Consensus designs and thermal stability determinants of a human glutamate transporter

Author:

Cirri Erica12,Brier Sébastien34ORCID,Assal Reda12,Canul-Tec Juan Carlos12,Chamot-Rooke Julia34,Reyes Nicolas12ORCID

Affiliation:

1. Molecular Mechanisms of Membrane Transport Laboratory, Institut Pasteur, Paris, France

2. UMR 3528, CNRS, Institut Pasteur, Paris, France

3. Mass Spectrometry for Biology Unit, Institut Pasteur, Paris, France

4. USR 2000, CNRS, Institut Pasteur, 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.

Funder

H2020 Excellent Science

Centre National de la Recherche Scientifique

Agence Nationale de la Recherche

Publisher

eLife Sciences Publications, Ltd

Subject

General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Medicine,General Neuroscience

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