Large domain movements through the lipid bilayer mediate substrate release and inhibition of glutamate transporters

  1. Xiaoyu Wang
  2. Olga Boudker  Is a corresponding author
  1. Weill Cornell Medicine, United States

Abstract

Glutamate transporters are essential players in glutamatergic neurotransmission in the brain, where they maintain extracellular glutamate below cytotoxic levels and allow for rounds of transmission. The structural bases of their function are well established, particularly within a model archaeal homologue, sodium and aspartate symporter GltPh. However, the mechanism of gating on the cytoplasmic side of the membrane remains ambiguous. We report Cryo-EM structures of GltPh reconstituted into nanodiscs, including those structurally constrained in the cytoplasm-facing state and either apo, bound to sodium ions only, substrate, or blockers. The structures show that both substrate translocation and release involve movements of the bulky transport domain through the lipid bilayer. They further reveal a novel mode of inhibitor binding and show how solutes release is coupled to protein conformational changes. Finally, we describe how domain movements are associated with the displacement of bound lipids and significant membrane deformations, highlighting the potential regulatory role of the bilayer.

Data availability

Cryo-EM coordinate files and electron density maps have been deposited in PDB under the following codes:GltPh OFS-TBOA: PDB 6X17, EMD-21991GltPh IFS-Asp: PDB 6X15, EMD-21989GltPh IFS-TBOA: PDB 6X16, EMD-21990GltPh IFS-TFB-TBOA: PDB 6X14, EMD-21988GltPh IFS-Na: PDB 6X13, EMD-21987GltPh IFS-Apo-open: PDB 6X12, EMD-21986

Article and author information

Author details

  1. Xiaoyu Wang

    Physiology and Biophysics, Weill Cornell Medicine, New York, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8745-8238
  2. Olga Boudker

    Department of Physiology and Biophysics, Weill Cornell Medicine, New York, United States
    For correspondence
    olb2003@med.cornell.edu
    Competing interests
    Olga Boudker, Senior editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6965-0851

Funding

National Institutes of Health (R37NS085318)

  • Olga Boudker

National Institutes of Health (R01NS064357)

  • Olga Boudker

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

Reviewing Editor

  1. Lucy R Forrest, National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States

Publication history

  1. Received: April 30, 2020
  2. Accepted: November 5, 2020
  3. Accepted Manuscript published: November 6, 2020 (version 1)
  4. Version of Record published: November 23, 2020 (version 2)

Copyright

© 2020, Wang & Boudker

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

  • 1,753
    Page views
  • 297
    Downloads
  • 21
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

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. Xiaoyu Wang
  2. Olga Boudker
(2020)
Large domain movements through the lipid bilayer mediate substrate release and inhibition of glutamate transporters
eLife 9:e58417.
https://doi.org/10.7554/eLife.58417

Further reading

    1. Structural Biology and Molecular Biophysics
    Amy Osterman, Alfonso Mondragón
    Research Article

    Topoisomerase V is a unique topoisomerase that combines DNA repair and topoisomerase activities. The enzyme has an unusual arrangement, with a small topoisomerase domain followed by 12 tandem (HhH)2 domains, which include three AP lyase repair domains. The uncommon architecture of this enzyme bears no resemblance to any other known topoisomerase. Here we present structures of topoisomerase V in complex with DNA. The structures show that the (HhH)2 domains wrap around the DNA and in this manner appear to act as a processivity factor. There is a conformational change in the protein to expose the topoisomerase active site. The DNA bends sharply to enter the active site, which melts the DNA and probably facilitates relaxation. The structures show a DNA binding mode not observed before and provide information on the way this atypical topoisomerase relaxes DNA. In common with type IB enzymes, topoisomerase V relaxes DNA using a controlled rotation mechanism, but the structures show that topoisomerase V accomplishes this in different manner. Overall, the structures firmly establish that type IC topoisomerases form a distinct type of topoisomerases, with no similarities to other types at the sequence, structural, or mechanistic level. They represent a completely different solution to DNA relaxation.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Sarah R Hansen et al.
    Research Article

    In eukaryotes, splice sites define the introns of pre-mRNAs and must be recognized and excised with nucleotide precision by the spliceosome to make the correct mRNA product. In one of the earliest steps of spliceosome assembly, the U1 small nuclear ribonucleoprotein (snRNP) recognizes the 5' splice site (5' SS) through a combination of base pairing, protein-RNA contacts, and interactions with other splicing factors. Previous studies investigating the mechanisms of 5' SS recognition have largely been done in vivo or in cellular extracts where the U1/5' SS interaction is difficult to deconvolute from the effects of trans-acting factors or RNA structure. In this work we used co-localization single-molecule spectroscopy (CoSMoS) to elucidate the pathway of 5' SS selection by purified yeast U1 snRNP. We determined that U1 reversibly selects 5' SS in a sequence-dependent, two-step mechanism. A kinetic selection scheme enforces pairing at particular positions rather than overall duplex stability to achieve long-lived U1 binding. Our results provide a kinetic basis for how U1 may rapidly surveil nascent transcripts for 5' SS and preferentially accumulate at these sequences rather than on close cognates.