Consensus designs and thermal stability determinants of a human glutamate transporter
- Institut Pasteur de Paris, France
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
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Author details
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.
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.
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Consensus designs and thermal stability determinants of a human glutamate transporter
eLife 7:e40110.
https://doi.org/10.7554/eLife.40110
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