Related Experiment Video
Updated: May 11, 2025
13:16
Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles
Published on: December 31, 2019
9.2K
Structural basis of excitatory amino acid transporter 3 substrate recognition
Biao Qiu1,2, Olga Boudker1,2
1Department of Physiology & Biophysics, Weill Cornell Medicine, New York, NY 10021.
Summary
Excitatory amino acid transporters (EAATs) like EAAT3 bind L-cysteine via specific structural changes. Cryo-EM reveals EAAT3 conformational states and substrate-dependent transport, but not R-2HG binding.
Area of Science:
- Structural biology
- Neuroscience
- Biochemistry
Background:
- Excitatory amino acid transporters (EAATs) regulate neurotransmitter levels and ion gradients.
- EAAT3 is unique in efficiently transporting L-cysteine, crucial for glutathione synthesis.
- EAAT3's role in transporting the oncometabolite R-2-hydroxyglutarate (R-2HG) is under investigation.
Purpose of the Study:
- To elucidate the structural mechanisms of substrate recognition and transport by EAAT3.
- To investigate the binding and transport of L-cysteine and R-2HG by EAAT3.
Main Methods:
- Cryogenic electron microscopy (cryo-EM) to determine the structures of EAAT3 bound to substrates.
- Biochemical assays to assess substrate binding and transport.
Main Results:
- Cryo-EM structures reveal EAAT3 binds L-cysteine in its thiolate form.
- EAAT3 utilizes fine-tuning of local conformations for substrate recognition.
- No R-2HG binding or transport was observed with purified human EAAT3.
- Structures show EAAT3 adopts various conformational states, including outward-facing and occluded states, influenced by substrate binding.
- Gate closure and sodium ion binding occur post-substrate binding.
Conclusions:
- EAAT3 exhibits substrate-dependent conformational dynamics affecting translocation rates.
- The structural basis for L-cysteine transport is elucidated.
- Further research is needed to clarify EAAT3's interaction with R-2HG.
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