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Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy
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Cryo-EM structures of the human glutamine transporter SLC1A5 (ASCT2) in the outward-facing conformation

Xiaodi Yu1, Olga Plotnikova1, Paul D Bonin1

  • 1Medicine Design, Pfizer Inc, Groton, United States.

Elife
|October 4, 2019
PubMed

Insights

Researchers visualized the Alanine-serine-cysteine transporter 2 (ASCT2, SLC1A5) in its outward-facing state, revealing its L-glutamine transport mechanism and potential cholesterol binding site.

Area of Science:

  • Structural Biology
  • Biochemistry
  • Cancer Biology

Background:

  • Alanine-serine-cysteine transporter 2 (ASCT2, SLC1A5) is crucial for glutamine uptake in cancer cells.
  • ASCT2 regulates the mTORC1 signaling pathway, a key factor in cancer progression.
  • The transporter's function relies on coordinated movements of its transport and scaffold domains.

Purpose of the Study:

  • To determine the cryo-electron microscopy (cryo-EM) structures of human SLC1A5 in its outward-facing conformation.
  • To elucidate the structural basis of L-glutamine transport and substrate recognition by SLC1A5.
  • To investigate the role of specific structural elements, like the ECL2a loop, in the transport cycle.

Main Methods:

  • Cryo-electron microscopy (cryo-EM) to obtain high-resolution structures.
  • Structural analysis of human SLC1A5 in complex with L-glutamine.
  • Comparative analysis with previously determined inward-facing SLC1A5 structures.

Main Results:

  • Presented cryo-EM structures of human SLC1A5 in an outward-facing conformation, bound to L-glutamine.
  • Revealed the conformation of the ECL2a loop, essential for domain movement during transport.
  • Identified conformational changes in the HP2 loop related to substrate recognition.
  • Discovered a potential cholesterol binding site near the domain interface.

Conclusions:

  • The structures provide detailed insights into the SLC1A5 transport mechanism and substrate binding.
  • Understanding SLC1A5 structure and function can inform cancer therapy targeting glutamine metabolism.
  • Comparison of outward- and inward-facing states offers a comprehensive view of the SLC1 transporter family's mechanism.
Keywords:
ASCT2SLC1A5cryo-EMglutamine transporterhumanmolecular biophysicsoutward-facing conformationsolute carrierstructural biology

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