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Consensus designs and thermal stability determinants of a human glutamate transporter.

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.

SUBMITTER: Cirri E

PROVIDER: S-EPMC6209432 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Consensus designs and thermal stability determinants of a human glutamate transporter.

Cirri Erica E Brier Sébastien S Assal Reda R Canul-Tec Juan Carlos JC Chamot-Rooke Julia J Reyes Nicolas N

eLife 20181018

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 native ...[more]

PMID: 30334738

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Project description:Introduction: The human organic cation transporter 2 (OCT2) is involved in the transport of endogenous quaternary amines and positively charged drugs across the basolateral membrane of proximal tubular cells. In the absence of a structure, the progress in unraveling the molecular basis of OCT2 substrate specificity is hampered by the unique complexity of OCT2 binding pocket, which seemingly contains multiple allosteric binding sites for different substrates. Here, we used the thermal shift assay (TSA) to better understand the thermodynamics governing OCT2 binding to different ligands. Methods: Molecular modelling and in silico docking of different ligands revealed two distinct binding sites at OCT2 outer part of the cleft. The predicted interactions were assessed by cis-inhibition assay using [3H]1-methyl-4-phenylpyridinium ([3H]MPP+) as a model substrate, or by measuring the uptake of radiolabeled ligands in intact cells. Crude membranes from HEK293 cells harboring human OCT2 (OCT2-HEK293) were solubilized in n-Dodecyl-β-D-Maltopyranoside (DDM), incubated with the ligand, heated over a temperature gradient, and then pelleted to remove heat-induced aggregates. The OCT2 in the supernatant was detected by western blot. Results: Among the compounds tested, cis-inhibition and TSA assays showed partly overlapping results. Gentamicin and methotrexate (MTX) did not inhibit [3H]MPP+ uptake but significantly increased the thermal stabilization of OCT2. Conversely, amiloride completely inhibited [3H]MPP+ uptake but did not affect OCT2 thermal stabilization. [3H]MTX intracellular level was significantly higher in OCT2-HEK293 cells than in wild type cells. The magnitude of the thermal shift (ΔTm) did not provide information on the binding. Ligands with similar affinity showed markedly different ΔTm, indicating different enthalpic and entropic contributions for similar binding affinities. The ΔTm positively correlated with ligand molecular weight/chemical complexity, which typically has high entropic costs, suggesting that large ΔTm reflect a larger displacement of bound water molecules. Discussion: In conclusion, TSA might represent a viable approach to expand our knowledge on OCT2 binding descriptors.

Dataset Information

Consensus designs and thermal stability determinants of a human glutamate transporter.

Publications

Consensus designs and thermal stability determinants of a human glutamate transporter.

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