Consensus designs and thermal stability determinants of a human glutamate transporter

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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.

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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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Erica CIrri

Structural Biology and Chemistry, Institut Pasteur de Paris, Paris, France

Competing interests
No competing interests declared.
Sébastien Brier

Structural Biology and Chemistry, Institut Pasteur de Paris, Paris, France

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0003-1758-8237
Reda Assal

Structural Biology and Chemistry, Institut Pasteur de Paris, Paris, France

Competing interests
No competing interests declared.
Juan Carlos Canul-Tec

Structural Biology and Chemistry, Institut Pasteur de Paris, Paris, France

Competing interests
No competing interests declared.
Julia Chamot-Rooke

Structural Biology and Chemistry, Institut Pasteur de Paris, Paris, France

Competing interests
No competing interests declared.
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.

"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.

Copyright

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.