, Methanococcus jannaschii, Q58715); animal transporters: SERT_Hs (Na+-dependent serotonin transporter, Homo sapiens, P31645), NET_Hs (Na+-dependent noradrenaline transporter, Homo sapiens, P23975), DAT_Dm (Na+-dependent dopamine transporter, Drosophila melanogaster, Q9NB97), GLYT-1_Rn (Na+- and chloride-dependent glycine transporter 1, Rattus norvegicus, P28572) AChT_Ce (Na+-dependent acetylcholine transporter, Caenorhabditis elegans, O76689), GAT-1_Hs (Na+- and chloride-dependent γ-aminobutyric transporter 1, Homo sapiens, P30531). Helix positions are shown according to the MhsT structure with the unwound region of TM5 in light blue. The Na+ coordinating residues of Na2 are marked by triangles: residues that coordinate Na+ by side chains (filled) and by backbone atoms (empty). The intracellular gate residues are indicated by circles: salt bridges and a hydrophobic plug (filled) and residues interacting with dipoles of TM5 (+) and TM4 (-) (empty). The kink between TM4-5 (empty stars) and the TM5 helix breaking motif Gly(X3-4)Gly(X9)Pro (filled stars) are conserved among all NSS family members. The highly conserved Trp33MhsT situated at the extracellular vestibule is indicated by diamond.

Supplementary Figure 3 Comparison of MhsT and LeuT substrate and Na+-binding sites.

a, L-tryptophan (2Fo-Fc map at 2 r.m.s.d.) carboxyl and amino groups are bound at the MhsT active site through hydrogen bonds to the side chains of Tyr108, Ser233 and backbone atoms of Ala26, Gly30, Phe230 and Thr231. In addition the L-tryptophan carboxyl group coordinates Na+ at the Na1 site. The indole ring is bound only through a hydrogen bond to Ser327Oγ. Met236 is moved away from the binding site, making space for the bulky L-tryptophan side chain. b, LeuT binding site with bound L-leucine in the occluded outward-facing state (PDB 2A65). c, LeuT mutation I359Q29 allows L-tryptophan to bind in an occluded outward-facing state (PDB 3QS5)29, but with a different rotamer than in the MhsT-Trp structure as seen in (a). d, Wild-type LeuT with bound L-tryptophan in an outward-open state (PDB 3F3A)8, where L-tryptophan pushes the LeuT binding site open. e-h, Na1 and Na2 binding sites in MhsT and LeuT. LeuT (PDB 2A657, grey) and MhsT (this study, light blue) superimposed by structural alignment of scaffold helices (TM3-4, TM8-9) (e and g), and simulated annealing omit maps contoured at 5.0 r.m.s.d. for Na+ sites in MhsT (f and h). e-f, Structures of the Na1 site of MhsT and LeuT are similar, but MhsT has an additional negative charge – Asp263 (Asn286LeuT). LeuT has a Glu290LeuT residue one helix turn away from the Na1 site that was associated with proton antiport in bacterial NSS members61. At the equivalent position MhsT has Ala267, with no such capacity, thus Asp263 is a likely other candidate for this role in proton antiport61. g-h, The Na2 site of MhsT in the occluded inward-facing state exhibits a more open structure than the Na2 site in the LeuT occluded outward-facing state: helix 8 residues Ala320 and Ser323 are moved away from helix 1 making space for an additional ligand - a water molecule, which is in contact with the intracellular environment. The Na+ coordination geometry changes from trigonal-bipyramidal (LeuT, grey dashed lines) to octahedral (MhsT, light green dashed lines).

Supplementary Figure 4 The intracellular-gate interactions in MhsT and LeuT.

Dense network of conserved residues (yellow sticks) of TM1a (red), TM5 (cyan) and neighboring helices forming the intracellular gate. a, LeuT occluded outward-facing (PDB 2A65)7, b, MhsT occluded inward-facing, and c, LeuT inward-open (PDB 3TT3)10 states.

Supplementary Figure 5 A conserved TM5 proline in LeuT-fold transporters.

a-b, Comparison of TMs 1, 4, 5 and 8 of MhsT (occluded inward-facing) and Mhp1 occluded outward-facing (PDB 2JLO)15 structures. c, Structural alignment of MhsT occluded inward-facing and Mhp1_Ml (benzyl-hydantoin transporter from Microbacterium liquefaciens, 210060745) occluded outward-facing (prepared by Chimera59, helix position shown for MhsT) states. TM5 helix breaking Gly/Pro of MhsT are marked by stars: conserved GlyX9Pro motif of the NSS family (filled) and the kink between TM4-5 (empty). d, Sequence alignment (generated with Muscle) of the nucleobase:cation symporter-1 (NCS1) family: HyuP_Aa (probable hydantoin permease of Arthrobacter aurescens, Q9F467); CodB_Ec (cytosine permease of Escherichia coli, P0AA82); PucI_Bs (allantoin permease of Bacillus subtilis, NP_391528.1); MtlP_Sf (putative mannitol transporter of Shewanella frigidimarina, Q082R8); FycB_An (cytosine-purine-scavenging protein of Aspergillus nidulans, B1PXD0); Fcy21_Ca (hypoxanthine/adenine/guanine (purine) transporter of Candida albicans, Q708J7); YbbW_Ec (allantoin permease of E. coli, P75712). Conserved Pro of the NCS1 family marked as filled squares, flexible Gly position as empty squares with helix positions shown for Mhp1. The sequence alignment was illustrated with Jalview59. Note the correspondence of Gly/Pro residues in TM4-TM5 of Mhp1/NCS1 family to the Gly(X3-4)Gly(X9)Pro motif of NSS that suggests a similar TM5 unwinding mechanism for Na2 release in the NCS1 family.

Supplementary Figure 6 Structural alignment of MhsT to ApcT, vSGLT and BetP prepared with DaliLite.

The structural alignment of MhsT to the LeuT-fold symporters: ApcT occluded inward-facing state (PDB 3GIA), vSGLT occluded inward-facing state (PDB 3DH4)16 and BetP fully-occluded state (PDB 4AIN chain B)17. TM1 is shown in red and pink, TM5 in cyan; substrates are shown as yellow spheres, Na+ ions as green spheres and proton binding residue for ApcT as orange spheres. The helix breaker in MhsT (Pro181) and potential helix breaker in vSGLT (Gly166-Gly167) are shown as cyan sphere. ApcT does not have any helix breaking residues in TM5, however the proposed H+ binding residue Lys158 is at a similar position (orange rectangle) to the Pro181 in MhsT (cyan rectangle). vSGLT in the crystal structure of occluded inward-facing state has missing residues at the TM4-TM5 turn (Tyr179-Gly180-Gly181-Leu182-Ser183-Ala184), probably due to their flexibility. Furthermore, vSGLT has two glycines (Gly166-Gly167, cyan rectangle) one helix-turn downstream from the Pro181 in MhsT. However, it is not clear if it would have similar effect as in MhsT. BetP does not have any glycines or prolines in TM5.

Supplementary Figure 7 Crystal packing of HiLiDe and LCP structures of MhsT.

a, Crystal packing of MhsT in the P2 crystal form from HiLiDe crystallization. The N-terminus of TM1a is unperturbed by the crystal packing and interacts with TM5. b, The C2 crystal form from lipid cubic phase crystallization. Crystal packing is tighter with the N-terminus displaced and disordered; TM5 adopts a less extended form as a near-continuous helix. c, Superpositioning of the two MhsT structures: the HiLiDe structure (grey, TM1a red, TM5 cyan) superimposed on the MhsTLCP (yellow). d, Superposition of MhsT HiLiDe structure (black) on LCP structure crystal packing (yellow) showing that the N-terminus is replaced by a loop of a neighboring molecule. e, Stereo view of TM5 electron density maps in MhsT HiLiDe and LCP structures.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Tables 1 and 2, and Supplementary Note (PDF 2563 kb)

Morph between MhsT HiLiDe and LCP structures.

The movie depicts the transition between the occluded inward-facing structures of MhsT obtained with HiLiDe and LCP structures. The movie was prepared by 'morphing' from the HiLiDe structure to the LCP and back. TM5 (cyan helix) is in an unwound conformation in the HiLiDe structure, stabilized by the N-terminus of TM1 (red helix). Upon dislocation of the N-terminal segment TM5 reforms a kinked helix as observed in the LeuT7,10 and dDAT11 structures. Na+ ions at the Na1 and Na2 sites are shown as green, and tryptophan at substrate binding site as yellow spheres. (MOV 3693 kb)

NSS family transport cycle.

The movie depicts the transport cycle of the NSS family going from outward-facing forms via the occluded inward-facing state (presented here) to inward-open form. The movie 'morphs' between available LeuT structures – outward-open and Na+-bound10 (PDB 3TT1), leucine and Na+-bound occluded outward-facing7 (PDB 2A65), inward open apo structures10 (PDB 3TT3). For compatibility in morphing procedures, a leucine and sodium-bound occluded inward-facing state was modeled for LeuT based on the MhsT structure using PHENIX SCULPTOR52. The structures were aligned to the scaffold domain (TMs 3-4, 8-9). TM5 is shown as cyan helix, TM1 as red helix, Na+ ions at Na1 and Na2 sites are shown as green, leucine as orange, Gly190 and Pro200 of GlyX9Pro as cyan and Leu29 (corresponding to Trp33MhsT) as red spheres. Outward closure occurs at the extracellular vestibule and inward opening at the Na2 site occurs by unwinding of TM5 at the conserved GlyX9Pro motif and most likely in concerted movements with the N-terminal segment. (MOV 8688 kb)

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Malinauskaite, L., Quick, M., Reinhard, L. et al. A mechanism for intracellular release of Na+ by neurotransmitter/sodium symporters. Nat Struct Mol Biol 21, 1006–1012 (2014). https://doi.org/10.1038/nsmb.2894

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