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

Consensus designs and thermal stability determinants of a human glutamate transporter

  1. Erica CIrri
  2. Sébastien Brier
  3. Reda Assal
  4. Juan Carlos Canul-Tec
  5. Julia Chamot-Rooke
  6. Nicolas Reyes  Is a corresponding author
  1. Institut Pasteur de Paris, France
Research Article
  • Cited 8
  • Views 1,626
  • Annotations
Cite this article as: eLife 2018;7:e40110 doi: 10.7554/eLife.40110

Abstract

Human excitatory amino acid transporters (EAATs) take up the neurotransmitter glutamate in the brain and are essential to maintain excitatory neurotransmission. Our understanding of the EAATs' molecular mechanisms has been hampered by the lack of stability of purified protein samples for biophysical analyses. Here, we present approaches based on consensus mutagenesis to obtain thermostable EAAT1 variants that share up to ~ 95% amino acid identity with the wild type transporters, and remain natively folded and functional. Structural analyses of EAAT1 and the consensus designs using hydrogen-deuterium exchange linked to mass spectrometry show that small and highly cooperative unfolding events at the inter-subunit interface rate-limit their thermal denaturation, while the transport domain unfolds at a later stage in the unfolding pathway. Our findings provide structural insights into the kinetic stability of human glutamate transporters, and introduce general approaches to extend the lifetime of human membrane proteins for biophysical analyses.

Data availability

All data generated or analysed during this study are included in the manuscript. Supporting files including the amino acid sequence alignments used in this study are also provided

Article and author information

Author details

  1. Erica CIrri

    Structural Biology and Chemistry, Institut Pasteur de Paris, Paris, France
    Competing interests
    No competing interests declared.

  2. Sébastien Brier

    Structural Biology and Chemistry, Institut Pasteur de Paris, Paris, France
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1758-8237

  3. Reda Assal

    Structural Biology and Chemistry, Institut Pasteur de Paris, Paris, France
    Competing interests
    No competing interests declared.

  4. Juan Carlos Canul-Tec

    Structural Biology and Chemistry, Institut Pasteur de Paris, Paris, France
    Competing interests
    No competing interests declared.

  5. Julia Chamot-Rooke

    Structural Biology and Chemistry, Institut Pasteur de Paris, Paris, France
    Competing interests
    No competing interests declared.

  6. Nicolas Reyes

    Structural Biology and Chemistry, Institut Pasteur de Paris, Paris, France
    For correspondence
    nreyes@pasteur.fr
    Competing interests
    Nicolas Reyes, Is inventor in PCT/FR2018/050371 describing the use of consensus mutagenesis to modify protein thermal stability.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6618-8307

Funding

H2020 Excellent Science (ERC Starting grant 309657)

  • Nicolas Reyes

Centre National de la Recherche Scientifique (UMR 3528)

  • Julia Chamot-Rooke
  • Nicolas Reyes

Agence Nationale de la Recherche (CACSICE grant ANR-11-EQPX-008)

  • Julia Chamot-Rooke
  • Nicolas Reyes

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

Publication history

  1. Received: July 14, 2018
  2. Accepted: October 17, 2018
  3. Accepted Manuscript published: October 18, 2018 (version 1)
  4. Version of Record published: October 31, 2018 (version 2)

Copyright

© 2018, CIrri 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

  • 1,626
    Page views
  • 259
    Downloads
  • 8
    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)

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

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

Categories and tags

Research organism

Further reading

    1. Biochemistry and Chemical Biology
    2. Chromosomes and Gene Expression

    Sumoylation of the human histone H4 tail inhibits p300-mediated transcription by RNA polymerase II in cellular extracts

    Calvin Jon A Leonen et al.
    Research Article Updated

    The post-translational modification of histones by the small ubiquitin-like modifier (SUMO) protein has been associated with gene regulation, centromeric localization, and double-strand break repair in eukaryotes. Although sumoylation of histone H4 was specifically associated with gene repression, this could not be proven due to the challenge of site-specifically sumoylating H4 in cells. Biochemical crosstalk between SUMO and other histone modifications, such as H4 acetylation and H3 methylation, that are associated with active genes also remains unclear. We addressed these challenges in mechanistic studies using an H4 chemically modified at Lys12 by SUMO-3 (H4K12su) and incorporated into mononucleosomes and chromatinized plasmids for functional studies. Mononucleosome-based assays revealed that H4K12su inhibits transcription-activating H4 tail acetylation by the histone acetyltransferase p300, as well as transcription-associated H3K4 methylation by the extended catalytic module of the Set1/COMPASS (complex of proteins associated with Set1) histone methyltransferase complex. Activator- and p300-dependent in vitro transcription assays with chromatinized plasmids revealed that H4K12su inhibits both H4 tail acetylation and RNA polymerase II-mediated transcription. Finally, cell-based assays with a SUMO-H4 fusion that mimics H4 tail sumoylation confirmed the negative crosstalk between histone sumoylation and acetylation/methylation. Thus, our studies establish the key role for histone sumoylation in gene silencing and its negative biochemical crosstalk with active transcription-associated marks in human cells.

    1. Biochemistry and Chemical Biology

    The molecular appearance of native TRPM7 channel complexes identified by high-resolution proteomics

    Astrid Kollewe et al.
    Research Article Updated

    The transient receptor potential melastatin-subfamily member 7 (TRPM7) is a ubiquitously expressed membrane protein consisting of ion channel and protein kinase domains. TRPM7 plays a fundamental role in the cellular uptake of divalent cations such as Zn2+, Mg2+, and Ca2+, and thus shapes cellular excitability, plasticity, and metabolic activity. The molecular appearance and operation of TRPM7 channels in native tissues have remained unresolved. Here, we investigated the subunit composition of endogenous TRPM7 channels in rodent brain by multi-epitope affinity purification and high-resolution quantitative mass spectrometry (MS) analysis. We found that native TRPM7 channels are high-molecular-weight multi-protein complexes that contain the putative metal transporter proteins CNNM1-4 and a small G-protein ADP-ribosylation factor-like protein 15 (ARL15). Heterologous reconstitution experiments confirmed the formation of TRPM7/CNNM/ARL15 ternary complexes and indicated that complex formation effectively and specifically impacts TRPM7 activity. These results open up new avenues towards a mechanistic understanding of the cellular regulation and function of TRPM7 channels.

    1. Biochemistry and Chemical Biology
    2. Chromosomes and Gene Expression

    Reversible phosphorylation of cyclin T1 promotes assembly and stability of P-TEFb

    Fang Huang et al.
    Research Article

    The positive transcription elongation factor b (P-TEFb) is a critical co-activator for transcription of most cellular and viral genes, including those of HIV. While P-TEFb is regulated by 7SK snRNA in proliferating cells, P-TEFb is absent due to diminished levels of CycT1 in quiescent and terminally differentiated cells, which has remained unexplored. In these cells, we found that CycT1 not bound to CDK9 is rapidly degraded. Moreover, productive CycT1:CDK9 interactions are increased by PKC mediated phosphorylation of CycT1 in human cells. Conversely, dephosphorylation of CycT1 by PP1 reverses this process. Thus, PKC inhibitors or removal of PKC by chronic activation results in P-TEFb disassembly and CycT1 degradation. This finding not only recapitulates P-TEFb depletion in resting CD4+ T cells but also in anergic T cells. Importantly, our studies reveal mechanisms of P-TEFb inactivation underlying T cell quiescence, anergy, and exhaustion as well as proviral latency and terminally differentiated cells.