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.
Highlights
Integral membrane proteins are essential to the life of any cell and constitute drug targets of paramount importance (Yildirim et al, 2007)
To increase the stability of the transporters in detergent solutions, we identified the most frequent amino acids among representative animal SoLute Carrier 1 (SLC1) structural homologs, and simultaneously exchanged all residues within the expected helical regions of EAAT1WT for consensus amino acids
Figure 1. | EAAT1 consensus mutants. (a-b) Residues exchanged for consensus amino acids in EAAT1CO (a) and EAAT1COCO (b) are mapped into the structure of the EAAT1CRYST (PDB 5LLM) trimer viewed from the extracellular medium, as well as the scaffold and the transport domains viewed from the membrane
Summary
Integral membrane proteins are essential to the life of any cell and constitute drug targets of paramount importance (Yildirim et al, 2007). Current methods to overcome this problem involve mainly engineering stability through mutagenesis using amino acid scanning and directed evolution (Scott et al, 2013), and have been successfully used to stabilize both prokaryotic (Zhou and Bowie, 2000; Faham et al, 2004) and animal integral membrane proteins (Magnani et al, 2008; Magnani et al, 2016; Sarkar et al, 2008; Scott and Pluckthun, 2013; Coleman et al, 2016). These approaches rely on screening of a large number of mutants and high-throughput methods to probe protein expression and stability, which makes them labor-intensive and sometimes impractical
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