1. Structural Biology and Molecular Biophysics

Binding and transport of D-aspartate by the glutamate transporter homologue GltTk

  1. Valentina Arkhipova
  2. Gianluca Trinco
  3. Thijs W Ettema
  4. Sonja Jensen
  5. Dirk Slotboom  Is a corresponding author
  6. Albert Guskov  Is a corresponding author
  1. University of Groningen, Netherlands
https://doi.org/10.7554/eLife.45286
Share this article
Cite this article
  1. Valentina Arkhipova
  2. Gianluca Trinco
  3. Thijs W Ettema
  4. Sonja Jensen
  5. Dirk Slotboom
  6. Albert Guskov
(2019)
Binding and transport of D-aspartate by the glutamate transporter homologue GltTk
eLife 8:e45286.
https://doi.org/10.7554/eLife.45286
  2. Download BibTeX
  3. Download .RIS

Abstract

Mammalian glutamate transporters are crucial players in neuronal communication as they perform neurotransmitter reuptake from the synaptic cleft. Besides L-glutamate and L-aspartate, they also recognize D-aspartate, which might participate in mammalian neurotransmission and/or neuromodulation. Much of the mechanistic insight in glutamate transport comes from studies of the archaeal homologues GltPh from Pyrococcus horikoshii and GltTk from Thermococcus kodakarensis. Here, we show that GltTk transports D-aspartate with identical Na+ : substrate coupling stoichiometry as L-aspartate, and that the affinities (Kd and Km) for the two substrates are similar. We determined a crystal structure of GltTk with bound D-aspartate at 2.8 Å resolution. Comparison of the L- and D-aspartate bound GltTk structures revealed that D-aspartate is accommodated with only minor rearrangements in the structure of the binding site. The structure explains how the geometrically different molecules L- and D-aspartate are recognized and transported by the protein in the same way.

Data availability

Diffraction data and the derived model have been deposited in PDB under the accession code 6R7R.

The following data sets were generated

Article and author information

Author details

  1. Valentina Arkhipova

    Groningen Biomolecular and Biotechnology Institute (GBB), University of Groningen, Groningen, Netherlands
    Competing interests
    The authors declare that no competing interests exist.

  2. Gianluca Trinco

    Groningen Biomolecular and Biotechnology Institute (GBB), University of Groningen, Groningen, Netherlands
    Competing interests
    The authors declare that no competing interests exist.

  3. Thijs W Ettema

    Groningen Biomolecular and Biotechnology Institute (GBB), University of Groningen, Groningen, Netherlands
    Competing interests
    The authors declare that no competing interests exist.

  4. Sonja Jensen

    Groningen Biomolecular and Biotechnology Institute (GBB), University of Groningen, Groningen, Netherlands
    Competing interests
    The authors declare that no competing interests exist.

  5. Dirk Slotboom

    Groningen Biomolecular and Biotechnology Institute (GBB), University of Groningen, Groningen, Netherlands
    For correspondence
    d.j.slotboom@rug.nl
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5804-9689

  6. Albert Guskov

    Groningen Biomolecular and Biotechnology Institute (GBB), University of Groningen, Groningen, Netherlands
    For correspondence
    a.guskov@rug.nl
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2340-2216

Funding

Nederlandse Organisatie voor Wetenschappelijk Onderzoek (723014.002)

  • Albert Guskov

Nederlandse Organisatie voor Wetenschappelijk Onderzoek (865.11.001)

  • Dirk Slotboom

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. José D Faraldo-Gómez, National Heart, Lung and Blood Institute, National Institutes of Health, United States

Version history

  1. Received: January 17, 2019
  2. Accepted: April 9, 2019
  3. Accepted Manuscript published: April 10, 2019 (version 1)
  4. Version of Record published: April 24, 2019 (version 2)

Copyright

© 2019, Arkhipova et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,436
    views
  • 281
    downloads
  • 23
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Valentina Arkhipova
  2. Gianluca Trinco
  3. Thijs W Ettema
  4. Sonja Jensen
  5. Dirk Slotboom
  6. Albert Guskov
(2019)
Binding and transport of D-aspartate by the glutamate transporter homologue GltTk
eLife 8:e45286.
https://doi.org/10.7554/eLife.45286

Share this article

https://doi.org/10.7554/eLife.45286

Categories and tags

Further reading

    1. Structural Biology and Molecular Biophysics

    LRMP inhibits cAMP potentiation of HCN4 channels by disrupting intramolecular signal transduction

    Colin H Peters, Rohit K Singh ... John R Bankston
    Research Article

    Lymphoid restricted membrane protein (LRMP) is a specific regulator of the hyperpolarization-activated cyclic nucleotide-sensitive isoform 4 (HCN4) channel. LRMP prevents cAMP-dependent potentiation of HCN4, but the interaction domains, mechanisms of action, and basis for isoform-specificity remain unknown. Here, we identify the domains of LRMP essential for this regulation, show that LRMP acts by disrupting the intramolecular signal transduction between cyclic nucleotide binding and gating, and demonstrate that multiple unique regions in HCN4 are required for LRMP isoform-specificity. Using patch clamp electrophysiology and Förster resonance energy transfer (FRET), we identified the initial 227 residues of LRMP and the N-terminus of HCN4 as necessary for LRMP to associate with HCN4. We found that the HCN4 N-terminus and HCN4-specific residues in the C-linker are necessary for regulation of HCN4 by LRMP. Finally, we demonstrated that LRMP-regulation can be conferred to HCN2 by addition of the HCN4 N-terminus along with mutation of five residues in the S5 region and C-linker to the cognate HCN4 residues. Taken together, these results suggest that LRMP inhibits HCN4 through an isoform-specific interaction involving the N-terminals of both proteins that prevents the transduction of cAMP binding into a change in channel gating, most likely via an HCN4-specific orientation of the N-terminus, C-linker, and S4-S5 linker.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics

    X-ray structure and enzymatic study of a bacterial NADPH oxidase highlight the activation mechanism of eukaryotic NOX

    Isabelle Petit-Hartlein, Annelise Vermot ... Franck Fieschi
    Research Article

    NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from Streptococcus pneumoniae, can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2’s requirement for activation.

    1. Cell Biology
    2. Structural Biology and Molecular Biophysics

    Effect of α-tubulin acetylation on the doublet microtubule structure

    Shun Kai Yang, Shintaroh Kubo ... Khanh Huy Bui
    Research Article

    Acetylation of α-tubulin at the lysine 40 residue (αK40) by αTAT1/MEC-17 acetyltransferase modulates microtubule properties and occurs in most eukaryotic cells. Previous literatures suggest that acetylated microtubules are more stable and damage resistant. αK40 acetylation is the only known microtubule luminal post-translational modification site. The luminal location suggests that the modification tunes the lateral interaction of protofilaments inside the microtubule. In this study, we examined the effect of tubulin acetylation on the doublet microtubule (DMT) in the cilia of Tetrahymena thermophila using a combination of cryo-electron microscopy, molecular dynamics, and mass spectrometry. We found that αK40 acetylation exerts a small-scale effect on the DMT structure and stability by influencing the lateral rotational angle. In addition, comparative mass spectrometry revealed a link between αK40 acetylation and phosphorylation in cilia.